Application of neoadjuvant chemotherapy improves survival rates and functional outcome in patients with non-metastatic, high-grade osteosarcoma of the extremities

Before consideration of limb preservation, the patient needs to be appropriately staged and assessed through a multidisciplinary approach. Some elements of the disease may warrant concern, including relative contraindications to such procedures. However, surgical treatment associated with a limb-sparing operation is also associated with a significant number of complications and requires extensive rehabilitation. The main risk of limbsalvage procedures is that complications sometimes may cause a delay of chemotherapy

Depending on cytological or histo-pathological features of the tumor matrix or tumor cells, osteosarcomas are divided into two groups. In the first group there are patients with *lowgrade osteosarcoma* and surgery alone has the primary role of treatment. In the second group there are patients with *high-grade osteosarcoma*. In this group of patients "sandwich therapy" is strongly preferred (neoadjuvant chemotherapy - surgery - adjuvant chemotherapy)

*Conventional or "classic"* osteosarcoma is the most prevalent type in children and adolescents (up to 80% of all cases). This type of osteosarcoma originates from the intramedullary cavity and is typically high-grade (Fig. 1a). An osteoblastic and/or osteolytic lesion with vast cortical destruction and various amount of soft tissue extension dominates on X-rays. Histopathologic examination demonstrates malignant mesenhimal cells, spindle to polyhedral in shape, with pleomorphyc nuclei and occasional mitotic figures. Evidence of direct bone or osteoid production from the mesenhim is crucial for diagnosis (Fig. 1b, 1c). World Health Organization has further subcategorized high-grade intramedullary osteosarcoma since 2002, depending on the predominant extra cellular matrix on: *osteoblastic* (approximately 50% of cases), *chondroblastic* (25% of cases) or *fibroblastic* (25% of cases). (Fletcher et al., 2002). *Teleangiectatic* osteosarcoma is a rare variant accounting for approximately 4% of all osteosarcoma cases in children and adolescents. Very often they are associated with pathological fracture of the first presentation. Eccentric osteolytic lesion on the metaphysis, with destruction and expansion of the eroded cortex dominates on x-ray (Fig. 1b). Histopathologic examination reveals a malignant tumor with multiple dilated hemorrhagic sinuses as well as a scarce amount of high-grade osteosarcoma cells and rare osteoid formation within the septa. These radiographic and histo-pathologic features resemble an

*Low-grade* intramedullary osteosarcoma constitutes 1 to 2% of all osteosarcoma cases and generally affects patients in the third or fourth decade. Lesions most commonly affect the distal femur and proximal tibia, with relatively unaggressive radiographic appearance, resembling fibrous dysplasia ("fibrous dysplasia-like" osteosarcoma). Histo-pathological features consist of well-differentiated cells dispersed within woven microtrabeculae of bone and fibrous stroma. Small amounts of osteoid, mitotic atypia and mitoses can also be seen

(Wittig et al., 2002).

(Sæter al., 1996).

**2. Osteosarcoma subtypes and characteristics**

(Enneking, 1975; Bacci et al., 1993; Messerschmitt et al., 2009)**.**

**2.1 Intramedullary osteosarcoma**

aneurismal bone cyst which is cdaracteristic.

(Fletcher et al., 2002).

*Small-cell* osteosarcoma is a rare variant constituting <1.5% of all osteosarcoma cases. This subtype is similar to the high-grade osteosarcoma, with the same site or age distribution and aggressive biologic behavior. The lesion is osteolytic with destruction of cortex and variable sclerosis. MRI reveals large spindle or circumferential tumor mass, similar to Ewing sarcoma. Small, round, malignant cells within an osteoid matrix make the histo-pathological diagnosis problematic. To differentiate this osteosarcoma from Ewing sarcoma, direct mesenhimal production of osteoid must be found, because this osteosarcoma is positive for CD 99 immuno-histochemical stains (Fletcher et al., 2002).

Fig. 1. **a)** X-ray of conventional intramedullary osteosarcoma (osteoblastic lesion with vast cortical destruction and soft tissue edema visible on x-rays); **b)** X-ray of teleangiectatic osteosarcoma with osteolytic lesion on the metaphysis of distal femur, destruction, expansion of the eroded cortex and Codman's periosteal reaction (arrow); **c)** Typical histopathological feature of osteosarcoma is osteoid formations directly from the mesenhime; **d)** Atypical osteoid formation in high-grade anaplastic osteosarcoma typifies the diagnosis.

A few osteosarcomas (less than 1% of all cases) have so many giant cells that they can be mistaken for giant cell tumors. Cytological atypia of the mononuclear cells can be very subtle and rare. It is important to remember the possibility of a *giant cell-rich osteosarcoma* when giant cell tumor-like lesion occurs in an unusual location and age, such as the metaphysis in children (Unni, 1998).

Effects of Neoadjuvant Chemotherapy in

**3. Imaging**

(Messerschmitt et al., 2009).

High-Grade Non-Metastatic Osteosarcoma of Extremities 217

and tumor extension into surrounding soft tissues. In earlier stages of the disease, destruction of the underlying cortex is absent, but with advanced lesions involvement of the medullary cavity is possible (Fig. 2c). The histological features are those of high-grade osteosarcoma, demonstrating spindle cells with atypia and a varying amount of osteoid**.** A high-grade surface osteosarcoma cannot be differentiated from a conventional osteosarcoma

There are various radiological imaging techniques available to achieve an accurate diagnosis and staging of osteosarcoma and to detect local recurrence or distant metastases. Most commonly used are: plain-film radiographs (as "gold" standard), Tc-99m bone scintigrapy, CT of the affected site or of the lungs and CT or conventional angiography. Positron emission tomography (PET-scan) and Thallium scintigraphy have been seldom used due to questionable results in evaluating early osteosarcoma metastases or due to their high-cost

in histological findings alone (Fletcher et al., 2002; Samardziski et al., 2009).

a) b)

confluent cloud-like densities, destroing the bone completely.

Fig. 3. **a)** X-ray in two orthogonal planes of typical mixed sclerotic end lytic osteosarcoma of the distal femur. Tumor has penetrated bone and formed a soft tissue mass with Codman's triangles. **b)** Frontal plane X-ray of osteosarcoma situated on proximal humerus with small,

Plain-film radiographs in two ortogonal plains show mixed osteosclerotic and osteolytic tumor, affecting the metaphysis of the bone (although primarily sclerotic or lytic osteosarcomas can occur). The lesion is ill defined from the surrounding bone, affecting and destroing the cortex, with typical small, irregular, confluent, cloud-like densities. If the cortex is completely eroded the lesion forms a soft tissue mass extruding from the bone into the surrounding tissue and may demonstrate ossification detectable on the

#### **2.2 Surface osteosarcoma**

*Parosteal osteosarcoma* arises on the outer surface of the long bone metaphysis, sparing the medullary canal (Fig. 2a). The peak incidence is in the second and third decade, affecting more females than males.(10) Parosteal osteosarcoma is most commonly seen as a juxtracortical variety and constitutes 1 to 6% of all osteosarcoma cases. Radiographs classically show densely ossified and lobulated mass on the posterior surface of the femur. Sometimes slow-growing tumors may encircle the bone. A low-grade, well differentiated fibrous stroma with osseous components is regularly seen on the histo-pathologic examination. Parallel orientation of trabeculae with additional cartilaginous differentiation is very common (Fig. 2d).

Fig. 2. **a)** Frontal and lateral x-ray of the periosteal osteosarcoma of right distal femur; **b)** X-ray in frontal and lateral view of parosteal osteosarcoma of the proximal tibia; **c)** Frontal and lateral x-ray of high-grade surface osteosarcoma on the right distal femur; **d)** Parosteal osteosarcoma showing parallel osteoid trabeculae embedded in fibroblastic stroma (HE, x100).

*Periosteal osteosarcoma* constitutes 1 to 2% of all osteosarcoma cases and is usually more aggressive than the parosteal variant. A radiolucent lesion is located on the distal femur or proximal tibia, sparing the medullar cavity (Fig. 2b). Codman triangle and "sunburst" periosteal reaction are common radiographic features. Histo-pathologic evaluation demonstrates an intermediate-grade tumor, rich with cartilaginous matrix and rare osteoid fields.

*High-grade surface osteosarcoma* constitutes <1% of all osteosarcomas with the predominant site around the knee. Radiographic analysis shows surface lesion with partial mineralization and tumor extension into surrounding soft tissues. In earlier stages of the disease, destruction of the underlying cortex is absent, but with advanced lesions involvement of the medullary cavity is possible (Fig. 2c). The histological features are those of high-grade osteosarcoma, demonstrating spindle cells with atypia and a varying amount of osteoid**.** A high-grade surface osteosarcoma cannot be differentiated from a conventional osteosarcoma in histological findings alone (Fletcher et al., 2002; Samardziski et al., 2009).

#### **3. Imaging**

216 Neoadjuvant Chemotherapy – Current Applications in Clinical Practice

*Parosteal osteosarcoma* arises on the outer surface of the long bone metaphysis, sparing the medullary canal (Fig. 2a). The peak incidence is in the second and third decade, affecting more females than males.(10) Parosteal osteosarcoma is most commonly seen as a juxtracortical variety and constitutes 1 to 6% of all osteosarcoma cases. Radiographs classically show densely ossified and lobulated mass on the posterior surface of the femur. Sometimes slow-growing tumors may encircle the bone. A low-grade, well differentiated fibrous stroma with osseous components is regularly seen on the histo-pathologic examination. Parallel orientation of trabeculae with additional cartilaginous differentiation

a) b)

c) d)

Fig. 2. **a)** Frontal and lateral x-ray of the periosteal osteosarcoma of right distal femur; **b)** X-ray in frontal and lateral view of parosteal osteosarcoma of the proximal tibia; **c)** Frontal and lateral x-ray of high-grade surface osteosarcoma on the right distal femur; **d)** Parosteal

osteosarcoma showing parallel osteoid trabeculae embedded in fibroblastic stroma (HE, x100). *Periosteal osteosarcoma* constitutes 1 to 2% of all osteosarcoma cases and is usually more aggressive than the parosteal variant. A radiolucent lesion is located on the distal femur or proximal tibia, sparing the medullar cavity (Fig. 2b). Codman triangle and "sunburst" periosteal reaction are common radiographic features. Histo-pathologic evaluation demonstrates an intermediate-grade tumor, rich with cartilaginous matrix and rare

*High-grade surface osteosarcoma* constitutes <1% of all osteosarcomas with the predominant site around the knee. Radiographic analysis shows surface lesion with partial mineralization

**2.2 Surface osteosarcoma**

is very common (Fig. 2d).

osteoid fields.

There are various radiological imaging techniques available to achieve an accurate diagnosis and staging of osteosarcoma and to detect local recurrence or distant metastases. Most commonly used are: plain-film radiographs (as "gold" standard), Tc-99m bone scintigrapy, CT of the affected site or of the lungs and CT or conventional angiography. Positron emission tomography (PET-scan) and Thallium scintigraphy have been seldom used due to questionable results in evaluating early osteosarcoma metastases or due to their high-cost (Messerschmitt et al., 2009).

Fig. 3. **a)** X-ray in two orthogonal planes of typical mixed sclerotic end lytic osteosarcoma of the distal femur. Tumor has penetrated bone and formed a soft tissue mass with Codman's triangles. **b)** Frontal plane X-ray of osteosarcoma situated on proximal humerus with small, confluent cloud-like densities, destroing the bone completely.

Plain-film radiographs in two ortogonal plains show mixed osteosclerotic and osteolytic tumor, affecting the metaphysis of the bone (although primarily sclerotic or lytic osteosarcomas can occur). The lesion is ill defined from the surrounding bone, affecting and destroing the cortex, with typical small, irregular, confluent, cloud-like densities. If the cortex is completely eroded the lesion forms a soft tissue mass extruding from the bone into the surrounding tissue and may demonstrate ossification detectable on the

Effects of Neoadjuvant Chemotherapy in

**4. Biopsy and histo-pathological diagnosis**

osteosarcoma) may be very delicate task (Fletcher et al. 2002).

G2 - High-grade

**T2**-Extraosseus; **M0**-No metastases; **M1**-With metastases.

**IIA**

**5. Staging**

High-Grade Non-Metastatic Osteosarcoma of Extremities 219

tumor is visible as well as reactive zone and tissue edema (Fig. 4b). Neurovascular structures and especially neurovascular encasement can be determined and it can help in the process of planning the definite method of treatment. (If encasement is present,

Obtaining an MRI prior to surgical resection permits accurate planning of the osteotomy and gross tumor excision (together with the reactive zone) for achieving a "wide" surgical margin. Skip metastases on MRI are easily detectable in the same bone or in the adjacent joint and then a more extensive resection is required. MRI studies are inferior to highdefinition CT scans for lung metastases detection (Di Caprio & Friedlander, 2003, 2003).

In spite of the risk for tumor spreading, biopsy is the key step in the diagnosis and treatment of osteosarcoma. Improperly performed biopsy may compromise the treatment plan. It is mandatory to place the biopsy in the line of definite surgical approach for osteosarcoma resection. A specimen taken from the necrotic tissue or from reactive zone (around the osteosarcoma) may be non informative. A large needle biopsy is sometimes preferable, because it is less invasive, with lower risk for skin necrosis, infection and pathological fracture. If no representative osteosarcoma tissue is obtained, an open biopsy will increase the risk of complications or local spreading of the tumor. The best results are achieved when all the biopsy samples are obtained by the same orthopedic oncologist (surgeon) who will perform the definite surgical procedure (Mankin et al., 1982; Campanacci, 1999). One must state that obtaining an accurate histo-pathological diagnosis of the tumor (especially of

**Stadium Grade Localisation Metastases IA** G1 - Low-grade T1 - Intraosseus M0 - No metastases **IB** T2 - Extraosseus M0 - No metastases

**IIB** T2 – Extraosseus M0 - No metastases **IIIA** G1-2 T1 -Intraosseus M1 - With metastases **IIIB** G1-2 T2 - Extraosseus M1 - With metastases

Table 1. Enneking's surgical staging system: **G1**-Low-grade; **G2**-High-grade; **T1**-Intraosseus;

The American National Comprehensive Cancer Network recommends plain radiographs of the lesion and lungs, MRI scan of the extremity, CT scan of the tumor site and of the lungs, and radionuclide bone scan. Technetium Tc-99 methylene diphosphonate scintigraphy will reveal increased metabolic activity at the site of the tumor, but also at the site of distant skip or bone metastases. Thallium (Tl-201) is a potassium analog, actively transported by the sodium-potassium adenosine triphosphatase (ATP) pump. This radioisotope is well accumulated in benign or malignant tumors, reflecting tumor activity. Nevertheless,

T1 - Intraosseus M0 - No metastases

amputation or wide resection with vascular reconstruction is obligatory).

radiographs (Fig. 3). The destruction may be so advanced that pathological fractures or complete bone erosion could be present (Fig. 3b). There is a typical periosteal reaction due to aggressive expansion of the tumor, forming hairy, sun-ray or velvet-like specula of neoplastic bone. In some cases "Codman's triangles" (arrows on Fig. 3a) are present**.** Plain-film radiographs are used in correlation with bone scintigraphy and CT to detect local recurrence or bone and lung metastases. Additional data for diagnosis and decisionmaking process can be obtained using a "computer assisted diagnosis" in analysis of the x-rays (Lodwick et al., 1963; Samardziski et al., 2004).

