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bacteria, which are essential to develop disease in animal models. Furthermore, accumulating evidence suggests that bacterial production of toxins, toxic metabolites and the direct influences on pro-carcinogenic pathways in host epithelial cells are contributing factors that promote the accumulation of mutations that may eventually lead to carcinomas. However, still many questions remain to be answered. For example, our knowledge on the on the impact of CRC on the local intestinal microbiota and *vice versa*, is still in its infancy. Future research should focus on the detailed mapping of the microbiota in close proximity of early adenomas and carcinomas. These local changes in microbiota may for instance provide clues in the understanding why only 10% of the adenomas progress into carcinomas. Such knowledge could give us new leads for cancer diagnosis, for example by using signaling bacteria, such as *S. gallolyticus* that benefit from the altered tumor environment, as diagnostic targets. Furthermore, this knowledge could provide leads for the selective removal of high-risk bacterial populations by health promoting species, as a new strategy in CRC prevention. Altogether, this Chapter points out that the colonic microbiota should be regarded as an important factor in intestinal carcinogenesis. Further research in this field is crucial to fully understand the etiology of CRC and has a high potential to lead to new diagnostic tools and therapeutic interventions.

#### **8. Acknowledgements**

We thank Albert Bolhuis, Dorine Swinkels, Bas Dutilh, Carla Muytjens, Guus Kortman, Ikuko Kato, Julian Marchesi, Philippe Glaser, Rian Roelofs, Shaynoor Dramsi & Wilbert Peters for inspiring discussions. Work in our laboratory was supported by the Dutch Cancer Society (KWF; project KUN 2006-3591) and the Dutch Digestive Diseases Foundation (MLDS; project WO10-53). Correspondence to: Harold Tjalsma, Department of Laboratory Medicine (LGEM 830), Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands<EMAIL_ADDRESS>
#### **9. References**

bacteria, which are essential to develop disease in animal models. Furthermore, accumulating evidence suggests that bacterial production of toxins, toxic metabolites and the direct influences on pro-carcinogenic pathways in host epithelial cells are contributing factors that promote the accumulation of mutations that may eventually lead to carcinomas. However, still many questions remain to be answered. For example, our knowledge on the on the impact of CRC on the local intestinal microbiota and *vice versa*, is still in its infancy. Future research should focus on the detailed mapping of the microbiota in close proximity of early adenomas and carcinomas. These local changes in microbiota may for instance provide clues in the understanding why only 10% of the adenomas progress into carcinomas. Such knowledge could give us new leads for cancer diagnosis, for example by using signaling bacteria, such as *S. gallolyticus* that benefit from the altered tumor environment, as diagnostic targets. Furthermore, this knowledge could provide leads for the selective removal of high-risk bacterial populations by health promoting species, as a new strategy in CRC prevention. Altogether, this Chapter points out that the colonic microbiota should be regarded as an important factor in intestinal carcinogenesis. Further research in this field is crucial to fully understand the etiology of CRC and has a high potential to lead

We thank Albert Bolhuis, Dorine Swinkels, Bas Dutilh, Carla Muytjens, Guus Kortman, Ikuko Kato, Julian Marchesi, Philippe Glaser, Rian Roelofs, Shaynoor Dramsi & Wilbert Peters for inspiring discussions. Work in our laboratory was supported by the Dutch Cancer Society (KWF; project KUN 2006-3591) and the Dutch Digestive Diseases Foundation (MLDS; project WO10-53). Correspondence to: Harold Tjalsma, Department of Laboratory Medicine (LGEM 830), Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500

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Holmes, E., Wang, Y. & Marchesi, J. R. (2008). Culture-independent analysis of the gut microbiota in colorectal cancer and polyposis. *Environl Microbiol* 10, 789-798. Schiffman, M. H., Van Tassell, R. L., Robinson, A. & other authors (1989). Case-control study of colorectal cancer and fecapentaene excretion. *Cancer Res* 49, 1322-1326. Sears, C. L. (2009). Enterotoxigenic Bacteroides fragilis: a rogue among symbiotes. *Clin*

D. M. & Sartor, R. B. (1998). Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient

mucosal adherent bacteria and associations with colorectal adenomas. *Gut Microbes*

8-hydroxydeoxyguanosine in DNA treated with fecapentaene-12 and -14. *Mutation*

Relationship between Helicobacter pylori CagA status and colorectal cancer. *Am J*

Adherence characteristics of endocarditis-derived Streptococcus gallolyticus ssp. gallolyticus (Streptococcus bovis biotype I) isolates to host extracellular matrix

adhesin, Acb, and 10 other putative MSCRAMM and pilus family proteins of Streptococcus gallolyticus subsp. gallolyticus (S. bovis biotype I). *J Bact* 191, 6643-

Tran Van Nhieu, J. & Furet, J. P. (2011). Microbial dysbiosis in colorectal cancer

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*Gastroenter* 96, 3406-3410.

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*immunol* 5, 569-573.

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**Part 5**

**Study Reports**

**Part 5**

**Study Reports**

410 Colorectal Cancer Biology – From Genes to Tumor

Wilkinson, S. M., Uhl, J. R., Kline, B. C. & Cockerill, F. R., 3rd (1998). Assessment of invasion

Winters, M. D., Schlinke, T. L., Joyce, W. A., Glore, S. R. & Huycke, M. M. (1998).

Wu, S., Morin, P. J., Maouyo, D. & Sears, C. L. (2003). Bacteroides fragilis enterotoxin induces c-Myc expression and cellular proliferation. *Gastroenterology* 124, 392-400. Wu, S., Rhee, K. J., Albesiano, E. & other authors (2009). A human colonic commensal

Xu, J., Mahowald, M. A., Ley, R. E. & other authors (2007). Evolution of symbiotic bacteria in

Yantiss, R. K., Goldman, H. & Odze, R. D. (2001). Hyperplastic polyp with epithelial

Zoetendal, E. G., von Wright, A., Vilpponen-Salmela, T., Ben-Amor, K., Akkermans, A. D. &

Zumkeller, N., Brenner, H., Zwahlen, M. & Rothenbacher, D. (2006). Helicobacter pylori infection and colorectal cancer risk: a meta-analysis. *Helicobacter* 11, 75-80.

immunohistochemical study of 19 cases. *Mod Pathol* 14, 869-875.

recovered from feces. *Appl Environ Microbiol* 68, 3401-3407.

an acridine orange assay. *J Clin Pathol* 51, 127-133.

the distal human intestine. *PLoS Biology* 5, e156.

*Am J Gastroenterol* 93, 2491-2500.

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frequencies of cultured HEp-2 cells by clinical isolates of Helicobacter pylori using

Prospective case-cohort study of intestinal colonization with enterococci that produce extracellular superoxide and the risk for colorectal adenomas or cancer.

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de Vos, W. M. (2002). Mucosa-associated bacteria in the human gastrointestinal tract are uniformly distributed along the colon and differ from the community

**19**

*1Department of Surgery, 2Institute of Pathology,*

*Czech Republic*

**Tumor Infiltrating Lymphocytes as Prognostic**

Sixty percent of patients with colorectal cancer (CRC) are afflicted with distant metastases (liver or lung metastatic process) or a local relapse of malignancy (Bird et al., 2006). The possibilities of surgical and oncological treatment of this disease offer us a large spectrum of treatments including the combination of surgical procedures and consecutive oncological treatments. In the case of radical surgical therapy we can consider the curative access. The main medical problem of CRC is the high rate of recurrences after radically performed surgical therapy. The operability of recurrence is only about 30% in the case of local relapse and 20% in the case of distant metastases (Coleman et al., 2008; Kobayashi et al., 2007). The second dominant problem is the early recurrence of CRC after radical surgical treatment, when the patients undergo a difficult and exhausting procedure with a high risk of perioperative complications without any significant differences in overall survival against

Contemporary clinical and histopathological prognostic factors (staging, grading, etc.) used for the detection of patients with a high risk of relapse and a short overall survival rate and for the indication of adjuvant oncological treatment after radical surgery are not sufficient. Tumor infiltrating lymphocytes (TIL) were described as a good prognostic factor for patients with a high risk of relapse. They are critical indicators of efficient antitumor immunological response. Their number, type and morphology of TIL cells determine resulting tumor prognosis (Atreya & Neurath, 2008; Galon et al., 2006). They could be connected also with the suppression of micrometastatical disease after radical surgery (Gajewski et al., 2006; Pages et al., 2005). We can recognize either the type of immune cells or distinguish their morphological aspects (infiltration of any part of tumor or surrounding of tumor or

modern palliative therapy (Van den Eynde & Hendlisz, 2009).

tributary lymph nodes) (Talmadge et al., 2007).

**1. Introduction**

**Factor of Early Recurrence and Poor**

Vaclav Liska1, Ondrej Daum2, Petr Novak1, Vladislav Treska1, Ondrej Vycital1, Jan Bruha1, Pavel Pitule1 and Lubos Holubec3

**Prognosis of Colorectal Cancer**

*3Department of Oncology, Charles University Prague,*

*Medical School and Teaching Hospital Pilsen,*

**After Radical Surgical Treatment**

### **Tumor Infiltrating Lymphocytes as Prognostic Factor of Early Recurrence and Poor Prognosis of Colorectal Cancer After Radical Surgical Treatment**

Vaclav Liska1, Ondrej Daum2, Petr Novak1, Vladislav Treska1, Ondrej Vycital1, Jan Bruha1, Pavel Pitule1 and Lubos Holubec3 *1Department of Surgery, 2Institute of Pathology, 3Department of Oncology, Charles University Prague, Medical School and Teaching Hospital Pilsen, Czech Republic*

#### **1. Introduction**

Sixty percent of patients with colorectal cancer (CRC) are afflicted with distant metastases (liver or lung metastatic process) or a local relapse of malignancy (Bird et al., 2006). The possibilities of surgical and oncological treatment of this disease offer us a large spectrum of treatments including the combination of surgical procedures and consecutive oncological treatments. In the case of radical surgical therapy we can consider the curative access. The main medical problem of CRC is the high rate of recurrences after radically performed surgical therapy. The operability of recurrence is only about 30% in the case of local relapse and 20% in the case of distant metastases (Coleman et al., 2008; Kobayashi et al., 2007). The second dominant problem is the early recurrence of CRC after radical surgical treatment, when the patients undergo a difficult and exhausting procedure with a high risk of perioperative complications without any significant differences in overall survival against modern palliative therapy (Van den Eynde & Hendlisz, 2009).

Contemporary clinical and histopathological prognostic factors (staging, grading, etc.) used for the detection of patients with a high risk of relapse and a short overall survival rate and for the indication of adjuvant oncological treatment after radical surgery are not sufficient. Tumor infiltrating lymphocytes (TIL) were described as a good prognostic factor for patients with a high risk of relapse. They are critical indicators of efficient antitumor immunological response. Their number, type and morphology of TIL cells determine resulting tumor prognosis (Atreya & Neurath, 2008; Galon et al., 2006). They could be connected also with the suppression of micrometastatical disease after radical surgery (Gajewski et al., 2006; Pages et al., 2005). We can recognize either the type of immune cells or distinguish their morphological aspects (infiltration of any part of tumor or surrounding of tumor or tributary lymph nodes) (Talmadge et al., 2007).

Tumor Infiltrating Lymphocytes as Prognostic Factor of Early

interval (DFI) after radical surgery for CRC.

**a**

**c**

Recurrence and Poor Prognosis of Colorectal Cancer After Radical Surgical Treatment 415

2a),intrastromal (ISL – intrastromal lymphocytes) (Fig. 2b), peritumoral (PTL – peritumoral lymphocytes) (Fig. 2c) and Crohn-like reaction (Crohn-like PTL)(Fig. 2d). We can also describe reactive histological changes in tributary lymph nodes (LN reactions). It means follicular hyperplasia (LN-FH) (Fig. 3a.), sinus histiocytosis (LN-SH) (Fig. 3b.) and the

The aim of this study was to analyze the relationship of contemporary clinical and histopathological factors and TIL to determine patients with a high risk of poor overall survival and tendency to early recurrence of malignancy with shortened disease free

**b**

**d**

Fig. 1. Examples of tumor infiltration by immune cells: a) CD8+ T lymphocytes; b) CD4+ T lymphocytes; c) Natural killer cell (CD 57 staining) and d) Dendritic cells (S 100 staining).

presence of granulomas (LN-GR) (Fig. 3c) (Ogino et al, 2009; Pages et al., 2005).