Computer tomography (CT) scann of the affected extremity is useful in visualization of the intra and extra-osseous extent of the tumor, especially when extensive necrosis and surrounding edema are present. In this case CT may be superior to MRI. High-definition CT scans can obtain a three dimensional view of the tumor in relation to adjacent neurovascular structures, especially when contrast medium is used (Fig. 4a). All patients with osteosarcoma should undergo CT scanning of the chest and lungs for detection of pulmonary metastases, for diagnosis and staging. After surgery has been performed in patients with non-metastatic osteosarcoma, CT scans of the lung should be repeated every three to six months for two years (Wittig et al., 2002).

Fig. 4. **a)** CT scans of proximal femur osteosarcoma, with visualization of the superficial and deep femoral artery **b)** T2-weighted MRI image of distal femur osteosarcoma with no extraosseous extension.

With magnetic resonance imaging (MRI), standard T1 and T2-weighted and fat-suppressed images are obtained to visualize the affected bone and surrounding tissue with osteosarcoma. Based on MRI studies, the intra-osseous and extra-osseous extent of the

radiographs (Fig. 3). The destruction may be so advanced that pathological fractures or complete bone erosion could be present (Fig. 3b). There is a typical periosteal reaction due to aggressive expansion of the tumor, forming hairy, sun-ray or velvet-like specula of neoplastic bone. In some cases "Codman's triangles" (arrows on Fig. 3a) are present**.** Plain-film radiographs are used in correlation with bone scintigraphy and CT to detect local recurrence or bone and lung metastases. Additional data for diagnosis and decisionmaking process can be obtained using a "computer assisted diagnosis" in analysis of the

Computer tomography (CT) scann of the affected extremity is useful in visualization of the intra and extra-osseous extent of the tumor, especially when extensive necrosis and surrounding edema are present. In this case CT may be superior to MRI. High-definition CT scans can obtain a three dimensional view of the tumor in relation to adjacent neurovascular structures, especially when contrast medium is used (Fig. 4a). All patients with osteosarcoma should undergo CT scanning of the chest and lungs for detection of pulmonary metastases, for diagnosis and staging. After surgery has been performed in patients with non-metastatic osteosarcoma, CT scans of the lung should be repeated every

a) b)

Fig. 4. **a)** CT scans of proximal femur osteosarcoma, with visualization of the superficial and deep femoral artery **b)** T2-weighted MRI image of distal femur osteosarcoma with no extra-

With magnetic resonance imaging (MRI), standard T1 and T2-weighted and fat-suppressed images are obtained to visualize the affected bone and surrounding tissue with osteosarcoma. Based on MRI studies, the intra-osseous and extra-osseous extent of the

x-rays (Lodwick et al., 1963; Samardziski et al., 2004).

three to six months for two years (Wittig et al., 2002).

osseous extension.

tumor is visible as well as reactive zone and tissue edema (Fig. 4b). Neurovascular structures and especially neurovascular encasement can be determined and it can help in the process of planning the definite method of treatment. (If encasement is present, amputation or wide resection with vascular reconstruction is obligatory).

Obtaining an MRI prior to surgical resection permits accurate planning of the osteotomy and gross tumor excision (together with the reactive zone) for achieving a "wide" surgical margin. Skip metastases on MRI are easily detectable in the same bone or in the adjacent joint and then a more extensive resection is required. MRI studies are inferior to highdefinition CT scans for lung metastases detection (Di Caprio & Friedlander, 2003, 2003).

### **4. Biopsy and histo-pathological diagnosis**

In spite of the risk for tumor spreading, biopsy is the key step in the diagnosis and treatment of osteosarcoma. Improperly performed biopsy may compromise the treatment plan. It is mandatory to place the biopsy in the line of definite surgical approach for osteosarcoma resection. A specimen taken from the necrotic tissue or from reactive zone (around the osteosarcoma) may be non informative. A large needle biopsy is sometimes preferable, because it is less invasive, with lower risk for skin necrosis, infection and pathological fracture. If no representative osteosarcoma tissue is obtained, an open biopsy will increase the risk of complications or local spreading of the tumor. The best results are achieved when all the biopsy samples are obtained by the same orthopedic oncologist (surgeon) who will perform the definite surgical procedure (Mankin et al., 1982; Campanacci, 1999). One must state that obtaining an accurate histo-pathological diagnosis of the tumor (especially of osteosarcoma) may be very delicate task (Fletcher et al. 2002).

Table 1. Enneking's surgical staging system: **G1**-Low-grade; **G2**-High-grade; **T1**-Intraosseus; **T2**-Extraosseus; **M0**-No metastases; **M1**-With metastases.

#### **5. Staging**

The American National Comprehensive Cancer Network recommends plain radiographs of the lesion and lungs, MRI scan of the extremity, CT scan of the tumor site and of the lungs, and radionuclide bone scan. Technetium Tc-99 methylene diphosphonate scintigraphy will reveal increased metabolic activity at the site of the tumor, but also at the site of distant skip or bone metastases. Thallium (Tl-201) is a potassium analog, actively transported by the sodium-potassium adenosine triphosphatase (ATP) pump. This radioisotope is well accumulated in benign or malignant tumors, reflecting tumor activity. Nevertheless,

Effects of Neoadjuvant Chemotherapy in

resection leaves a nonfunctional limb.

Caprio & Friedlander, 2003; Longhi et al., 2006).

**6.2 Surgery**

1996; Bacci et al., 2001; Messerschmitt et al., 2009).

High-Grade Non-Metastatic Osteosarcoma of Extremities 221

chemotherapeutic drugs to improve patient survival rates (Jaffe et al., 1989; Sæter et al.,

The two primary surgical options are tumor resection with limb-salvage, and amputation. Surgical margins in excision should encompass resection of tumor, pseudo capsule, and a cuff of normal tissue en block. Meticulous preoperative planning before the biopsy and definitive surgery will ensure better results. Prior to the emergence of limb-salvage surgery in the 1970s, amputation of the affected limb was considered the definitive surgical intervention. Amputation remains the indicated treatment when disease-free marginal

The limb-salvage surgery for osteosarcoma patients is possible due to the use of preoperative (neoadjuvant) chemotherapy and to advancement in musculoskeletal imaging, prosthetic implant design and surgical technique (Fig. 5). Today limb sparing surgery is possible for >85% of patients with extremities localized osteosarcomas (Bacci et al., 2006; Di

Surgical treatment has to be planed keeping in mind four basic principles of limb-salvage procedures: local recurrence should be no greater and survival no worse than by amputation; the procedure, or treatment of its complications, should not delay adjuvant therapy; reconstruction should be enduring and not associated with a large number of local complications requiring secondary procedures and frequent hospitalizations; function of the limb should approach that obtained by amputation, although body image, patient

There are a few relative contraindications to be taken in the consideration for limb-salvage surgery: wrong site or ill-planed biopsy; massive encasement of neurovascular bundles; extensive tumour involvement in soft tissue, muscles or skin; complex or complicated (i.e. with infection) pathological fractures; expected inequalities of the extremities more than 8 cm; and exceptionally poor effect of the neoadjuvant chemotherapy. In the process of decision making for limb-salvage surgery versus amputation the "rule of three" can be very helpful. For extremity survival the bone (1), nerves (2), blood vessels (3), and muscle and skin (4) are necessary to be preserved. If osteosarcoma involves one or two of the former structures, limb preserving is possible. If any three of the former are involved, amputation

When "negative" tumor margins are obtained, a large skeletal defect is often present, requiring reconstruction of the bone, muscles, other soft tissues, and the skin. Patient age, tumor location and extent of resection, determine the appropriate surgical alternatives. The extent of the disease, anatomical location of the tumor and the patient's age and psychological profile define the most appropriate surgical procedures. Several options for limb-sparing are available: resection arthrodesis and other similar techniques with special indications (Fig. 7c), modular or special expanding endoprostheses (Fig. 5), cortico-

For the patients who can't satisfy the principles of limb preservation, ablative surgery has to be taken into consideration. For these patients disarticulation of the hip or shoulder griddle, rotationplasty, femoral or below knee, humeral or other amputations are far more appropriate (González-Heranz et al., 1995; Sæter et al., 1996; Wittig et al., 2002;

preference and life style may influence the decision (González-Heranz et al. 1995).

must be taken in consideration (Di Caprio & Friedlander, 2003).

spongious or bulk auto graft.

Samardziski et al. 2009).

Thallium scanning is mostly used for monitoring the response to neoadjuvant chemotherapy (especially when MRI is not helpful).

Osteosarcoma can be divided into high-grade or low-grade variants, depending of cellularity, pleomorphism, anaplasia and number of mitoses (Fletcher et al. 2002). This fact and the data for presence or absence of osteosarcoma metastases will be enough to do the Enneking's surgical staging (Table 1). This staging system, first used by the American Musculoskeletal Tumor Society and International Symposium on Limb-Salvage is widely accepted. An alternative system, established by the American Joint Committee on Cancer, can be used with Enneking's staging system (Enneking et al., 1980).

### **6. Treatment**

A multidisciplinary approach is obligatory in the diagnosis and treatment of osteosarcoma. To achieve high standards in treatment there is a need for specialized radiologists, pathologists, orthopedic and other surgeons (specialized in oncology surgery), pediatric oncologists, specialized physical therapist and often social workers (Wittig et Al., 2002). When a proper chemotherapy and surgery protocol are followed, survival rates surpass 70%. High-grade osteosarcoma patients without clinically detectable lung metastases are presumed to have micro metastases. For these patients treatment consists of preoperative (neoadjuvant) chemotherapy, wide or radical surgical resection and postoperative (adjuvant) chemotherapy i.e. "sandwich therapy". Parosteal osteosarcoma or low-grade intra-medullar osteosarcoma patients are treated with wide or radical surgical resection alone. Chemotherapy is reserved only for cases with high-grade transformation. Periosteal osteosarcoma patients may be treated with preoperative (neoadjuvant) chemotherapy similar to that used for conventional osteosarcomas (Bacci et al., 1993; Bruland & Phil, 1999).

#### **6.1 Chemotherapy**

Advances in poly-chemotherapy protocols in the last 30 years have been responsible for improved survival rates and a possibility for limb salvage surgery. Since the beginning of the "odyssey" with Rosen and Jaffe, until now, chemotherapy has been shown to reduce the number of pulmonary metastases or to delay their appearance, facilitating surgical treatment (Rosen et al., 1976).

Standard modern regimens include drugs that have been shown to be the most effective against osteosarcoma: doxorubicin (Adriamycin), cisplatin (Platinol) ifosfamide (Ifex) with mesna (Mesnex) and high-dose methotrexate (Rheumatrex) with Leucoverin calcium rescue. Most standard protocols use doxorubicin and cisplatin with or without high-dose methotrexate for both neoadjuvant (preoperative) and adjuvant (postoperative) chemotherapy. The postoperative (adjuvant) chemotherapy is mostly dependent on the extent of tumor necrosis evaluated after surgical removal. The postoperative chemotherapy regimen is typically the same as the preoperative regimen when tumor necrosis is found to be ≥ 90% at the time of surgery. "Poor responders" to preoperative chemotherapy, defined as those with <90% tumor necrosis at the time of surgery, may benefit from postoperative chemotherapy. In these patients a salvage therapeutic regime is attempted with an increased dose of chemotherapy, an increased length of chemotherapy, or a change in chemotherapeutic agent. Recent trials have incorporated ifosfamide after conventional chemotherapeutic drugs to improve patient survival rates (Jaffe et al., 1989; Sæter et al., 1996; Bacci et al., 2001; Messerschmitt et al., 2009).

#### **6.2 Surgery**

220 Neoadjuvant Chemotherapy – Current Applications in Clinical Practice

Thallium scanning is mostly used for monitoring the response to neoadjuvant

Osteosarcoma can be divided into high-grade or low-grade variants, depending of cellularity, pleomorphism, anaplasia and number of mitoses (Fletcher et al. 2002). This fact and the data for presence or absence of osteosarcoma metastases will be enough to do the Enneking's surgical staging (Table 1). This staging system, first used by the American Musculoskeletal Tumor Society and International Symposium on Limb-Salvage is widely accepted. An alternative system, established by the American Joint Committee on Cancer,

A multidisciplinary approach is obligatory in the diagnosis and treatment of osteosarcoma. To achieve high standards in treatment there is a need for specialized radiologists, pathologists, orthopedic and other surgeons (specialized in oncology surgery), pediatric oncologists, specialized physical therapist and often social workers (Wittig et Al., 2002). When a proper chemotherapy and surgery protocol are followed, survival rates surpass 70%. High-grade osteosarcoma patients without clinically detectable lung metastases are presumed to have micro metastases. For these patients treatment consists of preoperative (neoadjuvant) chemotherapy, wide or radical surgical resection and postoperative (adjuvant) chemotherapy i.e. "sandwich therapy". Parosteal osteosarcoma or low-grade intra-medullar osteosarcoma patients are treated with wide or radical surgical resection alone. Chemotherapy is reserved only for cases with high-grade transformation. Periosteal osteosarcoma patients may be treated with preoperative (neoadjuvant) chemotherapy similar to that used for conventional osteosarcomas (Bacci et

Advances in poly-chemotherapy protocols in the last 30 years have been responsible for improved survival rates and a possibility for limb salvage surgery. Since the beginning of the "odyssey" with Rosen and Jaffe, until now, chemotherapy has been shown to reduce the number of pulmonary metastases or to delay their appearance, facilitating surgical

Standard modern regimens include drugs that have been shown to be the most effective against osteosarcoma: doxorubicin (Adriamycin), cisplatin (Platinol) ifosfamide (Ifex) with mesna (Mesnex) and high-dose methotrexate (Rheumatrex) with Leucoverin calcium rescue. Most standard protocols use doxorubicin and cisplatin with or without high-dose methotrexate for both neoadjuvant (preoperative) and adjuvant (postoperative) chemotherapy. The postoperative (adjuvant) chemotherapy is mostly dependent on the extent of tumor necrosis evaluated after surgical removal. The postoperative chemotherapy regimen is typically the same as the preoperative regimen when tumor necrosis is found to be ≥ 90% at the time of surgery. "Poor responders" to preoperative chemotherapy, defined as those with <90% tumor necrosis at the time of surgery, may benefit from postoperative chemotherapy. In these patients a salvage therapeutic regime is attempted with an increased dose of chemotherapy, an increased length of chemotherapy, or a change in chemotherapeutic agent. Recent trials have incorporated ifosfamide after conventional

chemotherapy (especially when MRI is not helpful).