We detail only short overview of their types and function. We recommend the readers with deeper interest in these problems to find comprehensive reviews in the cited papers (Jochems et al., 2011; Ohtani 2007). From this view we find CD8+ and CD4+ T lymphocytes (Fig. 1a & Fig. 1b), natural killer cells (Fig. 1c), dendritic cells (Fig. 1d), macrophages, etc. The exact function of these cells is under current discussion. We only know that they play main role in controlling tumor development and growth. CD8+ T lymphocytes within cancer cell nests of colorectal cancer have significant impact on the survival of patients. They contain the cytolytic enzyme granzyme-B. In case of increased proliferating activity of CD8+ T lymphocytes we observe their activated and cytotoxic phenotype that is significantly associated with the absence of early metastatic events (vascular emboli, lymphatic invasion or perineural invasion of tumor cells) and with a decreased rate of cancer recurrence (Atreya & Neurath, 2008, Pages et al., 2010). A high density of memory T lymphocytes within colorectal cancer tissue was more frequently observed in patients without early detectable signs of metastatic events and was associated with both improved disease free interval (DFI) and prolonged overall survival (OS) (Galon et al., 2006; Pages et al., 2005).

Natural killer cells (NK cells) mediate an effective lysis of cancer cells but the mechanism of detection of cancer cells is different from CD8+ T lymphocytes (Cooper et al., 2009). NK cells are mainly involved in the innate immune response and do not recognize specific tumor associated antigen on the surface of cancer cells as CD8+ T lymphocytes.  NK cells also respond to other signals as cytokines produced by antigen presenting cells, which allow them to mediate early host responses against pathogen (Moretta et al., 2006). Decreased preoperative number of NK cells was associated with increased frequency of postoperative recurrence of colorectal cancer (Atreya & Neurath, 2008; Cooper et al., 2001). Their crucial role in the elimination of haematological malignancies, primary and secondary tumors has been recognized (Lucas et al., 2007; Ljunggren & Malmberg, 2007, Stojanovic & Cerwenka, 2011). In the last year there are some signs that NK-cells have the capacity for memory-like responses, a property that was previously thought to be limited to adaptive immunity, but in this view the discussion still continues (Cooper et al., 2009).

Dendritic cells are considered to be most potent antigen presenting cells. They play key role in activation, stimulation and recruitment of T lymphocytes. They can also induce antigenspecific unresponsiveness or immune tolerance. Immature dendritic cells enter tumor tissue, uptake and process its antigens. Then after they migrate to lymph nodes, undergo maturation and interact with T-lymphocytes that are able to recognize presented antigen and so T-lymphocytes play effector role of this tumor-specific immunity (Atreya & Neurath, 2008; Pages et al., 2005; Sandel et al., 2005; Steinman et al., 2003).

Macrophages are important producers of different factors that have function during tumor progression and also during tumor progression control. Their function is not fully understand, but it was described that the number of tumor infiltrating macrophages correlates with overall survival of colorectal cancer patients (Atreya & Neurath, 2008; Pollard, 2004; Forssell et al., 2007). It seems that several types of tumor infiltrating macrophages influence the balance between pro- and anti-tumor properties of immune system (Forssell et al., 2007).

From the morphologic view we can observe TIL in the specific portions of tumor and so we detect lymphocytic infiltration intratumoral (ITL – intratumoral lymphocytes) (Fig.

We detail only short overview of their types and function. We recommend the readers with deeper interest in these problems to find comprehensive reviews in the cited papers (Jochems et al., 2011; Ohtani 2007). From this view we find CD8+ and CD4+ T lymphocytes (Fig. 1a & Fig. 1b), natural killer cells (Fig. 1c), dendritic cells (Fig. 1d), macrophages, etc. The exact function of these cells is under current discussion. We only know that they play main role in controlling tumor development and growth. CD8+ T lymphocytes within cancer cell nests of colorectal cancer have significant impact on the survival of patients. They contain the cytolytic enzyme granzyme-B. In case of increased proliferating activity of CD8+ T lymphocytes we observe their activated and cytotoxic phenotype that is significantly associated with the absence of early metastatic events (vascular emboli, lymphatic invasion or perineural invasion of tumor cells) and with a decreased rate of cancer recurrence (Atreya & Neurath, 2008, Pages et al., 2010). A high density of memory T lymphocytes within colorectal cancer tissue was more frequently observed in patients without early detectable signs of metastatic events and was associated with both improved disease free interval (DFI)

Natural killer cells (NK cells) mediate an effective lysis of cancer cells but the mechanism of detection of cancer cells is different from CD8+ T lymphocytes (Cooper et al., 2009). NK cells are mainly involved in the innate immune response and do not recognize specific tumor associated antigen on the surface of cancer cells as CD8+ T lymphocytes.  NK cells also respond to other signals as cytokines produced by antigen presenting cells, which allow them to mediate early host responses against pathogen (Moretta et al., 2006). Decreased preoperative number of NK cells was associated with increased frequency of postoperative recurrence of colorectal cancer (Atreya & Neurath, 2008; Cooper et al., 2001). Their crucial role in the elimination of haematological malignancies, primary and secondary tumors has been recognized (Lucas et al., 2007; Ljunggren & Malmberg, 2007, Stojanovic & Cerwenka, 2011). In the last year there are some signs that NK-cells have the capacity for memory-like responses, a property that was previously thought to be limited to adaptive immunity, but in this view the discussion

Dendritic cells are considered to be most potent antigen presenting cells. They play key role in activation, stimulation and recruitment of T lymphocytes. They can also induce antigenspecific unresponsiveness or immune tolerance. Immature dendritic cells enter tumor tissue, uptake and process its antigens. Then after they migrate to lymph nodes, undergo maturation and interact with T-lymphocytes that are able to recognize presented antigen and so T-lymphocytes play effector role of this tumor-specific immunity (Atreya & Neurath,

Macrophages are important producers of different factors that have function during tumor progression and also during tumor progression control. Their function is not fully understand, but it was described that the number of tumor infiltrating macrophages correlates with overall survival of colorectal cancer patients (Atreya & Neurath, 2008; Pollard, 2004; Forssell et al., 2007). It seems that several types of tumor infiltrating macrophages influence the balance between pro- and anti-tumor properties of immune

From the morphologic view we can observe TIL in the specific portions of tumor and so we detect lymphocytic infiltration intratumoral (ITL – intratumoral lymphocytes) (Fig.

and prolonged overall survival (OS) (Galon et al., 2006; Pages et al., 2005).

2008; Pages et al., 2005; Sandel et al., 2005; Steinman et al., 2003).

still continues (Cooper et al., 2009).

system (Forssell et al., 2007).

2a),intrastromal (ISL – intrastromal lymphocytes) (Fig. 2b), peritumoral (PTL – peritumoral lymphocytes) (Fig. 2c) and Crohn-like reaction (Crohn-like PTL)(Fig. 2d). We can also describe reactive histological changes in tributary lymph nodes (LN reactions). It means follicular hyperplasia (LN-FH) (Fig. 3a.), sinus histiocytosis (LN-SH) (Fig. 3b.) and the presence of granulomas (LN-GR) (Fig. 3c) (Ogino et al, 2009; Pages et al., 2005).

The aim of this study was to analyze the relationship of contemporary clinical and histopathological factors and TIL to determine patients with a high risk of poor overall survival and tendency to early recurrence of malignancy with shortened disease free interval (DFI) after radical surgery for CRC.

Fig. 1. Examples of tumor infiltration by immune cells: a) CD8+ T lymphocytes; b) CD4+ T lymphocytes; c) Natural killer cell (CD 57 staining) and d) Dendritic cells (S 100 staining).

Tumor Infiltrating Lymphocytes as Prognostic Factor of Early

diagnosed early recurrence of CRC.

postoperative oncological treatment.

and stained with hematoxylin-eosin.

manner as lymphocytic infiltration.

**2.2 Immunohistochemistry**

**2.3 Statistical evaluation**

**2. Methods**

**2.1 Histology**

Recurrence and Poor Prognosis of Colorectal Cancer After Radical Surgical Treatment 417

We analyzed 150 patients who underwent radical surgical procedure for CRC between the years 2004-2007 at the Department of Surgery, Medical School and Teaching Hospital in Pilsen, Charles University in Prague. We selected only patients who were operated on electively – our aim was to decrease the risk of inflammation that is often connected with the acute operation of CRC and does not depend on the immune reaction against a tumor but could be evoked by the distension of the bowel. We also excluded patients who had risk of understaging (for example low number of analysed lymph nodes) and patients with a synchronous metastatic process. The inclusion standard was also the entire follow-up of patients during the whole postoperative period to increase the number of patients with a

The following clinical parameters were statistically analysed in relation to the disease free interval (DFI) and the overall survival (OS): staging, grading, preoperative leukocytosis, type of surgical procedure (radical vs. palliative), postoperative complications and

We examined three different sections of each tumor and also sections of all found lymph nodes. Tissue for light microscopy was fixed in 4% formaldehyde and embedded in paraffin using routine procedures. Five micrometer-thick sections were cut from the tissue blocks

The type and grade of all tumors were determined according to WHO 2000 guidelines. The stage of tumors was established according to UICC 2002 guidelines. We evaluated endovascular (VI), endolymphatic (LI) and perineural infiltration (PI) by cancer cells (0 – none, 1 – yes). Lymphocytic infiltration was detected as intratumoral (ITL – intratumoral lymphocytes), intrastromal (ISL – intrastromal lymphocytes), peritumoral (PTL – peritumoral lymphocytes) and Crohn-like reaction (Crohn-like PTL), and scaled as none (0), mild (1), moderate (2) and severe (3). Reactive histological changes in lymph nodes (LN reactions) were detected as follicular hyperplasia (LN-FH), sinus histiocytosis (LN-SH) and the presence of granulomas (LN-GR), and all these parameters were quantified in the same

For immunohistochemical investigations the following primary antibodies were used: CD4 (clone 4B12, 1:50, Vector Laboratories, Burlingame, CA, USA) and CD8 (clone C8/144B, 1:50, Dako, Glostrup, Denmark). Microwave pretreatment was used in both cases. The primary antibodies were visualized using the supersensitive streptavidin-biotin-peroxidase complex (Biogenex, San Ramon, CA). Appropriate positive and negative control slides were employed. The density of intratumoral infiltration by lymphocytes was evaluated in five High power microscopical fields (HPF) and expressed as the number of immunopositive cells per HPF.

Statistical analysis was processed by the statistical software Statistica 9.0. The mean, median, standard deviation (SD), minimum, maximum, quartiles, frequencies and other basic statistical measurements were computed in given groups and subgroups of patients

corresponding to studied clinical and histopathological parameters.

Fig. 2. Different localisation of TIL within the tumor tissue: a) intratumoral lymphocytes; b) intrastromal lymphocytes; c) peritumoral lymphocytes and d) Crohn-like peritumoral lymphocytes. All sections stained with hematoxylin-eosin.

Fig. 3. Reactive histological changes in tributary lymph nodes: a) follicular hyperplasia; b) sinus histiocytosis and c) presence of granulomas. All sections stained with hematoxylin-eosin.

### **2. Methods**

416 Colorectal Cancer Biology – From Genes to Tumor

**b**

**d**

Fig. 2. Different localisation of TIL within the tumor tissue: a) intratumoral lymphocytes; b) intrastromal lymphocytes; c) peritumoral lymphocytes and d) Crohn-like peritumoral

Fig. 3. Reactive histological changes in tributary lymph nodes: a) follicular hyperplasia; b) sinus histiocytosis and c) presence of granulomas. All sections stained with hematoxylin-eosin.

lymphocytes. All sections stained with hematoxylin-eosin.

**a b c**

**a**

**c**

We analyzed 150 patients who underwent radical surgical procedure for CRC between the years 2004-2007 at the Department of Surgery, Medical School and Teaching Hospital in Pilsen, Charles University in Prague. We selected only patients who were operated on electively – our aim was to decrease the risk of inflammation that is often connected with the acute operation of CRC and does not depend on the immune reaction against a tumor but could be evoked by the distension of the bowel. We also excluded patients who had risk of understaging (for example low number of analysed lymph nodes) and patients with a synchronous metastatic process. The inclusion standard was also the entire follow-up of patients during the whole postoperative period to increase the number of patients with a diagnosed early recurrence of CRC.

The following clinical parameters were statistically analysed in relation to the disease free interval (DFI) and the overall survival (OS): staging, grading, preoperative leukocytosis, type of surgical procedure (radical vs. palliative), postoperative complications and postoperative oncological treatment.

#### **2.1 Histology**

We examined three different sections of each tumor and also sections of all found lymph nodes. Tissue for light microscopy was fixed in 4% formaldehyde and embedded in paraffin using routine procedures. Five micrometer-thick sections were cut from the tissue blocks and stained with hematoxylin-eosin.

The type and grade of all tumors were determined according to WHO 2000 guidelines. The stage of tumors was established according to UICC 2002 guidelines. We evaluated endovascular (VI), endolymphatic (LI) and perineural infiltration (PI) by cancer cells (0 – none, 1 – yes). Lymphocytic infiltration was detected as intratumoral (ITL – intratumoral lymphocytes), intrastromal (ISL – intrastromal lymphocytes), peritumoral (PTL – peritumoral lymphocytes) and Crohn-like reaction (Crohn-like PTL), and scaled as none (0), mild (1), moderate (2) and severe (3). Reactive histological changes in lymph nodes (LN reactions) were detected as follicular hyperplasia (LN-FH), sinus histiocytosis (LN-SH) and the presence of granulomas (LN-GR), and all these parameters were quantified in the same manner as lymphocytic infiltration.