**6. Treatment**

al., 1993; Bruland & Phil, 1999).

treatment (Rosen et al., 1976).

**6.1 Chemotherapy**

can be used with Enneking's staging system (Enneking et al., 1980).

The two primary surgical options are tumor resection with limb-salvage, and amputation. Surgical margins in excision should encompass resection of tumor, pseudo capsule, and a cuff of normal tissue en block. Meticulous preoperative planning before the biopsy and definitive surgery will ensure better results. Prior to the emergence of limb-salvage surgery in the 1970s, amputation of the affected limb was considered the definitive surgical intervention. Amputation remains the indicated treatment when disease-free marginal resection leaves a nonfunctional limb.

The limb-salvage surgery for osteosarcoma patients is possible due to the use of preoperative (neoadjuvant) chemotherapy and to advancement in musculoskeletal imaging, prosthetic implant design and surgical technique (Fig. 5). Today limb sparing surgery is possible for >85% of patients with extremities localized osteosarcomas (Bacci et al., 2006; Di Caprio & Friedlander, 2003; Longhi et al., 2006).

Surgical treatment has to be planed keeping in mind four basic principles of limb-salvage procedures: local recurrence should be no greater and survival no worse than by amputation; the procedure, or treatment of its complications, should not delay adjuvant therapy; reconstruction should be enduring and not associated with a large number of local complications requiring secondary procedures and frequent hospitalizations; function of the limb should approach that obtained by amputation, although body image, patient preference and life style may influence the decision (González-Heranz et al. 1995).

There are a few relative contraindications to be taken in the consideration for limb-salvage surgery: wrong site or ill-planed biopsy; massive encasement of neurovascular bundles; extensive tumour involvement in soft tissue, muscles or skin; complex or complicated (i.e. with infection) pathological fractures; expected inequalities of the extremities more than 8 cm; and exceptionally poor effect of the neoadjuvant chemotherapy. In the process of decision making for limb-salvage surgery versus amputation the "rule of three" can be very helpful. For extremity survival the bone (1), nerves (2), blood vessels (3), and muscle and skin (4) are necessary to be preserved. If osteosarcoma involves one or two of the former structures, limb preserving is possible. If any three of the former are involved, amputation must be taken in consideration (Di Caprio & Friedlander, 2003).

When "negative" tumor margins are obtained, a large skeletal defect is often present, requiring reconstruction of the bone, muscles, other soft tissues, and the skin. Patient age, tumor location and extent of resection, determine the appropriate surgical alternatives. The extent of the disease, anatomical location of the tumor and the patient's age and psychological profile define the most appropriate surgical procedures. Several options for limb-sparing are available: resection arthrodesis and other similar techniques with special indications (Fig. 7c), modular or special expanding endoprostheses (Fig. 5), corticospongious or bulk auto graft.

For the patients who can't satisfy the principles of limb preservation, ablative surgery has to be taken into consideration. For these patients disarticulation of the hip or shoulder griddle, rotationplasty, femoral or below knee, humeral or other amputations are far more appropriate (González-Heranz et al., 1995; Sæter et al., 1996; Wittig et al., 2002; Samardziski et al. 2009).

Effects of Neoadjuvant Chemotherapy in

**6.3 Postoperative follow-up**

High-Grade Non-Metastatic Osteosarcoma of Extremities 223

The current recommendation for detectable metastases is to excise as many lesions as technically feasible following surgical treatment of the primary tumor. The survival rate for patients can be as high as 60%-75% when both the primary tumor and the solitary lung

The rate of surgical site recurrence is 4% to 6% for both limb-salvage and amputations. Complications following limb-salvage reconstructions include wound complications, infections, mechanical failure, and nonunion. The reported incidence of complications with

After chemotherapy, the patient should be closely followed by the orthopedic oncology surgeon and the medical oncologist. The patient should be monitored for local recurrence, distant or systemic metastases and complications related to reconstruction of the extremity. CT scanning of the chest, plain film radiographs of the reconstructed extremity and meticulous physical examinations are recommended every three months for the first two years and at least every six months from the second year through to the fifth year, and subsequently on a yearly basis. Also, annual bone scintigraphy is mandatory for the first

Dramatic changes over the past few decades have occurred with neoadjuvant (preoperative) and adjuvant (postoperative) poly chemotherapy protocols. This improved the ability to perform safe limb-sparing resection of the tumor in more than 85% of the osteosarcoma patients. Today, as reported in the literature, 60-80% of the patients with extremity localized

metastasis are adequately resected (Yonemoto et al., 1997; Bacci et al., 2006).

limb-salvage surgical techniques is 4% to 38% (Kotz et al., 2002).

two years after completion of the chemotherapy.

Fig. 6. Scandinavian Sarcoma Group Protocol XIV

non metastatic osteosarcomas are long term survivors.

**7. Neoadjuvant chemotherapy**

Fig. 5. **a)** x-ray of high-grade chondroblastic osteosarcoma of right distal femur in a girl of 17; **b)** anterior and lateral MRI view of the lesion; **c)** photo of the resected tumor; **d)** tumor site ready for reconstruction; **e, f)** reconstructed right femur and knee (Link modular endoprosthesis).

The current recommendation for detectable metastases is to excise as many lesions as technically feasible following surgical treatment of the primary tumor. The survival rate for patients can be as high as 60%-75% when both the primary tumor and the solitary lung metastasis are adequately resected (Yonemoto et al., 1997; Bacci et al., 2006).

The rate of surgical site recurrence is 4% to 6% for both limb-salvage and amputations. Complications following limb-salvage reconstructions include wound complications, infections, mechanical failure, and nonunion. The reported incidence of complications with limb-salvage surgical techniques is 4% to 38% (Kotz et al., 2002).

#### **6.3 Postoperative follow-up**

222 Neoadjuvant Chemotherapy – Current Applications in Clinical Practice

a) b)

c) d) Fig. 5. **a)** x-ray of high-grade chondroblastic osteosarcoma of right distal femur in a girl of 17; **b)** anterior and lateral MRI view of the lesion; **c)** photo of the resected tumor; **d)** tumor site ready for reconstruction; **e, f)** reconstructed right femur and knee (Link modular

endoprosthesis).

a) b)

After chemotherapy, the patient should be closely followed by the orthopedic oncology surgeon and the medical oncologist. The patient should be monitored for local recurrence, distant or systemic metastases and complications related to reconstruction of the extremity. CT scanning of the chest, plain film radiographs of the reconstructed extremity and meticulous physical examinations are recommended every three months for the first two years and at least every six months from the second year through to the fifth year, and subsequently on a yearly basis. Also, annual bone scintigraphy is mandatory for the first two years after completion of the chemotherapy.

Fig. 6. Scandinavian Sarcoma Group Protocol XIV

#### **7. Neoadjuvant chemotherapy**

Dramatic changes over the past few decades have occurred with neoadjuvant (preoperative) and adjuvant (postoperative) poly chemotherapy protocols. This improved the ability to perform safe limb-sparing resection of the tumor in more than 85% of the osteosarcoma patients. Today, as reported in the literature, 60-80% of the patients with extremity localized non metastatic osteosarcomas are long term survivors.

Effects of Neoadjuvant Chemotherapy in

bone cement.

High-Grade Non-Metastatic Osteosarcoma of Extremities 225

(Fig. 7) by reducing its neo-vascularity and promoting tumor demarcation from surrounding tissue with pseudo-capsule (Fig 5b). This makes limb-salvage surgery technically more

a) b) c)

Fig. 7. **a)** Fifteen years old female osteosarcoma patient with pathological fracture of the

(neoadjuvant) chemotherapy with Swedish Sarcoma Protocol XIV. **b)** Excellent response (>90% tumor necrosis) with sclerosation after neoadjuvant chemotherapy (arrow shows the site of the pathological fracture). **c)** Radiograph of the humerus after wide resection of the osteosarcoma, and first stage reconstruction of the bone with intramedullary rod and

The primary goal of neoadjuvant chemotherapy is to treat undetectable (or micro) metastases. It is reasonable to believe that neoadjuvant chemotherapy may decrease the risk of spreading viable tumor cells after biopsy, and therefore, decrease the possibility of distant metastases and of local recurrence. This is only possible with optimal serum concentration of methotrexate (at least 1000 µM) at the end of a 6 hour infusion. All of these advantages of neoadjuvant chemotherapy enables more options for wide or near-marginal resection of the

**7.3 Macedonian long-term follow-up experiences with the effects of neoadjuvant chemotherapy in patients with extremity localized high-grade osteosarcoma**

Following the "wave of modern" poly-chemotherapy, in the period 2000-2008, a prospective study was done at the University Clinic for Orthopedic Surgery and Institute of Radiology

left proximal humerus at the first presentation. The patient had preoperative

tumor and for a limb-sparing surgery (Bruland & Phil, 1999).

feasible, even if a marginal resection is obtained (Messershmitt et al., 2009).

Multidrug neoadjuvant chemotherapy, popularized for patients with osteosarcoma by Rosen and later by Jaffe in the late 1970's, is usually initiated as appropriate after histopathological diagnosis and staging. Neoadjuvant chemotherapy protocols with high-dose methotrexate, and cisplatin and doxorubicin dramatically improved long-term survival rates in patients with osteosarcoma sensitive to chemotherapy. Using high dose ifosfamide or different additional and more aggressive therapeutic agents for less sensitive in postoperative chemotherapy (as in: Cooperative Osteosarcomstudiengruppe 96 protocol, Scandinavian Sarcoma Group Protocol XIV and European bone over 40 sarcoma study) improved the results and overall survival of these patients (Kotz et al., 2002). Because of the aggressive nature of the protocols, rescue with Leucoverin (as antidote), bone marrow stimulation with Neupogen and renal protection with Uromitexan are essential. Maximal hydration followed by diuretic forced renal clearance further improves patient's chemotherapy tolerance. During chemotherapy, antiemetics, including: dexamethasone, diphenylhydramine and lorazepam are routinely used in all patients (Bacci et al., 1993; Bruland, 1999; Messershmitt et al., 2009).

#### **7.1 Various neoadjuvant chemotherapy protocols**

There are various poly-chemotherapy protocols (some in regular practice, other in experimental phase). Basic science is making continuous advance that may yield more specific, less-toxic drugs that will further improve survival rates. The use of high dose Ifosfamide or different, additional and more aggressive, therapeutic agents for less sensitive osteosarcoma patients in postoperative modern chemotherapy becomes a rule. There are many chemotherapy regimens, but most commonly reported are: Cooperative Osteosarcomstudiengruppe 96 protocol (COSS 96), Scandinavian Sarcoma Group Protocol (SSG) XIV, European bone over 40 sarcoma study (EURO-B.O.S.S/COSS), Italian Sarcoma Group protocol (ISG), Sloan-Kettering Center protocol T-10 (SSG III), American Society of Clinical Oncology (ASCO) protocol, etc. Introducing more aggressive chemotherapy for poor responders, improved the results and overall survival of these patients (Brulnad, 1999; Di Caprio & Friedlander, 2003).

#### **7.2 Effects of neoadjuvant chemotherapy in high-grade non-metastatic osteosarcoma of extremities**

Various effects of neoadjuvant therapy, such as: remission of pain, reduction of the size of the tumor, sclerosation, pseudo capsule formation, decreasing of neo-vascularisation, tumor necrosis and decrease of the elevated alkaline phosphathase and lactate dehydrogenase levels are widely reported. After neoadjuvant chemotherapy a clinical and radiological response of the tumor has been observed (Bacci et al., 1993). There was reduction, or more often complete remission of pain. This was usually followed with normalization of serum alkaline phosphathase and lactate dehydrogenase levels (if elevated). Bacci further reported an increased density (as seen on Fig. 7b) by the bone lesion on plain radiographs associated with decreased vascularity on angiograms.

Clinical and radiographic reduction in tumor size was observed in more than half of the patients. This was more due to a decrease of the surrounding inflammatory and reactive tissue than to an actual reduction in tumor size. Bacci reported that reduction in vascularity, was the one, most predictable criterion to assess the response of the tumor after neoadjuvant chemotherapy. Neoadjuvant chemotherapy may also decrease the size of the primary tumor

Multidrug neoadjuvant chemotherapy, popularized for patients with osteosarcoma by Rosen and later by Jaffe in the late 1970's, is usually initiated as appropriate after histopathological diagnosis and staging. Neoadjuvant chemotherapy protocols with high-dose methotrexate, and cisplatin and doxorubicin dramatically improved long-term survival rates in patients with osteosarcoma sensitive to chemotherapy. Using high dose ifosfamide or different additional and more aggressive therapeutic agents for less sensitive in postoperative chemotherapy (as in: Cooperative Osteosarcomstudiengruppe 96 protocol, Scandinavian Sarcoma Group Protocol XIV and European bone over 40 sarcoma study) improved the results and overall survival of these patients (Kotz et al., 2002). Because of the aggressive nature of the protocols, rescue with Leucoverin (as antidote), bone marrow stimulation with Neupogen and renal protection with Uromitexan are essential. Maximal hydration followed by diuretic forced renal clearance further improves patient's chemotherapy tolerance. During chemotherapy, antiemetics, including: dexamethasone, diphenylhydramine and lorazepam are routinely used in all patients (Bacci et al., 1993;

There are various poly-chemotherapy protocols (some in regular practice, other in experimental phase). Basic science is making continuous advance that may yield more specific, less-toxic drugs that will further improve survival rates. The use of high dose Ifosfamide or different, additional and more aggressive, therapeutic agents for less sensitive osteosarcoma patients in postoperative modern chemotherapy becomes a rule. There are many chemotherapy regimens, but most commonly reported are: Cooperative Osteosarcomstudiengruppe 96 protocol (COSS 96), Scandinavian Sarcoma Group Protocol (SSG) XIV, European bone over 40 sarcoma study (EURO-B.O.S.S/COSS), Italian Sarcoma Group protocol (ISG), Sloan-Kettering Center protocol T-10 (SSG III), American Society of Clinical Oncology (ASCO) protocol, etc. Introducing more aggressive chemotherapy for poor responders, improved the results and overall survival of these patients (Brulnad, 1999;

**7.2 Effects of neoadjuvant chemotherapy in high-grade non-metastatic osteosarcoma**

Various effects of neoadjuvant therapy, such as: remission of pain, reduction of the size of the tumor, sclerosation, pseudo capsule formation, decreasing of neo-vascularisation, tumor necrosis and decrease of the elevated alkaline phosphathase and lactate dehydrogenase levels are widely reported. After neoadjuvant chemotherapy a clinical and radiological response of the tumor has been observed (Bacci et al., 1993). There was reduction, or more often complete remission of pain. This was usually followed with normalization of serum alkaline phosphathase and lactate dehydrogenase levels (if elevated). Bacci further reported an increased density (as seen on Fig. 7b) by the bone lesion on plain radiographs associated

Clinical and radiographic reduction in tumor size was observed in more than half of the patients. This was more due to a decrease of the surrounding inflammatory and reactive tissue than to an actual reduction in tumor size. Bacci reported that reduction in vascularity, was the one, most predictable criterion to assess the response of the tumor after neoadjuvant chemotherapy. Neoadjuvant chemotherapy may also decrease the size of the primary tumor

Bruland, 1999; Messershmitt et al., 2009).