#### **2.2 Immunohistochemistry**

For immunohistochemical investigations the following primary antibodies were used: CD4 (clone 4B12, 1:50, Vector Laboratories, Burlingame, CA, USA) and CD8 (clone C8/144B, 1:50, Dako, Glostrup, Denmark). Microwave pretreatment was used in both cases. The primary antibodies were visualized using the supersensitive streptavidin-biotin-peroxidase complex (Biogenex, San Ramon, CA). Appropriate positive and negative control slides were employed. The density of intratumoral infiltration by lymphocytes was evaluated in five High power microscopical fields (HPF) and expressed as the number of immunopositive cells per HPF.

#### **2.3 Statistical evaluation**

Statistical analysis was processed by the statistical software Statistica 9.0. The mean, median, standard deviation (SD), minimum, maximum, quartiles, frequencies and other basic statistical measurements were computed in given groups and subgroups of patients corresponding to studied clinical and histopathological parameters.

Tumor Infiltrating Lymphocytes as Prognostic Factor of Early

significance of CD8/CD4 ratio.

FH positivity decreased the risk of shorter overall survival 3.3times.

Recurrence and Poor Prognosis of Colorectal Cancer After Radical Surgical Treatment 419

Endovascular infiltration (VI) was proved as a negative prognostic factor of shorter overall survival (Fig.5.). Patients with positive histopathological findings of VI have 3.1x increased risk for shorter overall survival. The presence of peritumoral lymphocytes (PTL) (Fig.6.) and of Crohn-like PTL (Fig.7.) was proved as a positive prognostic factor of OS. Patients with a positive histopathological finding of PTL and Crohn-like PTL have a decreased risk for shorter overall survival (2.3x and 2.3x respectively). Lymph node follicular hyperplasia (LN-FH) was verified as a positive prognostic factor for longer overall survival (Fig.8.). The statistical significance of LN-FH increased also with the raised density of infiltration. LN-

The severity of CD8+ lymphocytic infiltration was proved by the Cox regression hazard model as a positive prognostic factor enlarging overall survival (cut off 30 cells/HPF). The severity of CD4+ lymphocytic infiltration was proved as a significant factor for the prognosis of overall survival (cut off 4cells/HPF) with 2.5x increased hazard ratio in patients over the cut off (Fig.9.). Statistical analysis did not confirm the statistical

Fig. 5. Endovascular infiltration by cancer cells (VI) was proved as a statistically significant

factor for the prognosis of overall survival (p<0.05). The patients with a positive histopathological finding of VI have 3.1x higher risk ratio for shorter overall survival.

The relationships between the variables were described by Spearman rank correlation coefficients.

The analyses of Overall survival (OS) and Disease free interval (DFI) were performed by Kaplan-Meier´s survival functions. The influence of given covariates (clinical and histopathological factors) was tested by the Log-Rank test and Wilcoxon test. The Cox regression hazard model, hazard ratio (HR) and 95% confidence interval (CI) for HR were computed for the evaluation of given clinical and histopathological factors to OS or DFI. Multivariate analysis was performed by the use of classification and regression trees (CART). The Cox regression hazard model (stepwise regression) was applied to find the predictors in CART.

#### **3. Results**

The statistical analysis of the studied cohort of patients after surgical treatment for colorectal cancer demonstrated an acceptable distribution of basic statistical description parameters (gender ratio 93:57 (male vs. female)). 1, 3 and 5 years overall survival was 92.2%, 76.5% and 70.2% and 1, 3 and 5 year DFI was 85.3%, 64.3% and 49.4%.

The Spearman rank correlation coefficient did not prove any stronger correlation than a moderate correlation at endolymphatic invasion (LI) and lymph node infiltration by metastatic process (Spearman rank correlation coefficient 0.56, p<0,05). All the other studied factors were independent factors or factors with a low correlation.

Statistical analysis proved lymph node infiltration by metastatic process as statistically significant for the prognosis of overall survival (p<0.05) and N2 status of lymph nodes increased the risk of shorter overall survival 9.3x (Fig. 4.).

Fig. 4. Statistical analysis proved the lymph node infiltration by a metastatic process as statistically significant for the prognosis of overall survival (p<0.05), and the N2 status of lymph nodes increased the risk of a shorter overall survival 9.3x.

The relationships between the variables were described by Spearman rank correlation

The analyses of Overall survival (OS) and Disease free interval (DFI) were performed by Kaplan-Meier´s survival functions. The influence of given covariates (clinical and histopathological factors) was tested by the Log-Rank test and Wilcoxon test. The Cox regression hazard model, hazard ratio (HR) and 95% confidence interval (CI) for HR were computed for the evaluation of given clinical and histopathological factors to OS or DFI. Multivariate analysis was performed by the use of classification and regression trees (CART). The Cox regression hazard model (stepwise regression) was applied to find the

The statistical analysis of the studied cohort of patients after surgical treatment for colorectal cancer demonstrated an acceptable distribution of basic statistical description parameters (gender ratio 93:57 (male vs. female)). 1, 3 and 5 years overall survival was 92.2%, 76.5% and

The Spearman rank correlation coefficient did not prove any stronger correlation than a moderate correlation at endolymphatic invasion (LI) and lymph node infiltration by metastatic process (Spearman rank correlation coefficient 0.56, p<0,05). All the other studied

Statistical analysis proved lymph node infiltration by metastatic process as statistically significant for the prognosis of overall survival (p<0.05) and N2 status of lymph nodes

Fig. 4. Statistical analysis proved the lymph node infiltration by a metastatic process as statistically significant for the prognosis of overall survival (p<0.05), and the N2 status of

lymph nodes increased the risk of a shorter overall survival 9.3x.

70.2% and 1, 3 and 5 year DFI was 85.3%, 64.3% and 49.4%.

increased the risk of shorter overall survival 9.3x (Fig. 4.).

factors were independent factors or factors with a low correlation.

coefficients.

predictors in CART.

**3. Results**

Endovascular infiltration (VI) was proved as a negative prognostic factor of shorter overall survival (Fig.5.). Patients with positive histopathological findings of VI have 3.1x increased risk for shorter overall survival. The presence of peritumoral lymphocytes (PTL) (Fig.6.) and of Crohn-like PTL (Fig.7.) was proved as a positive prognostic factor of OS. Patients with a positive histopathological finding of PTL and Crohn-like PTL have a decreased risk for shorter overall survival (2.3x and 2.3x respectively). Lymph node follicular hyperplasia (LN-FH) was verified as a positive prognostic factor for longer overall survival (Fig.8.). The statistical significance of LN-FH increased also with the raised density of infiltration. LN-FH positivity decreased the risk of shorter overall survival 3.3times.

The severity of CD8+ lymphocytic infiltration was proved by the Cox regression hazard model as a positive prognostic factor enlarging overall survival (cut off 30 cells/HPF). The severity of CD4+ lymphocytic infiltration was proved as a significant factor for the prognosis of overall survival (cut off 4cells/HPF) with 2.5x increased hazard ratio in patients over the cut off (Fig.9.). Statistical analysis did not confirm the statistical significance of CD8/CD4 ratio.

Fig. 5. Endovascular infiltration by cancer cells (VI) was proved as a statistically significant factor for the prognosis of overall survival (p<0.05). The patients with a positive histopathological finding of VI have 3.1x higher risk ratio for shorter overall survival.

Tumor Infiltrating Lymphocytes as Prognostic Factor of Early

overall survival 3.3x.

ratio in patients over the cut off (p<0.05).

Recurrence and Poor Prognosis of Colorectal Cancer After Radical Surgical Treatment 421

Fig. 8. Lymph node follicular hyperplasia (LN-FH) was verified as a positive prognostic factor for a longer overall survival (p<0.05). The statistical significance of LN-FH increased also with the raised density of infiltration. LN-FH positivity decreased the risk of a shorter

Fig. 9. Severity of CD4+ lymphocytic intratumoral infiltration was proved as a significant factor for the prognosis of overall survival (cut off 4cells/HPF) with a 2.5x increased hazard

Fig. 6. Presence of peritumoral lymphocytes (PTL) was proved as a statistically significant positive factor for the prognosis of overall survival (p<0.05). The patients with a positive histopathological finding of PTL have 2.3x lower risk ratio for a shorter overall survival.

Fig. 7. Presence of Crohn-like PTL was proved as a statistically significant positive factor for the prognosis of overall survival (p<0.05). The patients with a positive histopathological finding of PTL have 2.3x lower risk ratio for a shorter overall survival.

Fig. 6. Presence of peritumoral lymphocytes (PTL) was proved as a statistically significant positive factor for the prognosis of overall survival (p<0.05). The patients with a positive histopathological finding of PTL have 2.3x lower risk ratio for a shorter overall survival.

Fig. 7. Presence of Crohn-like PTL was proved as a statistically significant positive factor for the prognosis of overall survival (p<0.05). The patients with a positive histopathological

finding of PTL have 2.3x lower risk ratio for a shorter overall survival.

Fig. 8. Lymph node follicular hyperplasia (LN-FH) was verified as a positive prognostic factor for a longer overall survival (p<0.05). The statistical significance of LN-FH increased also with the raised density of infiltration. LN-FH positivity decreased the risk of a shorter overall survival 3.3x.

Fig. 9. Severity of CD4+ lymphocytic intratumoral infiltration was proved as a significant factor for the prognosis of overall survival (cut off 4cells/HPF) with a 2.5x increased hazard ratio in patients over the cut off (p<0.05).

Tumor Infiltrating Lymphocytes as Prognostic Factor of Early

increased risk for shorter DFI.

shorter DFI 5x (Fig. 13.).

Recurrence and Poor Prognosis of Colorectal Cancer After Radical Surgical Treatment 423

Fig. 11. Perineural infiltration (PI) was proved as a negative prognostic factor of an earlier recurrence (p<0.05). Patients with a positive histopathological finding of PI have a 3.8x

Statistical analysis proved lymph node infiltration by a metastatic process as statistically significant for the prognosis of DFI and N2 status of lymph nodes increased the risk of

Fig. 12. Severity of CD8+ lymphocytic intratumoral infiltration was proved as a positive prognostic factor enlarging DFI (cut off 30 cells/HPF) by the Cox regression hazard model

(p<0.05). Patients over the cut off have a 2.2x increased risk of an early recurrence.

The Multivariate Cox Regression Hazard Model proved the combination of the severity of lymph node infiltration by metastatic process and LN-FH as the best prognostic factors for the prediction of the risk of shorter overall survival. This situation is demonstrated in the Classification and Regression Tree (CART)(p<0.05) (Fig.10.). All other studied parameters were not proved as statistically significant for the prognosis of overall survival.

Fig. 10. Multivariate Cox Regression Hazard Model proved the combination of the severity of lymph node infiltration by a metastatic process and LN-FH as the best prognostic factors for the prediction of risk of a shorter overall survival (p<0.05). This situation is demonstrated in the Classification and Regression Tree (CART).

Perineural infiltration (PI) was proved as a negative prognostic factor of an earlier recurrence (Fig.11.). Patients with a positive histopathological finding of PI have 3.8x increased risk for shorter DFI.

The severity of CD8+ lymphocytic infiltration was proved by the Cox regression hazard model as a positive prognostic factor enlarging DFI (cut off 30cells/HPF) (Fig.12.). Patients over the cut off have 2.2x increased risk of an early recurrence. The severity of CD4+ lymphocytic infiltration was not proved as a significant factor for the prognosis of DFI. Statistical analysis did not confirm the statistical significance of the CD8/CD4 ratio.

The Multivariate Cox Regression Hazard Model proved the combination of the severity of lymph node infiltration by metastatic process and LN-FH as the best prognostic factors for the prediction of the risk of shorter overall survival. This situation is demonstrated in the Classification and Regression Tree (CART)(p<0.05) (Fig.10.). All other studied parameters

Fig. 10. Multivariate Cox Regression Hazard Model proved the combination of the severity of lymph node infiltration by a metastatic process and LN-FH as the best prognostic factors

Perineural infiltration (PI) was proved as a negative prognostic factor of an earlier recurrence (Fig.11.). Patients with a positive histopathological finding of PI have 3.8x

The severity of CD8+ lymphocytic infiltration was proved by the Cox regression hazard model as a positive prognostic factor enlarging DFI (cut off 30cells/HPF) (Fig.12.). Patients over the cut off have 2.2x increased risk of an early recurrence. The severity of CD4+ lymphocytic infiltration was not proved as a significant factor for the prognosis of DFI.

Statistical analysis did not confirm the statistical significance of the CD8/CD4 ratio.

for the prediction of risk of a shorter overall survival (p<0.05). This situation is

demonstrated in the Classification and Regression Tree (CART).

increased risk for shorter DFI.

were not proved as statistically significant for the prognosis of overall survival.

Fig. 11. Perineural infiltration (PI) was proved as a negative prognostic factor of an earlier recurrence (p<0.05). Patients with a positive histopathological finding of PI have a 3.8x increased risk for shorter DFI.