Di Caprio & Friedlander, 2003).

with decreased vascularity on angiograms.

**of extremities**

**7.1 Various neoadjuvant chemotherapy protocols**

(Fig. 7) by reducing its neo-vascularity and promoting tumor demarcation from surrounding tissue with pseudo-capsule (Fig 5b). This makes limb-salvage surgery technically more feasible, even if a marginal resection is obtained (Messershmitt et al., 2009).

Fig. 7. **a)** Fifteen years old female osteosarcoma patient with pathological fracture of the left proximal humerus at the first presentation. The patient had preoperative (neoadjuvant) chemotherapy with Swedish Sarcoma Protocol XIV. **b)** Excellent response (>90% tumor necrosis) with sclerosation after neoadjuvant chemotherapy (arrow shows the site of the pathological fracture). **c)** Radiograph of the humerus after wide resection of the osteosarcoma, and first stage reconstruction of the bone with intramedullary rod and bone cement.

The primary goal of neoadjuvant chemotherapy is to treat undetectable (or micro) metastases. It is reasonable to believe that neoadjuvant chemotherapy may decrease the risk of spreading viable tumor cells after biopsy, and therefore, decrease the possibility of distant metastases and of local recurrence. This is only possible with optimal serum concentration of methotrexate (at least 1000 µM) at the end of a 6 hour infusion. All of these advantages of neoadjuvant chemotherapy enables more options for wide or near-marginal resection of the tumor and for a limb-sparing surgery (Bruland & Phil, 1999).

#### **7.3 Macedonian long-term follow-up experiences with the effects of neoadjuvant chemotherapy in patients with extremity localized high-grade osteosarcoma**

Following the "wave of modern" poly-chemotherapy, in the period 2000-2008, a prospective study was done at the University Clinic for Orthopedic Surgery and Institute of Radiology

Effects of Neoadjuvant Chemotherapy in

up (m.)

Response to neoa-d. chemoth.

Decrease of pain

(y.) Gender Follow-

Patient No.

Age

yes; **0**-no or none.

with neoadjuvant chemotherapy.

High-Grade Non-Metastatic Osteosarcoma of Extremities 227

Decrease in diameter

Table 2. Clinical data of patients with high-grade osteosarcoma of the extremities, treated

Local recurrence appeared in 17/29 patients (58.6%). The examineеs without local relapse had significantly longer overall survival time than the examined persons with no relapse. Ten percent of the patients with relapse survived more than 100 months, while 48% of the examined with no local relapse were alive even after 100 months (Log-Rank test p=0.0002).

Pseudocapsule Sclerosis Recurrence (m.)

Metastases (m.)

Deceased after (m.)

and Oncology in Skopje. In this period, 47 patients with high-grade osteosarcoma, were treated (Samardziski et al., 2009a).

Selection of patients for neoadjuvant chemotherapy and limb-salvage surgery was based on the following criteria:

*Inclusion criteria:*

*Exclusion criteria:*

According to the exclusion criteria, 8/47 patients were excluded, owing to lung metastases at first presentation or pelvic localization. Another 10/39 patients were excluded from the study due to primary indication for ablative surgery (amputation or disarticulation). Seventy five percent of the patients (29/39) were treated with limb-sparing surgery (Table 2). Fourteen (48%) patients were male and 15 (52%) were female. The mean age was 23.4 ± 14.5 years (range 8-63). Mean follow-up was 49.9 ± 23.1 months (range 23-108).

All patients received to the Scandinavian Sarcoma Group XIV neoadjuvant chemotherapy protocol (SSG XIV). Patients received 2 cycles of preoperative chemotherapy (high dose methotrexate 1200 mg/m2, cisplatin 45 mg/m2/day ×2 days, and doxorubicin 75 mg/m2), (Fig. 6).

After resection, a detailed histopathological assessment of the specimen was done to determine the extent of necrosis of the tumor tissue. Considering the percentage of necrotic tumor tissue, patients were classified into two groups. The first group experienced good response to chemotherapy (>90% necrosis of the tumor). The second group had a poor response to chemotherapy (>10% viable tumor). Regarding good or poor response of the tumor to chemotherapy, patients followed different branches of the protocol (Fig. 6). All 29 patients received 3 courses of postoperative chemotherapy (the same as preoperative). Patients with poor response received 3 more cycles of chemotherapy with high dose ifosfamide (2000 mg/m2/ day ×5 days plus Mesna) every 3 weeks (Fig. 6). Histopathological assessment of the specimen did not only identify the extent of tumor necrosis, but information on tumor-free margins, too.

We have analyzed the following parameters of the clinical and radiological data after neoadjuvant chemotherapy: age, gender, time of follow-up, necrosis of the resected tumor after neoadjuvant chemotherapy (poor or good response), decrease of pain, decrease in tumor diameter, tumor pseudo-capsule seen on MRI, sclerosis seen on radiographs or CT, local recurrence and metastases (Table 2).

Response to neoadjuvant chemotherapy was good (more than 90% necrosis of the tumor) in 16/29 patients (55.2%). The examinees with good response to neoadjuvant therapy had significantly longer overall survival time than the patients with poor response (fig. 8). Ten percents of the patients with poor response survived for more than 65 months, while 58% of the patients with good response survived for more than 100 months (Log-Rank test=3,74 p=0.0002).

#### Effects of Neoadjuvant Chemotherapy in High-Grade Non-Metastatic Osteosarcoma of Extremities 227

226 Neoadjuvant Chemotherapy – Current Applications in Clinical Practice

and Oncology in Skopje. In this period, 47 patients with high-grade osteosarcoma, were

Selection of patients for neoadjuvant chemotherapy and limb-salvage surgery was based on

According to the exclusion criteria, 8/47 patients were excluded, owing to lung metastases at first presentation or pelvic localization. Another 10/39 patients were excluded from the study due to primary indication for ablative surgery (amputation or disarticulation). Seventy five percent of the patients (29/39) were treated with limb-sparing surgery (Table 2). Fourteen (48%) patients were male and 15 (52%) were female. The mean age was 23.4 ± 14.5 years

All patients received to the Scandinavian Sarcoma Group XIV neoadjuvant chemotherapy protocol (SSG XIV). Patients received 2 cycles of preoperative chemotherapy (high dose methotrexate 1200 mg/m2, cisplatin 45 mg/m2/day ×2 days, and doxorubicin 75 mg/m2),

After resection, a detailed histopathological assessment of the specimen was done to determine the extent of necrosis of the tumor tissue. Considering the percentage of necrotic tumor tissue, patients were classified into two groups. The first group experienced good response to chemotherapy (>90% necrosis of the tumor). The second group had a poor response to chemotherapy (>10% viable tumor). Regarding good or poor response of the tumor to chemotherapy, patients followed different branches of the protocol (Fig. 6). All 29 patients received 3 courses of postoperative chemotherapy (the same as preoperative). Patients with poor response received 3 more cycles of chemotherapy with high dose ifosfamide (2000 mg/m2/ day ×5 days plus Mesna) every 3 weeks (Fig. 6). Histopathological assessment of the specimen did not only identify the extent of tumor necrosis, but

We have analyzed the following parameters of the clinical and radiological data after neoadjuvant chemotherapy: age, gender, time of follow-up, necrosis of the resected tumor after neoadjuvant chemotherapy (poor or good response), decrease of pain, decrease in tumor diameter, tumor pseudo-capsule seen on MRI, sclerosis seen on radiographs or CT,

Response to neoadjuvant chemotherapy was good (more than 90% necrosis of the tumor) in 16/29 patients (55.2%). The examinees with good response to neoadjuvant therapy had significantly longer overall survival time than the patients with poor response (fig. 8). Ten percents of the patients with poor response survived for more than 65 months, while 58% of the patients with good response survived for more than 100 months (Log-Rank

(range 8-63). Mean follow-up was 49.9 ± 23.1 months (range 23-108).

treated (Samardziski et al., 2009a).

diagnosis of osteosarcoma.

information on tumor-free margins, too.

local recurrence and metastases (Table 2).

test=3,74 p=0.0002).

the following criteria: *Inclusion criteria:*

*Exclusion criteria:*

(Fig. 6).

**M**: male; **F:** female; **G:** good response after neoadjuvant chemotherapy (necrosis >90% of the tumor); **P**: poor response after neoadjuvant chemotherapy (>10% viable tumor); **U** -unknown or missing data; **1** yes; **0**-no or none.

Table 2. Clinical data of patients with high-grade osteosarcoma of the extremities, treated with neoadjuvant chemotherapy.

Local recurrence appeared in 17/29 patients (58.6%). The examineеs without local relapse had significantly longer overall survival time than the examined persons with no relapse. Ten percent of the patients with relapse survived more than 100 months, while 48% of the examined with no local relapse were alive even after 100 months (Log-Rank test p=0.0002).

Effects of Neoadjuvant Chemotherapy in

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 1,1 1,2

is promising and encouraging.

**7.4 Toxic effects of neoadjuvant chemotherapy regimen**

Cumulative Proportion Surviving

High-Grade Non-Metastatic Osteosarcoma of Extremities 229

Survival Function Complete Censored

0 20 40 60 80 100 120 Survival Time

Fig. 9. Disease and event free survival time of the patients with extremity localized highgrade osteosarcoma treated with SSG XIV neoadjuvant chemotherapy protocol and surgery. Using high dose ifosfamide for poor responders in postoperative chemotherapy should improve the results and overall survival time of these patients. If treatment and management principles of high-grade osteosarcoma are followed, limb-sparing with 60-80% survival rates could be achieved. Our preliminary results are slightly different from those published in the literature. There was a significantly different overall survival time in our study between the group of patients with good response to neoadjuvant chemotherapy compared to the group of patients with bad response. Furthermore, overall survival time in our group of patients was shorter than the time reported in the literature. In spite of the recorded differences in the results, the treatment regimen with neoadjuvant chemotherapy

Most often hematologic toxicity is seen after chemotherapy. Various authors report Grade 3 or Grade 4 hematologic toxicity in 10-15% of the treated patients. Severe leucopenia and/or thrombocytopenia are the two conditions for readmitting patients in hospital. In that case

Most of these patients with myelotoxicity have fever and microbiologically proven bacteremia during their granulocytopenic phase. Wide spectrum antibiotics in the beginning

Neupogen or Leucoverin rescue treatment is beneficial (Bacci & Picci, 1994).

and specific antibiotics after microbiological assessment are necessary.

Most of the tumor relapses were seen in the patients by 22 months after surgery. The 3 patients with early local recurrences had secondary extirpation of the relapsed tumor and one of them had to be amputated.

Fig. 8. Response of the patients after neoadjuvant chemotherapy treated with SSG XIV chemotherapy protocol.

Lung metastases appeared in 18/29 patients or 62.1%. The examinees with metastases had significantly shorter overall survival time than the metastasis-free patients. Four percent of the examined patients with metastases survived longer than 100 months, while 90% of the examined with no metastases were alive even after 100 months (Log-Rank test p=0.0002).

Plain radiograph or CT-scan sclerosis of the tumor after neoadjuvant chemotherapy was seen in 18/29 patients (62.1%). Pseudo-capsule was seen in 19/29 patients (65.5%), but in 3/29 (10.3%) MRI imaging showed inconclusive data. Cystic necrosis after neoadjuvant chemotherapy was seen in 14/29 patients (48.3%). Inconclusive results for cystic necrosis were found in 3 and data was missing for 1 patient.

Up to date 10/29 patients (34.5%) are disease or event free. Mean survival time of the patients was 53 months, and 20% of the examinees survived longer than 60 months (Fig. 9).

Most of the tumor relapses were seen in the patients by 22 months after surgery. The 3 patients with early local recurrences had secondary extirpation of the relapsed tumor and

good response 0 10 20 30 40 50 60 70 80 90 100 110 120

Time

Fig. 8. Response of the patients after neoadjuvant chemotherapy treated with SSG XIV

Lung metastases appeared in 18/29 patients or 62.1%. The examinees with metastases had significantly shorter overall survival time than the metastasis-free patients. Four percent of the examined patients with metastases survived longer than 100 months, while 90% of the examined with no metastases were alive even after 100 months (Log-Rank test

Plain radiograph or CT-scan sclerosis of the tumor after neoadjuvant chemotherapy was seen in 18/29 patients (62.1%). Pseudo-capsule was seen in 19/29 patients (65.5%), but in 3/29 (10.3%) MRI imaging showed inconclusive data. Cystic necrosis after neoadjuvant chemotherapy was seen in 14/29 patients (48.3%). Inconclusive results for cystic necrosis

Up to date 10/29 patients (34.5%) are disease or event free. Mean survival time of the patients was 53 months, and 20% of the examinees survived longer than 60 months (Fig. 9).

bad response

Cumulative Proportion Surviving (Kaplan-Meier) Complete Censored

one of them had to be amputated.

p=0.0002).

chemotherapy protocol.

were found in 3 and data was missing for 1 patient.

0,0

0,2

0,4

Cumulative Proportion Surviving

0,6

0,8

1,0

Fig. 9. Disease and event free survival time of the patients with extremity localized highgrade osteosarcoma treated with SSG XIV neoadjuvant chemotherapy protocol and surgery.

Using high dose ifosfamide for poor responders in postoperative chemotherapy should improve the results and overall survival time of these patients. If treatment and management principles of high-grade osteosarcoma are followed, limb-sparing with 60-80% survival rates could be achieved. Our preliminary results are slightly different from those published in the literature. There was a significantly different overall survival time in our study between the group of patients with good response to neoadjuvant chemotherapy compared to the group of patients with bad response. Furthermore, overall survival time in our group of patients was shorter than the time reported in the literature. In spite of the recorded differences in the results, the treatment regimen with neoadjuvant chemotherapy is promising and encouraging.

#### **7.4 Toxic effects of neoadjuvant chemotherapy regimen**

Most often hematologic toxicity is seen after chemotherapy. Various authors report Grade 3 or Grade 4 hematologic toxicity in 10-15% of the treated patients. Severe leucopenia and/or thrombocytopenia are the two conditions for readmitting patients in hospital. In that case Neupogen or Leucoverin rescue treatment is beneficial (Bacci & Picci, 1994).

Most of these patients with myelotoxicity have fever and microbiologically proven bacteremia during their granulocytopenic phase. Wide spectrum antibiotics in the beginning and specific antibiotics after microbiological assessment are necessary.

Effects of Neoadjuvant Chemotherapy in

exists for the recurrence of the tumor.

**8.1 Prognosis**

Friedlander, 2003).

**8.2 Future considerations**

High-Grade Non-Metastatic Osteosarcoma of Extremities 231

treatment of non-metastatic osteosarcoma in terms of cure rate and quality of life for survivors. These advances are due mainly to the development of effective adjuvant and neoadjuvant chemotherapeutic regimens. Reports on the progress and controversies in the treatment of osteosarcoma occurred with respect to the construct, expirimental design and interpretation of

Poor prognostic factors for patients with osteosarcoma include metastases at first presentation, extremely large primary tumor, increased alkaline phosphatase and lactate dehydrogenase levels, poor response to neoadjuvant chemotherapy, tumor discontinuous from bone, pathologic fractures and lymph node involvement (Longhi, et al. 2006). Despite current surgical and chemotherapeutic treatment regimens, 30% to 40% of osteosarcoma patients experience relapse within 3 years of treatment. Pulmonary recurrence is most common secondary to micro-metastatic disease. Regardless of poor prognosis, repeated tumor excisions can be performed (of primary site or metastatic one), because many studies have shown improved survival rates (Yonemoto, 1997; Bacci et al., 2001). The role of "second-line" chemotherapy regimen remains controversial because no standard regimen

The evaluation variables influencing systemic and local recurrence and final outcome are extremely important in defining risk-adapted treatments for patients with nonmetastatic osteosarcoma of the extremity. Upon multivariate analysis, age ≤ 14 years, high serum levels of alkaline phosphatase, tumor volume >200 mL, a dual-drug regimen chemotherapy, inadequate surgical margins, and poor histologic response to treatment maintained independent prognostic values on the outcome of nonmetastatic osteosarcoma of the extremities. These factors must be considered when deciding risk-adapted treatments for osteosarcoma patients (Pochanugool, 1997; Bacci, 2006; Yu & Wang, 2009). Amputation remains the indicated treatment when these factors are taken into consideration or tumor resection to disease-free margins leaves a nonfunctional limb (Enneking, 1975; Di Caprio &

A logical development of chemotherapy was introduction of local (or loco-regional) intraarterial methods of chemotherapy. The obvious limitations are complicated intra-arterial techniques of application of chemotherapeutics and uncontrolled risk of tissue necrosis. Intra-arterial administration of cisplatin has been investigated for achieving improved histological response following chemotherapy. Since the originalr attempts to administer intra-arterial cisplatin from the 1980's, major advance in imaging and surgical techniques have improved the results and made it easier and safer. Reported studies demonstrate an increase in long-term survival up to 93%. Thus, a consensus on the routine use of intraarterial chemotherapy does not exist (Jaffe, 1989; Bacci et al., 2001; Messershmit et al., 2009). Basic science is making continuous advance in molecular mechanisms and biologic pathways that may yield more specific, less-toxic drugs that will further improve survival rates. Inhibition of tyrosine kinase signaling is known to regulate cell growth, cell proliferation, angiogenesis, and apoptosis and is an area of current interest (Messershmit et al., 2008). Liposomal muramyl tripeptide phosphatidilethanolamine (L-MTP-PE) is a

the studies. Never the les, this sdudyes led to remarkable results (Bacci, 2008).

Cardiotoxicity is less often reported in patients with neoadjuvant chemotherapy. Unfortunately this side effect of the treatment is most serious and cardiopathy following the treatment may become a chronic life treating condition.

Due to general toxic effects to the human body and profound systemic reaction of all organs and systems to chemotherapeutics, sickness, malaise and weakness are general side effects. A common problem is abdominal pain associated with mild asscites, easily visualised on ultrasonography. Therefore antiemetics and corticosteroids, including dexamethasone, diphenylhydramine and lorazepam are routinely used in all patients (Bacci et al., 1993; Bruland, 1999; Messershmitt et al., 2009).

Skin necrosis, tender local swelling, inflammation, due to local intra-arterial cisplatin or venous trombophlebitis occurres in some patients. This lesions usually do not cause a major problem and usually heal in 2-3 weeks with pigmented scars.

#### **8. Discussion**

Prior to the introduction of chemotherapy, when amputation was the primary treatment for patients with osteosarcoma, the predicted long-term survival was 15-20%. Dismal survival rates were presumably attributable to pulmonary metastatic disease, whether clinically obvious or occult (Enneking, 1975). Survival rates dramatically increased during 1970's and 1980's with the pioneering work of Rosen and Jaffe. Currently, long-term survival rates are 60% to 70% for patients with localized osteosarcoma and for extremity localized up to 80% (Meyers et al., 2008). Despite the use of modern neoadjuvant chemotherapy the 10-year survival rates decline significantly to 20% in patients with clinically detectable metastases.  Excluding high-grade surface osteosarcoma, which has similar prognosis to that of conventional osteosarcoma, the surface (parosteal and periosteal) osteosarcoma variants have the best prognosis of all. The 10-year survival rates for this group of patients is up to 85% (Samardziski et al., 2009b).

The site of the lesion has prognostic importance. The best survival rates are expected in patients with appendicular localization of the osteosarcoma. Central localization (pelvis, ribs and vertebrae) are less common sites of osteosarcoma, and have poorest prognosis. Osteosarcoma of the jaw is associated with an especially good prognosis, whereas osteosarcoma involving the skull has a very poor prognosis (Unni, 1998; Yu & Wang, 2009). Badly planned and ill preformed biopsy can complicate the final surgery and may decrease survival rates due to local spreading or risk for early metastatic disease (Mankin et al., 1982; Campanacci, 1999).

The overall treatment results in high-grade osteosarcoma are less impressive than widely presumed. Whereas classical osteosarcoma survival has indeed increased, in other subgroups, comprising more than 40% of the entire osteosarcoma population , the prognosis has been modestly improved. Today still more than half of an unselected osteosarcoma population eventualy succumbs to the disease, despite the current multimodal primary treatment as well as second-line chemotherapy and surgical metastatectomies (Bruland, 1999).

Neoadjuvant chemotherapy enables limb-sparing in the majority of patients with extremity localised ostesarcoma. During the past 20 years dramatic advances have been made in the treatment of non-metastatic osteosarcoma in terms of cure rate and quality of life for survivors. These advances are due mainly to the development of effective adjuvant and neoadjuvant chemotherapeutic regimens. Reports on the progress and controversies in the treatment of osteosarcoma occurred with respect to the construct, expirimental design and interpretation of the studies. Never the les, this sdudyes led to remarkable results (Bacci, 2008).

#### **8.1 Prognosis**

230 Neoadjuvant Chemotherapy – Current Applications in Clinical Practice

Cardiotoxicity is less often reported in patients with neoadjuvant chemotherapy. Unfortunately this side effect of the treatment is most serious and cardiopathy following the

Due to general toxic effects to the human body and profound systemic reaction of all organs and systems to chemotherapeutics, sickness, malaise and weakness are general side effects. A common problem is abdominal pain associated with mild asscites, easily visualised on ultrasonography. Therefore antiemetics and corticosteroids, including dexamethasone, diphenylhydramine and lorazepam are routinely used in all patients (Bacci et al., 1993;

Skin necrosis, tender local swelling, inflammation, due to local intra-arterial cisplatin or venous trombophlebitis occurres in some patients. This lesions usually do not cause a major

Prior to the introduction of chemotherapy, when amputation was the primary treatment for patients with osteosarcoma, the predicted long-term survival was 15-20%. Dismal survival rates were presumably attributable to pulmonary metastatic disease, whether clinically obvious or occult (Enneking, 1975). Survival rates dramatically increased during 1970's and 1980's with the pioneering work of Rosen and Jaffe. Currently, long-term survival rates are 60% to 70% for patients with localized osteosarcoma and for extremity localized up to 80% (Meyers et al., 2008). Despite the use of modern neoadjuvant chemotherapy the 10-year survival rates decline significantly to 20% in patients with clinically detectable metastases.  Excluding high-grade surface osteosarcoma, which has similar prognosis to that of conventional osteosarcoma, the surface (parosteal and periosteal) osteosarcoma variants have the best prognosis of all. The 10-year survival rates for this group of patients is up to 85%

The site of the lesion has prognostic importance. The best survival rates are expected in patients with appendicular localization of the osteosarcoma. Central localization (pelvis, ribs and vertebrae) are less common sites of osteosarcoma, and have poorest prognosis. Osteosarcoma of the jaw is associated with an especially good prognosis, whereas osteosarcoma involving the skull has a very poor prognosis (Unni, 1998; Yu & Wang, 2009). Badly planned and ill preformed biopsy can complicate the final surgery and may decrease survival rates due to local spreading or risk for early metastatic disease (Mankin et al., 1982;

The overall treatment results in high-grade osteosarcoma are less impressive than widely presumed. Whereas classical osteosarcoma survival has indeed increased, in other subgroups, comprising more than 40% of the entire osteosarcoma population , the prognosis has been modestly improved. Today still more than half of an unselected osteosarcoma population eventualy succumbs to the disease, despite the current multimodal primary treatment as well as second-line chemotherapy and surgical

Neoadjuvant chemotherapy enables limb-sparing in the majority of patients with extremity localised ostesarcoma. During the past 20 years dramatic advances have been made in the

treatment may become a chronic life treating condition.

problem and usually heal in 2-3 weeks with pigmented scars.

Bruland, 1999; Messershmitt et al., 2009).

**8. Discussion**

(Samardziski et al., 2009b).

Campanacci, 1999).

metastatectomies (Bruland, 1999).

Poor prognostic factors for patients with osteosarcoma include metastases at first presentation, extremely large primary tumor, increased alkaline phosphatase and lactate dehydrogenase levels, poor response to neoadjuvant chemotherapy, tumor discontinuous from bone, pathologic fractures and lymph node involvement (Longhi, et al. 2006). Despite current surgical and chemotherapeutic treatment regimens, 30% to 40% of osteosarcoma patients experience relapse within 3 years of treatment. Pulmonary recurrence is most common secondary to micro-metastatic disease. Regardless of poor prognosis, repeated tumor excisions can be performed (of primary site or metastatic one), because many studies have shown improved survival rates (Yonemoto, 1997; Bacci et al., 2001). The role of "second-line" chemotherapy regimen remains controversial because no standard regimen exists for the recurrence of the tumor.

The evaluation variables influencing systemic and local recurrence and final outcome are extremely important in defining risk-adapted treatments for patients with nonmetastatic osteosarcoma of the extremity. Upon multivariate analysis, age ≤ 14 years, high serum levels of alkaline phosphatase, tumor volume >200 mL, a dual-drug regimen chemotherapy, inadequate surgical margins, and poor histologic response to treatment maintained independent prognostic values on the outcome of nonmetastatic osteosarcoma of the extremities. These factors must be considered when deciding risk-adapted treatments for osteosarcoma patients (Pochanugool, 1997; Bacci, 2006; Yu & Wang, 2009). Amputation remains the indicated treatment when these factors are taken into consideration or tumor resection to disease-free margins leaves a nonfunctional limb (Enneking, 1975; Di Caprio & Friedlander, 2003).

#### **8.2 Future considerations**

A logical development of chemotherapy was introduction of local (or loco-regional) intraarterial methods of chemotherapy. The obvious limitations are complicated intra-arterial techniques of application of chemotherapeutics and uncontrolled risk of tissue necrosis. Intra-arterial administration of cisplatin has been investigated for achieving improved histological response following chemotherapy. Since the originalr attempts to administer intra-arterial cisplatin from the 1980's, major advance in imaging and surgical techniques have improved the results and made it easier and safer. Reported studies demonstrate an increase in long-term survival up to 93%. Thus, a consensus on the routine use of intraarterial chemotherapy does not exist (Jaffe, 1989; Bacci et al., 2001; Messershmit et al., 2009). Basic science is making continuous advance in molecular mechanisms and biologic pathways that may yield more specific, less-toxic drugs that will further improve survival rates. Inhibition of tyrosine kinase signaling is known to regulate cell growth, cell proliferation, angiogenesis, and apoptosis and is an area of current interest (Messershmit et al., 2008). Liposomal muramyl tripeptide phosphatidilethanolamine (L-MTP-PE) is a

Effects of Neoadjuvant Chemotherapy in

111, (1975), pp. (2-4).

63), PMID: 3874932.

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1082364.

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(515-527), PMID: 19652033.

High-Grade Non-Metastatic Osteosarcoma of Extremities 233

Campanacci, M. (1999). Errors in the diagnosis and treatment of the musculoskeletal tumors: What must not be done. *Chir Organi Mov*, Vol. 84, (1999), pp. (1-17). Di Caprio, MR. & Friedlander, GE. (2003). Malignant Bone Tumours: Limb Sparing Versus Amputation. *J Am Acad Ort Surg,* Vol.11, No.1, pp. (125-129), PMID: 12699369 . Enneking, WF. (1975). Osteosarcoma (editorial comment). *Clin Orthop Rel Research*, Vol.