Statistical analysis proved lymph node infiltration by a metastatic process as statistically significant for the prognosis of DFI and N2 status of lymph nodes increased the risk of shorter DFI 5x (Fig. 13.).

Fig. 12. Severity of CD8+ lymphocytic intratumoral infiltration was proved as a positive prognostic factor enlarging DFI (cut off 30 cells/HPF) by the Cox regression hazard model (p<0.05). Patients over the cut off have a 2.2x increased risk of an early recurrence.

Tumor Infiltrating Lymphocytes as Prognostic Factor of Early

Recurrence and Poor Prognosis of Colorectal Cancer After Radical Surgical Treatment 425

Fig. 14. Multivariate Cox Regression Hazard Model proved the combination of the severity of lymph node infiltration by a metastatic process and the severity of CD8 positivity as the best prognostic factors for the prediction of risk of an early recurrence (p<0.05). This

The combination of the severity of the lymph node infiltration by a metastatic process and LN-FH were proved as the best prognostic factors for the prediction of risk of a shorter

We also demonstrated PI, lymph node metastatic infiltration and the N2 status of lymph nodes as negative prognostic factors of an earlier recurrence, and the severity of CD8+ lymphocytic intratumoral infiltration as a positive prognostic factor enlarging DFI. The combination of the severity of the lymph node infiltration by a metastatic process and the severity of CD8+ lymphocytic intratumoral infiltration were proved as the best prognostic factors for the prediction of the risk of an early recurrence by the Multivariate Cox

Our results support the hypothesis that the adaptive immunological response in tumor tissue and its reaction in regional lymph nodes can influence the behavior of CRC and so affect the prognosis of patients (Atreya & Neurath, 2008; Galon et al., 2006). CD4 and CD8 positivity of ITL was demonstrated as a key histopathological sign of tumor-specific immune response that could reflect the contemporary clinical situation and a tendency to relapse (CD4+) or the larger overall survival (CD8+) (Chiba et al. 2004; Koch et al., 2006,

situation is demonstrated in the Classification and Regression Tree (CART).

overall survival by the Multivariate Cox Regression Hazard Model.

Regression Hazard Model.

Pages et al., 2010).

Fig. 13. Statistical analysis proved lymph node infiltration by a metastatic process as statistically significant for the prognosis of DFI, and a N2 status of lymph nodes 5x increased the risk of a shorter DFI (p<0.05).

The Multivariate Cox Regression Hazard Model proved the combination of the severity of the lymph node infiltration by a metastatic process and the severity of CD8 positivity of infiltrating lymphocytes as the best prognostic factors for the prediction of risk of early recurrence (p<0.05). This situation is demonstrated in the Classification and Regression Tree (CART) (Fig. 14). All other studied parameters were not proved as statistically significant for the prognosis of DFI.

#### **4. Discussion**

The role of the adaptive immunological response in controlling the growth and relapse of CRC remains controversial and contemporary studies have not answered all the questions about the prognosis of patients after radical surgical treatment of CRC (Galon et al., 2006; Ohtani, 2007; Van den Eynde & Hendlisz, 2009). We analysed our large cohort of patients of CRC with consideration to detect the negative and also positive prognostic factors of early recurrence of the disease and the poor overall survival after radical surgery. It was stimulated by the unsatisfactory situation and some dilemmas in the indication of surgical and oncological treatment, when early recurrence depreciates our effort to radical surgery with a high risk of complications and the long time of the decreased quality of life of our patients.

In the presented clinico-pathological study we demonstrated that lymph node infiltration by a metastatic process, N2 status of lymph nodes, VI, and extent of CD4+ lymphocytic intratumoral infiltration as negative prognostic factors of OS. In contrary PTL, Crohn-like PTL, LN-FH, and severity of CD8+ lymphocytic intratumoral infiltration were proved as positive prognostic factors of the overall survival.

Fig. 13. Statistical analysis proved lymph node infiltration by a metastatic process as statistically significant for the prognosis of DFI, and a N2 status of lymph nodes 5x

The Multivariate Cox Regression Hazard Model proved the combination of the severity of the lymph node infiltration by a metastatic process and the severity of CD8 positivity of infiltrating lymphocytes as the best prognostic factors for the prediction of risk of early recurrence (p<0.05). This situation is demonstrated in the Classification and Regression Tree (CART) (Fig. 14). All other studied parameters were not proved as statistically significant for

The role of the adaptive immunological response in controlling the growth and relapse of CRC remains controversial and contemporary studies have not answered all the questions about the prognosis of patients after radical surgical treatment of CRC (Galon et al., 2006; Ohtani, 2007; Van den Eynde & Hendlisz, 2009). We analysed our large cohort of patients of CRC with consideration to detect the negative and also positive prognostic factors of early recurrence of the disease and the poor overall survival after radical surgery. It was stimulated by the unsatisfactory situation and some dilemmas in the indication of surgical and oncological treatment, when early recurrence depreciates our effort to radical surgery with a high risk of complications and the long time of the decreased quality of life of our

In the presented clinico-pathological study we demonstrated that lymph node infiltration by a metastatic process, N2 status of lymph nodes, VI, and extent of CD4+ lymphocytic intratumoral infiltration as negative prognostic factors of OS. In contrary PTL, Crohn-like PTL, LN-FH, and severity of CD8+ lymphocytic intratumoral infiltration were proved as

increased the risk of a shorter DFI (p<0.05).

positive prognostic factors of the overall survival.

the prognosis of DFI.

**4. Discussion**

patients.

Fig. 14. Multivariate Cox Regression Hazard Model proved the combination of the severity of lymph node infiltration by a metastatic process and the severity of CD8 positivity as the best prognostic factors for the prediction of risk of an early recurrence (p<0.05). This situation is demonstrated in the Classification and Regression Tree (CART).

The combination of the severity of the lymph node infiltration by a metastatic process and LN-FH were proved as the best prognostic factors for the prediction of risk of a shorter overall survival by the Multivariate Cox Regression Hazard Model.

We also demonstrated PI, lymph node metastatic infiltration and the N2 status of lymph nodes as negative prognostic factors of an earlier recurrence, and the severity of CD8+ lymphocytic intratumoral infiltration as a positive prognostic factor enlarging DFI. The combination of the severity of the lymph node infiltration by a metastatic process and the severity of CD8+ lymphocytic intratumoral infiltration were proved as the best prognostic factors for the prediction of the risk of an early recurrence by the Multivariate Cox Regression Hazard Model.