Enneking, WF. et al. (1980). A surgical staging of musculoskeletal sarcomas*. J Bone Joint*

Fletcher, CDM. et al. (2002). Classification of Tumours. Pathology and Genetics of Tumours of Soft Tissue and Bone. IARC Press, Lyon, ISBN: 92 832 2413 2. González-Heranz, P. et al. (1995). The Management of Limb-Length Discrepancies in

Jaffe, N. et al. (1985). Analysis of efficacy of the intra-arterial cis-Diammine-

Kotz, R. et al. (2002). Advances in bone tumor treatment in 30 years with respect to

Lodwick, GS. et al. (1963). Computer Diagnosis of Primary Bone Tumours, A Preliminary

Longhi, A. et al. (2006). Primary bone osteosarcoma in the pediatric age: State of the art. *Cancer Treat Rev,*Vol. 32, (2006), pp. (423-436), DOI:10.1016/j.ctrv.2006.05.005. Mankin, HJ. et al. (1982). The Hazards of Biopsy in Patients with Malignant Primary Bone

Messershmitt, PJ. Et al. (2008). Specific tyrosine kinase inhibition regulated human

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(2002), pp. (197-206), DOI: 10.1007/s00264-002-0365-1.

promising drug in clinical trial that functions to stimulate the formation of tumoricidal macrophages (Meyers et al., 2008).

#### **9. Conclusion**

With advances in neoadjuvant chemotherapy, radiographic imaging, and reconstructive surgery, most patients with osteosarcoma can now be offered limb-sparing treatment. A multidisciplinary approach in diagnosis and treatment is mandatory. Surgical resection with wide margins after neoadjuvant and adjuvant chemotherapy after surgery is a current standard of care. Osteoarticular allografts, modular prostheses, or composites of these two approaches form the basis for most current reconstructions. However, amputation still plays an important role and offers a standard to which other approaches must be compared. Basic science is making continuous advance in molecular mechanisms and more specific, lesstoxic drugs will further improve survival rates. Current research into the cell biology of osteosarcoma may lead to improved and more target-selective treatment with the intent of improved overall survival.

Applying neoadjuvant chemotherapy followed by appropriate surgery requires responsible, trained and highly engaged medical staff. If treatment and management principles of highgrade osteosarcoma with neoadjuvant therapy are followed, long-term 60-80% overall survival rates could be easily achieved.

#### **10. Acknowledgement**

Special thanks to Mrs. Marija Tanevska-Pulios for English language editing of the paper.

#### **11. References**

promising drug in clinical trial that functions to stimulate the formation of tumoricidal

With advances in neoadjuvant chemotherapy, radiographic imaging, and reconstructive surgery, most patients with osteosarcoma can now be offered limb-sparing treatment. A multidisciplinary approach in diagnosis and treatment is mandatory. Surgical resection with wide margins after neoadjuvant and adjuvant chemotherapy after surgery is a current standard of care. Osteoarticular allografts, modular prostheses, or composites of these two approaches form the basis for most current reconstructions. However, amputation still plays an important role and offers a standard to which other approaches must be compared. Basic science is making continuous advance in molecular mechanisms and more specific, lesstoxic drugs will further improve survival rates. Current research into the cell biology of osteosarcoma may lead to improved and more target-selective treatment with the intent of

Applying neoadjuvant chemotherapy followed by appropriate surgery requires responsible, trained and highly engaged medical staff. If treatment and management principles of highgrade osteosarcoma with neoadjuvant therapy are followed, long-term 60-80% overall

Special thanks to Mrs. Marija Tanevska-Pulios for English language editing of the paper.

Bacci, G. et al. (1993). Primary chemotherapy and delayed surgery for non-metastatic

Bacci, G. et al. (2001). A comparison of methods of loco-regional chemotherapy combined

extremity. *Eur J Surg Oncol*, Vol. 27, (2001), pp. (98-104), PMID: 11237499. Bacci, G. et al. (2006). Prognostic factors for osteosarcoma of the extremity treated

Bacci, G. et al. (2008). High-grade osteosarcoma of the extremities with lung metastases at

Bruland, ØS. & Phil, A. (1999) On the current management of osteosarcoma. A critical

Osteosarcoma. Review. *Forum,* Vol. 4, No.1,(1994), pp. (52-64).

osteosarcoma of the extremities. *Cancer,* Vol. 72, No.11, pp. (3227-3238), DOI: 10.1002/1097-0142(19931201)72:11<3227::AID-CNCR2820721116>3.0.CO;2-C. Bacci, G. & Picci, P. (1994). Analysis of Factors Influencing Treatment Options in

with systemic chemotherapy as neoadjuvant treatment of osteosarcoma of the

with neoadjuvant chemotherapy: 15-Year experience in 789 patients treated at a single institution. *Cancer*, Vol. 106, No. 5, (2006), pp. (1154-1161), ISSN 0008-

presentation: Treatment with neoadjuvant chemotherapy and simultaneous resection of primary and metastatic lesions. *J Surg Oncol*, Vol. 98, (2008), pp. (415-

evaluation and a proposal for modified treatment surgery. *Eur J Cancer*, Vol. 33,

macrophages (Meyers et al., 2008).

**9. Conclusion**

improved overall survival.

**10. Acknowledgement**

**11. References**

543X.

420) PMID: 18792969.

pp. (1725-1731), PMID: 9470825.

survival rates could be easily achieved.

**15**

*Brasil*

**Chemotherapy and Mechanisms of**

Andre Lima de Oliveira1, Roberto Euzebio dos Santos1

*1DOGI General Gynecology Clinic, Brotherhood of the Santa Casa de São Paulo 2DOGI Pelvic Oncology Clinic, Brotherhood of the Santa Casa de São Paulo*

In the mid 1950s, we started to have a much better understanding of the biological mechanisms of establishment of metastases and the role of regional lymph nodes as an effective barrier to tumor spread, because malignant cells have been observed in the

Early studies with adjuvant chemotherapy after surgery in solid tumors (breast adenocarcinoma implanted in mice) began in 1957 (Shapiro, Fugman, 1957). Based on these findings, Bernard Fisher and colleagues began in 1958, the first collaborative study with the objective of evaluating the response to systemic administration of perioperative chemotherapy in patients with operable breast cancer (Fisher et al, 1958). Good results were obtained in relation to disease-free interval and overall survival in premenopausal women (Fisher et al, 1968). Similar results were also observed by other authors, with the use of multidrug therapy (cyclophosphamide, methotrexate and fluorouracil (CMF) with or without prednisone) in advanced breast cancer (BC)(Canellos et al, 1974 and 1976, Bonadonna et al, 1976). Therefore, the addition of adjuvant polychemotherapy in BC showed gain by controlling survival of micrometastases in patients with lymph nodes affected by cancer or not (Fisher et al, 1975; Bonadonna et al, 1976; Early Breast Cancer Trialists Collaborative Group (EBCTCG ), 1988; Bonadonna, Valagussa, 1983,1985,1987, Henderson, 1987, Fisher et al, 1989; Bonadonna et al, 1995; Mansour et al, 1998, Carlson et al,

Neoadjuvant chemotherapy is defined as a treatment option where chemotherapy is introduced before local treatment, either surgery or radiotherapy (Bear, 1998). This was introduced by De Lena et al (1978) who administered adriablastin and vincristine in 110

The biological rationale for using neoadjuvant chemotherapy was based on observations in animal models where the removal of primary tumor growth accelerated due to changes in metastatic tumor kinetics, suggesting that growth factors derived from tumor influence the

women with advanced BC, achieving response rates of 70% partial.

**1. Introduction**

2000 and NIH 2000).

**1.2 Neoadjuvant therapy**

bloodstream (Fisher, Turnbull, 1955).

**1.1 Adjuvant**

**Resistance in Breast Cancer**

and Fabio Francisco Oliveira Rodrigues2

### **Chemotherapy and Mechanisms of Resistance in Breast Cancer**

Andre Lima de Oliveira1, Roberto Euzebio dos Santos1 and Fabio Francisco Oliveira Rodrigues2 *1DOGI General Gynecology Clinic, Brotherhood of the Santa Casa de São Paulo 2DOGI Pelvic Oncology Clinic, Brotherhood of the Santa Casa de São Paulo Brasil*

#### **1. Introduction**

#### **1.1 Adjuvant**

234 Neoadjuvant Chemotherapy – Current Applications in Clinical Practice

Sæter, G. et al. (1996), Extremity and non-extremity high-grade osteosarcoma, *Acta Oncol,*

Samardziski, M. et al. (2004). Computer assisted diagnosis of benign bone tumors. *Radiol*

Samardziski, M. et al. (2009a). Limb-sparing in patients with non-metastatic high-grade osteosarcoma. *J. BUON*, Vol. 14, (2009) pp. (63-69), PubMed ID: 19373949. Samardziski, M. et al. (2009b). Parosteal Osteosarcoma. *Bratisl Lek Listy*. Vol. 110, (2009),

Unni, KK. (1998). Osteosarcoma of bone. *J Orthop Sci,* Vol. 3, (1998), pp. (287-294), DOI:

Wittig, JC. et al. (2002). Osteosarcoma: A Multidisciplinary Approach to Diagnosis and

Yonemoto, T. et al. (1997). Prognosis of Osteosarcoma with Pulmonary Metastases at

Yu, XC & Wang, W. (2009). Multivariate analysis of factors influencing on preoperative

*Treatment,* Vol. 36, No.10, (2009) pp. 863-868, ISSN 1000-8578.

Treatment. *Am Fam Physician*, Vol. 65, No. 6, (2002), pp. (1123-1132), PMID:

Initial Presentation is not Dismal. *Clin Orth Rel Res,* Vol. 349, (1997), pp. (194-199),

chemotherapy response for osteosarcoma. *Cancer Research on Prevention and*

Vol. 35, (1996), pp. (129-134), PMID: 9073059 .

pp. (240-244), PubMed ID: 19507652.

10.1007/s007760050055.

11925089.

PMID: 9584383.

*Oncol,*Vol. 38, (2004), pp. (165-169), ISSN: 1581-3207.

In the mid 1950s, we started to have a much better understanding of the biological mechanisms of establishment of metastases and the role of regional lymph nodes as an effective barrier to tumor spread, because malignant cells have been observed in the bloodstream (Fisher, Turnbull, 1955).

Early studies with adjuvant chemotherapy after surgery in solid tumors (breast adenocarcinoma implanted in mice) began in 1957 (Shapiro, Fugman, 1957). Based on these findings, Bernard Fisher and colleagues began in 1958, the first collaborative study with the objective of evaluating the response to systemic administration of perioperative chemotherapy in patients with operable breast cancer (Fisher et al, 1958). Good results were obtained in relation to disease-free interval and overall survival in premenopausal women (Fisher et al, 1968). Similar results were also observed by other authors, with the use of multidrug therapy (cyclophosphamide, methotrexate and fluorouracil (CMF) with or without prednisone) in advanced breast cancer (BC)(Canellos et al, 1974 and 1976, Bonadonna et al, 1976). Therefore, the addition of adjuvant polychemotherapy in BC showed gain by controlling survival of micrometastases in patients with lymph nodes affected by cancer or not (Fisher et al, 1975; Bonadonna et al, 1976; Early Breast Cancer Trialists Collaborative Group (EBCTCG ), 1988; Bonadonna, Valagussa, 1983,1985,1987, Henderson, 1987, Fisher et al, 1989; Bonadonna et al, 1995; Mansour et al, 1998, Carlson et al, 2000 and NIH 2000).

#### **1.2 Neoadjuvant therapy**

Neoadjuvant chemotherapy is defined as a treatment option where chemotherapy is introduced before local treatment, either surgery or radiotherapy (Bear, 1998). This was introduced by De Lena et al (1978) who administered adriablastin and vincristine in 110 women with advanced BC, achieving response rates of 70% partial.

The biological rationale for using neoadjuvant chemotherapy was based on observations in animal models where the removal of primary tumor growth accelerated due to changes in metastatic tumor kinetics, suggesting that growth factors derived from tumor influence the

Chemotherapy and Mechanisms of Resistance in Breast Cancer 237

pharmacology, activation and inactivation of drugs, modification of specific targets and regulatory pathways of apoptosis) (Leonessa et al, 2003, Riddick et al, 2005). Identification of factors that affect cell metabolism, which are related to drug resistance, will enable the

Among the biochemical and molecular mechanisms of drug resistance, we stress: changes in the activity of topoisomerase II, alterations in the DNA repair mechanism, overexpression of P-glycoprotein; high intracellular concentrations of enzymes purification of cellular metabolism - among them enzymes the family of glutathione S-transferases (GSTs) and changes in the mechanisms of signaling via c-Jun N-terminal kinase 1 (JNK1) -and "apoptosis signal-regulating kinase (ASK1) required for activation of the" mitogenactivated protein (MAP kinases) in apoptosis and cellular restoration. These pathways are also mediated by proteins encoded by genes of GSTs (O'Brien, Tew, 1996; Burg, Mulder,

Different response rates to particular chemotherapy regimens, as observed in patient groups with the same biological characteristics and stage, suggest the existence of different mechanisms of drug resistance, probably induced by genetic alterations (Hayes, Pulford,

Among the mechanisms of purification of cellular metabolism involved in the inactivation of toxic substances to the cell there is the action of the enzyme family of GSTs in phase II metabolism of cell purification. The first evidence of their involvement in resistance to drugs used in chemotherapy have emerged from research published by scientific groups Schisswelbauer et al (1990), Tew (1994) and Hayes, Pulford, (1995). However, the relationship between GSTs and resistance to chemotherapy remains inconsistent (Riddick et al, 2005). This mechanism of resistance is related to the ability to regulate the action of enzymes involved in catalyzing electrophilic compounds harmful to cells from activation by cytochrome P-450 1A1 and 1B1 (Phase I). These compounds in turn are substrates for phase II enzyme systems, represented here by the family of GSTs, which are involved on two fronts in the process of drug resistance: production of protective enzymes of metabolism and cellular apoptotic processes via inhibition of JNK1 and ASK1 (Townsed, Cowan, 1989, Hamada et al, 1994; Tew, 1994; O'Brien, Tew, 1996; Gaudiano et al, 2000, O'Brien et al, 2000;

The family of GSTs consists of eight classes termed cytosolic and symbolized by the Greek alphabet: Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta and Zeta. They are highly polymorphic, with about 30% homology between their base sequences. Each of these classes has several alleles that reach 50% similarity between their base sequences, and are able to

Cellular purification occurs through the ability to regulate the action of protein kinases involved in the catalysis of electrophilic compounds harmful to cells (xenobiotics) from the activation by cytochrome P-450 1A1 and 1B1, such as genotoxic chemical carcinogens and cytotoxic agents chemotherapeutic drugs and their metabolites by means of connection to glutathione (Fig. 1) (Townsed, Cowan, 1989, Shea et al, 1990; Tew, 1994, Shimada et al, 1996; Townsend, Tew, 2003a, b; Daly, 2003). The enzymes of the GST family represents about 5 to

Studies have shown that the GST enzyme complex participates in the JNK1 and ASK1 pathways necessary for activation of MAP kinase signaling processes involved in apoptosis

identification of which patients are at particular risk of treatment failure.

2002, L'Ecuyer et al, 2004).

1995; O'Brien , Tew, 1996; Pakunlu et al, 2003).

**1.3.1 Glutathione S-transferases (GSTs)**

produce enzymes of phase II cell purification.

10% of all cellular proteins (Burg, Mulder, 2002).

Tashiro et al, 2001; Harbottle et al, 2001; Townsend, Tew, 2003b).

development of micrometastases. The prior addition of chemotherapy such as cyclophosphamide in mice transplanted with murine mammary tumor cells showed a significant reduction in the proliferation rate of residual tumor and metastases, and prolonged their survival (Gunduz et al, 1979, Fisher et al, 1989b; Fisher et al, 1989c).

The use of neoadjuvant chemotherapy has additional advantages in patients with locally advanced carcinoma, enhancing the possibility of performing conservative surgery due to the reduction of tumor size, as well as providing evidence in vivo of sensitivity to therapy and providing early treatment of micrometastases (Bonadonna et al. 1990; Wolff, Davidson, 2000, Kafka et al 2003, Hutcheon, Heys, 2004).

Studies demonstrated a significant increase in survival in patients with stage III breast carcinoma, influenced by neoadjuvant chemotherapy associated with local therapy (Canellos, 1976; Jacquillat et al, 1987 Valagussa et al, 1990). Six randomized trials compared the use of adjuvant and neoadjuvant therapy with the aim of measuring the survival of patients with complete clinical response rates from 6.6 to 41% and pathological complete rates of 3 to 29%, with high rates of breast conservation in patients undergoing neoadjuvant chemotherapy (Mauriac et al, 1991; Semiglazov et al, 1994; Scholl, 1994; Powles et al, 1995, van der Hage et al, 2001; Wolmark et al, 2001). One of the major studies related to neoadjuvant chemotherapy was the National Surgical Adjuvant Breast and Bowel Project B18 (NSABP B18), which showed no significant difference between the rates of disease-free survival and survival free of distant disease (among those who received neoadjuvant chemotherapy and those who received postoperative adjuvant chemotherapy). However, neoadjuvant chemotherapy allowed higher rates of conservative surgery and the study in vivo of tumor biology (Fisher et al, 1998). Further analysis, with a follow up of nine years, showed that patients under 49 years experienced a significant advantage in terms of survival rates and disease-free survival when they were submitted to primary chemotherapy in relation to patients 50 years or more, suggesting that age could influence the indication of neoadjuvant chemotherapy, continuing the strong correlation between the clinical primary tumor response to chemotherapy and prognosis (Wolmark et al, 2001).

The neoadjuvant therapy was extended for the treatment of patients with operable breast tumors initially with different chemotherapy regimens and variable rates of clinical response (Scholl et al, 1994; Ragaz et al, 1997, Fisher et al, 1997, Fisher et al, 1998). The clinical response to neoadjuvant administration of chemotherapy, namely the reduction of tumor size, was 10 to 75% in several studies (Kafka et al, 2003).

#### **1.3 Mechanisms of resistance to chemotherapeutic agents**

The main reasons responsible for treatment failure in cancer patients are the mechanisms of drug resistance and emergence of disseminated disease (Terek et al, 2003). We identified two types of resistance most relevant to BC: primary resistance, which corresponds to the clinical situation where the patient showed no response to therapy, and secondary or acquired resistance in which, initially, there is an observed response and a subsequent failure of the treatment regimen (Kroger et al, 1999).

Several mechanisms may cause the phenotype of multidrug resistance to chemotherapy drugs and are well characterized in in vitro experiments, including alterations in systemic pharmacology (pharmacokinetics and metabolism), extracellular mechanisms (tumor environment, multicellular drug resistance), and cellular mechanisms (cellular

development of micrometastases. The prior addition of chemotherapy such as cyclophosphamide in mice transplanted with murine mammary tumor cells showed a significant reduction in the proliferation rate of residual tumor and metastases, and

The use of neoadjuvant chemotherapy has additional advantages in patients with locally advanced carcinoma, enhancing the possibility of performing conservative surgery due to the reduction of tumor size, as well as providing evidence in vivo of sensitivity to therapy and providing early treatment of micrometastases (Bonadonna et al. 1990; Wolff, Davidson,

Studies demonstrated a significant increase in survival in patients with stage III breast carcinoma, influenced by neoadjuvant chemotherapy associated with local therapy (Canellos, 1976; Jacquillat et al, 1987 Valagussa et al, 1990). Six randomized trials compared the use of adjuvant and neoadjuvant therapy with the aim of measuring the survival of patients with complete clinical response rates from 6.6 to 41% and pathological complete rates of 3 to 29%, with high rates of breast conservation in patients undergoing neoadjuvant chemotherapy (Mauriac et al, 1991; Semiglazov et al, 1994; Scholl, 1994; Powles et al, 1995, van der Hage et al, 2001; Wolmark et al, 2001). One of the major studies related to neoadjuvant chemotherapy was the National Surgical Adjuvant Breast and Bowel Project B18 (NSABP B18), which showed no significant difference between the rates of disease-free survival and survival free of distant disease (among those who received neoadjuvant chemotherapy and those who received postoperative adjuvant chemotherapy). However, neoadjuvant chemotherapy allowed higher rates of conservative surgery and the study in vivo of tumor biology (Fisher et al, 1998). Further analysis, with a follow up of nine years, showed that patients under 49 years experienced a significant advantage in terms of survival rates and disease-free survival when they were submitted to primary chemotherapy in relation to patients 50 years or more, suggesting that age could influence the indication of neoadjuvant chemotherapy, continuing the strong correlation between the clinical primary tumor response to

The neoadjuvant therapy was extended for the treatment of patients with operable breast tumors initially with different chemotherapy regimens and variable rates of clinical response (Scholl et al, 1994; Ragaz et al, 1997, Fisher et al, 1997, Fisher et al, 1998). The clinical response to neoadjuvant administration of chemotherapy, namely the reduction of

The main reasons responsible for treatment failure in cancer patients are the mechanisms of drug resistance and emergence of disseminated disease (Terek et al, 2003). We identified two types of resistance most relevant to BC: primary resistance, which corresponds to the clinical situation where the patient showed no response to therapy, and secondary or acquired resistance in which, initially, there is an observed response and a subsequent

Several mechanisms may cause the phenotype of multidrug resistance to chemotherapy drugs and are well characterized in in vitro experiments, including alterations in systemic pharmacology (pharmacokinetics and metabolism), extracellular mechanisms (tumor environment, multicellular drug resistance), and cellular mechanisms (cellular

prolonged their survival (Gunduz et al, 1979, Fisher et al, 1989b; Fisher et al, 1989c).

2000, Kafka et al 2003, Hutcheon, Heys, 2004).

chemotherapy and prognosis (Wolmark et al, 2001).

failure of the treatment regimen (Kroger et al, 1999).

tumor size, was 10 to 75% in several studies (Kafka et al, 2003).

**1.3 Mechanisms of resistance to chemotherapeutic agents**

pharmacology, activation and inactivation of drugs, modification of specific targets and regulatory pathways of apoptosis) (Leonessa et al, 2003, Riddick et al, 2005). Identification of factors that affect cell metabolism, which are related to drug resistance, will enable the identification of which patients are at particular risk of treatment failure.

Among the biochemical and molecular mechanisms of drug resistance, we stress: changes in the activity of topoisomerase II, alterations in the DNA repair mechanism, overexpression of P-glycoprotein; high intracellular concentrations of enzymes purification of cellular metabolism - among them enzymes the family of glutathione S-transferases (GSTs) and changes in the mechanisms of signaling via c-Jun N-terminal kinase 1 (JNK1) -and "apoptosis signal-regulating kinase (ASK1) required for activation of the" mitogenactivated protein (MAP kinases) in apoptosis and cellular restoration. These pathways are also mediated by proteins encoded by genes of GSTs (O'Brien, Tew, 1996; Burg, Mulder, 2002, L'Ecuyer et al, 2004).

Different response rates to particular chemotherapy regimens, as observed in patient groups with the same biological characteristics and stage, suggest the existence of different mechanisms of drug resistance, probably induced by genetic alterations (Hayes, Pulford, 1995; O'Brien , Tew, 1996; Pakunlu et al, 2003).

Among the mechanisms of purification of cellular metabolism involved in the inactivation of toxic substances to the cell there is the action of the enzyme family of GSTs in phase II metabolism of cell purification. The first evidence of their involvement in resistance to drugs used in chemotherapy have emerged from research published by scientific groups Schisswelbauer et al (1990), Tew (1994) and Hayes, Pulford, (1995). However, the relationship between GSTs and resistance to chemotherapy remains inconsistent (Riddick et al, 2005). This mechanism of resistance is related to the ability to regulate the action of enzymes involved in catalyzing electrophilic compounds harmful to cells from activation by cytochrome P-450 1A1 and 1B1 (Phase I). These compounds in turn are substrates for phase II enzyme systems, represented here by the family of GSTs, which are involved on two fronts in the process of drug resistance: production of protective enzymes of metabolism and cellular apoptotic processes via inhibition of JNK1 and ASK1 (Townsed, Cowan, 1989, Hamada et al, 1994; Tew, 1994; O'Brien, Tew, 1996; Gaudiano et al, 2000, O'Brien et al, 2000; Tashiro et al, 2001; Harbottle et al, 2001; Townsend, Tew, 2003b).

#### **1.3.1 Glutathione S-transferases (GSTs)**

The family of GSTs consists of eight classes termed cytosolic and symbolized by the Greek alphabet: Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta and Zeta. They are highly polymorphic, with about 30% homology between their base sequences. Each of these classes has several alleles that reach 50% similarity between their base sequences, and are able to produce enzymes of phase II cell purification.

Cellular purification occurs through the ability to regulate the action of protein kinases involved in the catalysis of electrophilic compounds harmful to cells (xenobiotics) from the activation by cytochrome P-450 1A1 and 1B1, such as genotoxic chemical carcinogens and cytotoxic agents chemotherapeutic drugs and their metabolites by means of connection to glutathione (Fig. 1) (Townsed, Cowan, 1989, Shea et al, 1990; Tew, 1994, Shimada et al, 1996; Townsend, Tew, 2003a, b; Daly, 2003). The enzymes of the GST family represents about 5 to 10% of all cellular proteins (Burg, Mulder, 2002).

Studies have shown that the GST enzyme complex participates in the JNK1 and ASK1 pathways necessary for activation of MAP kinase signaling processes involved in apoptosis

Chemotherapy and Mechanisms of Resistance in Breast Cancer 239

the enzymes of the family of GSTs (Arrick, Nathan, 1984; O'Brien, Tew, 1996; Burg,

GSH has four functions in the anti-cancer therapy: cell protection by blockade of toxic substances to the cell, mediating the formation of toxic to cells, cellular targeting, allowing the efflux and influx of substances through association with enzyme systems membrane and therapeutic interaction through changes in the effectiveness of certain drugs (Arrick, Nathan, 1984). Among the substrates for the cytosolic enzymes of the family of GSTs are anti-neoplastic drugs such as melphalan, chlorambucil, adriamycin, cyclophosphamide, and platinum salts among others (Table 1), which, in the presence of these enzymes, have a lower intracellular

**Substances not characterized as direct substrates of GSTs**

Table 1. Nonsteroidal anti-neoplastic agents associated with increased levels of GST and

as breast, bladder and colon are also substrates for the enzymes of the GST family of

**<IP_ADDRESS> The glutathione S-transferases (GSTs) and breast cancer**

further information in relation to adjuvant chemotherapy and CM.

Cytotoxic and carcinogenic substances from the environment such as tobacco, alcohol and red meat, which are possibly related tocarcinogenesis in various organs such

The classes of GSTs are related to the BC classes Alpha, Theta, and Pi Mu. In this review we approach the last three, as they are most frequently studied and their analysis has provided

The proteins that belong to the Mu class are encoded by a group of genes located on chromosome 1 (GSTM 1-5). These genes are related to various diseases and susceptibility to

concentration (Dirven, 1994; Paumi et al, 2001; Townsend, Tew, 2003a, b).

Inhibitors of topoisomerases I and II \*

**Direct substrates of GSTs**

Chlorambucil Melphalan Nitrogen mustard Mustard Phosphoramide

Antimetabolites \* Antitubulin drugs \*

Bleomycin Hepsulfan Mitomycin C \* Adriamycin \* Cisplatin \* Carboplatin \* Requires activation of JNK for cytotoxicity (Adapted from Townsend, Tew, 2003b)

cellular resistance.

Acrolein Carmustine Hidroxialquilantes Ethacrynic acid Steroids

Mulder, 2002).

and cellular restoration. They also participate in and catalyze the conjugation of electrophilic compounds and free radicals to the tri-peptide glutathione (γ-glu-cys-gly, or GSH), produced by GSH-reductase. Thus, they become less chemically reactive and more soluble, and its excretion facilitated by membrane enzyme complexes, among which stands out the GP1 enzyme encoded by the MRP1 gene family of ABC transporters (Arrick, Nathan, 1984; Townsed Cowan, 1989, Hamada et al, 1994; Tew, 1994; O'Brien, Tew, 1996, Morrow et al, 1998, Gaudiano et al, 2000; Harbottle et al, 2001; Burg, Mulder, 2002; Townsend, Tew, 2003a, b; Parl, 2005).

Fig. 1. Conjugation of glutathione to a generic xenobiotic (X) via catalysis by GSTs to form a conjugate of GST.

Glutathione (GSH) is a major intracellular non-protein substance present in the process of activation and inactivation of toxic substrates to the cell cycle. These reactions begins in the presence of free radicals and products released by the oxide-reactive phenomena of stress and inflammation than healthy and tumor cells are subjected (Arrick, Nathan, 1984; Russo, Mitchell, 1985, Asakura et al, 1999, Adler et al, 1999; Burg, Mulder, 2002; McIlwain et al, 2006). Thus, GSH plays an important role in cell survival and can be found in high concentrations in tumor tissue, where the highest enzyme activity of GSTs family exists (O'Brien, Tew, 1996, O'Brien et al, 2000).

GSH may present itself in several ways, most commonly its reduced sulfhydryl which is related to reactions with substances or reduced-oxide reactions with electrophilic substances. These reactions may be reversible or irreversible, spontaneous or mediated by

and cellular restoration. They also participate in and catalyze the conjugation of electrophilic compounds and free radicals to the tri-peptide glutathione (γ-glu-cys-gly, or GSH), produced by GSH-reductase. Thus, they become less chemically reactive and more soluble, and its excretion facilitated by membrane enzyme complexes, among which stands out the GP1 enzyme encoded by the MRP1 gene family of ABC transporters (Arrick, Nathan, 1984; Townsed Cowan, 1989, Hamada et al, 1994; Tew, 1994; O'Brien, Tew, 1996, Morrow et al, 1998, Gaudiano et al, 2000; Harbottle et al, 2001; Burg, Mulder, 2002; Townsend, Tew, 2003a,

**+**

O

<sup>O</sup> COOH

Fig. 1. Conjugation of glutathione to a generic xenobiotic (X) via catalysis by GSTs to form a

Glutathione (GSH) is a major intracellular non-protein substance present in the process of activation and inactivation of toxic substrates to the cell cycle. These reactions begins in the presence of free radicals and products released by the oxide-reactive phenomena of stress and inflammation than healthy and tumor cells are subjected (Arrick, Nathan, 1984; Russo, Mitchell, 1985, Asakura et al, 1999, Adler et al, 1999; Burg, Mulder, 2002; McIlwain et al, 2006). Thus, GSH plays an important role in cell survival and can be found in high concentrations in tumor tissue, where the highest enzyme activity of GSTs family exists

GSH may present itself in several ways, most commonly its reduced sulfhydryl which is related to reactions with substances or reduced-oxide reactions with electrophilic substances. These reactions may be reversible or irreversible, spontaneous or mediated by

NH

**GLUTATIONA CONJUGADO**

**XENOBIÓTICO (X)**

COOH

NH S-**X**

b; Parl, 2005).

NH²

conjugate of GST.

(O'Brien, Tew, 1996, O'Brien et al, 2000).

COOH

**GLUTATIONA**

O

NH

O

O

NH SH

COOH

the enzymes of the family of GSTs (Arrick, Nathan, 1984; O'Brien, Tew, 1996; Burg, Mulder, 2002).

GSH has four functions in the anti-cancer therapy: cell protection by blockade of toxic substances to the cell, mediating the formation of toxic to cells, cellular targeting, allowing the efflux and influx of substances through association with enzyme systems membrane and therapeutic interaction through changes in the effectiveness of certain drugs (Arrick, Nathan, 1984). Among the substrates for the cytosolic enzymes of the family of GSTs are anti-neoplastic drugs such as melphalan, chlorambucil, adriamycin, cyclophosphamide, and platinum salts among others (Table 1), which, in the presence of these enzymes, have a lower intracellular concentration (Dirven, 1994; Paumi et al, 2001; Townsend, Tew, 2003a, b).

\* Requires activation of JNK for cytotoxicity (Adapted from Townsend, Tew, 2003b)

Table 1. Nonsteroidal anti-neoplastic agents associated with increased levels of GST and cellular resistance.

Cytotoxic and carcinogenic substances from the environment such as tobacco, alcohol and red meat, which are possibly related tocarcinogenesis in various organs such as breast, bladder and colon are also substrates for the enzymes of the GST family of

#### **<IP_ADDRESS> The glutathione S-transferases (GSTs) and breast cancer**

The classes of GSTs are related to the BC classes Alpha, Theta, and Pi Mu. In this review we approach the last three, as they are most frequently studied and their analysis has provided further information in relation to adjuvant chemotherapy and CM.

The proteins that belong to the Mu class are encoded by a group of genes located on chromosome 1 (GSTM 1-5). These genes are related to various diseases and susceptibility to

Chemotherapy and Mechanisms of Resistance in Breast Cancer 241

The enzymes produced by gene GSTP1 \* A prevails as a marker of carcinogenesis, since they are present in many tumor cells (Townsend, Tew, 2003a). Their relationship with cell protection is more related to performance in the apoptotic process. While the proteins encoded by the form "wild" GSTP1 \* A are related to proteins of the JNK pathway, which inhibits apoptosis. This action will cease as the intensity of the phenomena of stress to which the cell is subjected to increase (fig 2), a phenomenon that occurs independently of its catalytic action. Since the polymorphic forms do not have the capacity to synthesize proteins that coalesce to JNK pathway enzymes and therefore do not have the ability to inhibit this

> Substancias oxido-reativas

> > **+**

P P

**Apoptose**

Fig. 2. Action of GSTM1 and GSTP1 on pathways of apoptosis. While related enzymes ASK1 and JNK1, the proteins encoded by the genes GSTM1 and GSTP1 exert an inhibitory effect of the corresponding pathways of apoptosis. Once exposed to substances oxide - reactive is the dissociation of the complex and phosphorylation of enzymes ASK1 and JNK1 pathways that

The involvement of enzymes encoded by the gene GSTP1 \* A in cell survival processes by catalyzing GSH seems to be a secondary response or consequence of the phenomena of stress to which the cells are submitted and occur in two ways of acting. The first one is

GSTP1

JNK C-Jun

P

JNK C-Jun

GSTP1

Drogas anti-neoplásicas

pathway of apoptosis (Adler et al, 1999, Dang et al, 2005).

P

GSTM1

ASK1

GSTM1 ASK1

ASK1: "Apoptosis Signal-Regulating Kinase" JNK / Cjun: "c-Jun N-terminal Kinase 1"

pass the signal to the apoptosis pathway.

P: phosphorus atom

various forms of cancer (Townsend, Tew, 2003a). The GSTM1 gene (Genbank access number AY532926) is the most studied and has four different allelic forms (GSTM 1 \* A, B 1 \* 1 \* 1 \* 0 null and Ax2 that are related to a variety of malignancies, as lung, colorectal, oropharyngeal, bladder and breast cancers (Bell et al, 1993; Ambrosone et al, 1995; Saarikoski et al, 1998; Helzlsouer et al, 1998; Jourenkova-Mironova et al, 1999, Dunning et al, 1999; Ambrosone et al, 2001; Loktionov et al, 2001 and Sgambati et al, 2002; Townsend, Tew, 2003a). However, some authors failed to demonstrate such a relationship (Bailey et al, 1998; Lizard-Nacolia et al , 1999; Garcia-Close et al, 1999).

The enzymes encoded by the gene GSTM1 catalyze the conjugation of electrophilic compounds and free radicals by GSH and still exert an inhibitory effect on apoptosis via ASK1, independent of its catalytic action. This inhibition occurs while the enzyme complexes of GSTM1/ASK1 are related. In the presence of high concentrations of oxidereactive substances, this complex dissociates, releasing enzymes to ASK1 phosphorylation and signaling of apoptosis (Fig. 2) (Cho et al, 2001).

The GSTM1 null genotype polymorphism results from the absence of the two alleles that determine gene expression. Thus, individuals with this genotype do not have the capacity to produce the enzymes necessary to catalyze the conjugation with GSH. Moreover, they also do not synthesize the proteins that coalesce to the ASK1 pathway proteins necessary for the inhibition of this pathway of apoptosis (Cho et al, 2001; McIlwain et al, 2006). The null genotype is present in 40 to 50% of the population (Tew, 1994), ranging from 22% in Nigeria, 58% among Chinese, 45% in Western Europe and up to 67% in Australia, and it is related to better response to some classes of chemotherapeutic agents against various types of cancer (Alpert et al, 1997; Ambrosone et al, 2001; Sgambati et al, 2002; Autrup et al, 2002; Townsend, Tew, 2003a; Khedhaier et al, 2003; Parl , 2005).

The proteins of the class Theta are encoded by two genes (T1 and T2), which are located on chromosome 22. The class GSTT1 (accession number AB057594 in Genbank) has three allelic forms: \* The T1, T1 and T1 \* B \* 0 or null, but the latter is present between 10 and 30% in African populations, 10% in European and American populations, and 64% in Asian populations (Townsend, Tew, 2003a). The T1 null allele is associated with a predisposition to some cancers (Townsend, Tew, 2003a; Saarikoski, 1998; Helzlsouer et al, 1998; Jourenkova-Mironova et al, 1999 and Ambrosone et al, 2001), among them breast cancer in postmenopausal women, users of large quantities of alcoholic and longtime smokers, as well as in premenopausal women or nulliparous women who gave birth after age 30 (Park et al, 2000, Zheng et al, 2002, Zheng et al, 2003; Park et al, 2003), although some studies have not shown this relationship (Bailey et al, 1998; Garcia-Close et al, 1999; Millikan et al, 2000). The presence of the GSTT1 null form, in which there is not production of the enzymes, was associated with a better response to chemotherapy in patients with BC and the greater toxicity of some chemotherapeutic agents (Howells et al, 2001;Naoe et al, 2002; Khedhaier et al, 2003).

The class Pi, in turn, consists of only one protein encoded by a gene located on chromosome 11 and called GSTP1 (GenBank access number AY324387). The GSTP1 gene has three allelic forms. The wild GSTP1 \* A (Ile105Ile/Ala113Ala) genotype results in the replacement of Ile by Val at least one amino acid at codon 105 and) and two polymorphic forms, GSTP1 \* B (Val 105Ile Val/113 Val) where, in addition to the alteration observed in GSTP1B \* there is also replacement of Ala by Val in at least one amino acid codon 113. GSTP1 \* C (105 Ile Ala. These forms are represented, respectively, in 68%, 26% and 7% of the Caucasian population (Townsend, Tew, 2003a).

various forms of cancer (Townsend, Tew, 2003a). The GSTM1 gene (Genbank access number AY532926) is the most studied and has four different allelic forms (GSTM 1 \* A, B 1 \* 1 \* 1 \* 0 null and Ax2 that are related to a variety of malignancies, as lung, colorectal, oropharyngeal, bladder and breast cancers (Bell et al, 1993; Ambrosone et al, 1995; Saarikoski et al, 1998; Helzlsouer et al, 1998; Jourenkova-Mironova et al, 1999, Dunning et al, 1999; Ambrosone et al, 2001; Loktionov et al, 2001 and Sgambati et al, 2002; Townsend, Tew, 2003a). However, some authors failed to demonstrate such a relationship (Bailey et al, 1998; Lizard-Nacolia et

The enzymes encoded by the gene GSTM1 catalyze the conjugation of electrophilic compounds and free radicals by GSH and still exert an inhibitory effect on apoptosis via ASK1, independent of its catalytic action. This inhibition occurs while the enzyme complexes of GSTM1/ASK1 are related. In the presence of high concentrations of oxidereactive substances, this complex dissociates, releasing enzymes to ASK1 phosphorylation

The GSTM1 null genotype polymorphism results from the absence of the two alleles that determine gene expression. Thus, individuals with this genotype do not have the capacity to produce the enzymes necessary to catalyze the conjugation with GSH. Moreover, they also do not synthesize the proteins that coalesce to the ASK1 pathway proteins necessary for the inhibition of this pathway of apoptosis (Cho et al, 2001; McIlwain et al, 2006). The null genotype is present in 40 to 50% of the population (Tew, 1994), ranging from 22% in Nigeria, 58% among Chinese, 45% in Western Europe and up to 67% in Australia, and it is related to better response to some classes of chemotherapeutic agents against various types of cancer (Alpert et al, 1997; Ambrosone et al, 2001; Sgambati et al, 2002; Autrup et al, 2002;

The proteins of the class Theta are encoded by two genes (T1 and T2), which are located on chromosome 22. The class GSTT1 (accession number AB057594 in Genbank) has three allelic forms: \* The T1, T1 and T1 \* B \* 0 or null, but the latter is present between 10 and 30% in African populations, 10% in European and American populations, and 64% in Asian populations (Townsend, Tew, 2003a). The T1 null allele is associated with a predisposition to some cancers (Townsend, Tew, 2003a; Saarikoski, 1998; Helzlsouer et al, 1998; Jourenkova-Mironova et al, 1999 and Ambrosone et al, 2001), among them breast cancer in postmenopausal women, users of large quantities of alcoholic and longtime smokers, as well as in premenopausal women or nulliparous women who gave birth after age 30 (Park et al, 2000, Zheng et al, 2002, Zheng et al, 2003; Park et al, 2003), although some studies have not shown this relationship (Bailey et al, 1998; Garcia-Close et al, 1999; Millikan et al, 2000). The presence of the GSTT1 null form, in which there is not production of the enzymes, was associated with a better response to chemotherapy in patients with BC and the greater toxicity of some chemotherapeutic agents (Howells et al,

The class Pi, in turn, consists of only one protein encoded by a gene located on chromosome 11 and called GSTP1 (GenBank access number AY324387). The GSTP1 gene has three allelic forms. The wild GSTP1 \* A (Ile105Ile/Ala113Ala) genotype results in the replacement of Ile by Val at least one amino acid at codon 105 and) and two polymorphic forms, GSTP1 \* B (Val 105Ile Val/113 Val) where, in addition to the alteration observed in GSTP1B \* there is also replacement of Ala by Val in at least one amino acid codon 113. GSTP1 \* C (105 Ile Ala. These forms are represented, respectively, in 68%, 26% and 7% of the Caucasian

al , 1999; Garcia-Close et al, 1999).

and signaling of apoptosis (Fig. 2) (Cho et al, 2001).

Townsend, Tew, 2003a; Khedhaier et al, 2003; Parl , 2005).

2001;Naoe et al, 2002; Khedhaier et al, 2003).

population (Townsend, Tew, 2003a).

The enzymes produced by gene GSTP1 \* A prevails as a marker of carcinogenesis, since they are present in many tumor cells (Townsend, Tew, 2003a). Their relationship with cell protection is more related to performance in the apoptotic process. While the proteins encoded by the form "wild" GSTP1 \* A are related to proteins of the JNK pathway, which inhibits apoptosis. This action will cease as the intensity of the phenomena of stress to which the cell is subjected to increase (fig 2), a phenomenon that occurs independently of its catalytic action. Since the polymorphic forms do not have the capacity to synthesize proteins that coalesce to JNK pathway enzymes and therefore do not have the ability to inhibit this pathway of apoptosis (Adler et al, 1999, Dang et al, 2005).

ASK1: "Apoptosis Signal-Regulating Kinase" JNK / Cjun: "c-Jun N-terminal Kinase 1" P: phosphorus atom

Fig. 2. Action of GSTM1 and GSTP1 on pathways of apoptosis. While related enzymes ASK1 and JNK1, the proteins encoded by the genes GSTM1 and GSTP1 exert an inhibitory effect of the corresponding pathways of apoptosis. Once exposed to substances oxide - reactive is the dissociation of the complex and phosphorylation of enzymes ASK1 and JNK1 pathways that pass the signal to the apoptosis pathway.

The involvement of enzymes encoded by the gene GSTP1 \* A in cell survival processes by catalyzing GSH seems to be a secondary response or consequence of the phenomena of stress to which the cells are submitted and occur in two ways of acting. The first one is

Chemotherapy and Mechanisms of Resistance in Breast Cancer 243