Our results support the hypothesis that the adaptive immunological response in tumor tissue and its reaction in regional lymph nodes can influence the behavior of CRC and so affect the prognosis of patients (Atreya & Neurath, 2008; Galon et al., 2006). CD4 and CD8 positivity of ITL was demonstrated as a key histopathological sign of tumor-specific immune response that could reflect the contemporary clinical situation and a tendency to relapse (CD4+) or the larger overall survival (CD8+) (Chiba et al. 2004; Koch et al., 2006, Pages et al., 2010).

Tumor Infiltrating Lymphocytes as Prognostic Factor of Early

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We assessed several types of tumor infiltrating lymphocytes and clearly identified their relationships to relapse and the overall survival as positive or negative prognostic factors in contrary to previous publications that evaluated only the infiltration of the tumor but not the quality and type of infiltration (Ogino et al., 2009).

Tumor infiltration by lymphocytes seems to be a promising prognostic factor reflecting the risk of patients to early recurrence or poor overall survival. Future work has to be focused on the molecular-biological background of tumor infiltration by lymphocytes to understand their pathophysiological functions (Pages et al., 2005, Zbar, 2004).

#### **5. Conclusion**

Tumor infiltrating lymphocytes seem to be promising prognostic factors that could find their use in colorectal surgery and consecutive oncological treatment as an indicator of the type or combinations of therapies reflecting the risk of patients to early recurrence or poor overall survival. The TIL status corresponds to immune control of cancer progression.

### **6. Acknowledgment**

This article was supported by research project MSM<PHONE_NUMBER> (Replacement of and support to some vital organs) and grant IGA MZ CR 10230 and IGA MZ CR 12025 and specific students research grant of Charles University SVV-2011- 262 806.

#### **7. References**

We assessed several types of tumor infiltrating lymphocytes and clearly identified their relationships to relapse and the overall survival as positive or negative prognostic factors in contrary to previous publications that evaluated only the infiltration of the tumor but not

Tumor infiltration by lymphocytes seems to be a promising prognostic factor reflecting the risk of patients to early recurrence or poor overall survival. Future work has to be focused on the molecular-biological background of tumor infiltration by lymphocytes to understand

Tumor infiltrating lymphocytes seem to be promising prognostic factors that could find their use in colorectal surgery and consecutive oncological treatment as an indicator of the type or combinations of therapies reflecting the risk of patients to early recurrence or poor overall survival. The TIL status corresponds to immune control of cancer progression.

This article was supported by research project MSM<PHONE_NUMBER> (Replacement of and support to some vital organs) and grant IGA MZ CR 10230 and IGA MZ CR 12025 and

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**20**

*1Latvia 2Ukraine*

**Fluorescent Biomarker**

E. Kirilova1, I. Kalnina1, G. Kirilov1 and G. Gorbenko2

*2N. Karazin University of National Academy of Sciences, Kharkov,*

Patients with gastrointestinal cancer exhibit a poorly functioning immune system that is characterized, in part, by decreases in T-lymphocyte proliferation (Greenstein, et al., 1991; Milasiene et al., 2007) and reduced CD4+:CD8+ ratios (Arista et al., 1994; Franciosi et al., 2002). In different pathologies (including these and other types of cancers), membrane damage in immune cells (and other cell types) often evolves as a consequence of alterations that are induced in cell-associated lipids and proteins in the affected patients (Gryzunov and Dobretsov, 1994, 1998; Rolinsky et al., 2007). It is now widely accepted that the dynamics (actual rate of occurrence - not only incidence) of these changes, along with the types of alterations in structure(s) of the immune system cells' lipids/proteins themselves, play a critical role in the maintenance of the immune status of any given organism (Lakowicz, 2000). As a result of the potential importance of changes in the structural integrity of cells of the immune system, it is important for clinicians to receive information on the biophysical status of these cells via quick, reliable, reproducible methods. In this regard, fluorescent probes have shown to be excellent tools for use in such protocols (Lakowicz, 2000, 2006). The work reported here, which built upon earlier findings reported by our laboratories, investigated the possibility of using the fluorescent probe ABM (an amine derivative of benzanthrone) for the detection of structural/functional alterations in blood plasma albumin and among immunocompetent cells in patients with select types of pathologies, i.e,. cancers. Such an analysis has a great potential for use not only for helping to comprehend mechanisms of immunomodulation associated with the induction/progression of malignancies, but might also have the potential to serve as a very important prognostic

indicator of long-term survival among patients with such pathologies.

In the work reported here, ABM fluorescence intensity in blood plasma and following combination with cell suspensions from colorectal cancer patients was examined in the context of the host's immunological parameters and state of cancer progression. For study patients with colorectal cancer were examined: 1) 1 day before and 10 days after their surgical treatment (Stages II-III) (Kalnina et al., 2009); 2) as disease worsened (Stages IIa, IIIb, IV) (Kalnina et al., 2010b); 3) advanced cancer patients, they were divided into two groups in accordance of its survival rate (0-6 months and > 24 months) (Kalnina et al., 2011). Apart from the aforementioned potential benefits from these types of studies to clinicians in general, this type of research is very important in Latvia itself. This is because, in the context

**1. Introduction**

**in Colorectal Cancer**

*1Daugavpils University, Daugavpils,*

### **Fluorescent Biomarker in Colorectal Cancer**

E. Kirilova1, I. Kalnina1, G. Kirilov1 and G. Gorbenko2 *1Daugavpils University, Daugavpils, 2N. Karazin University of National Academy of Sciences, Kharkov, 1Latvia 2Ukraine*

#### **1. Introduction**

428 Colorectal Cancer Biology – From Genes to Tumor

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der Burg, S. H., Janssen-van Rhijn, C. M., Ensink, N. G. , Tollenaar, R. A., van de Velde, C. J., & Kuppen, P. J. (2005). Prognostic value of tumor-infiltrating dendritic cells in colorectal cancer: role of maturation status and intratumoral localization. *Clinical Cancer Research*, Vol.11, No.7, (April 2005), pp. 2576-2582, ISSN 1078-0432 Steinman, R. M., Hawiger, D., & Nussenzweig, M. C. (2003). Tolerogenic dendritic cells.

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Patients with gastrointestinal cancer exhibit a poorly functioning immune system that is characterized, in part, by decreases in T-lymphocyte proliferation (Greenstein, et al., 1991; Milasiene et al., 2007) and reduced CD4+:CD8+ ratios (Arista et al., 1994; Franciosi et al., 2002). In different pathologies (including these and other types of cancers), membrane damage in immune cells (and other cell types) often evolves as a consequence of alterations that are induced in cell-associated lipids and proteins in the affected patients (Gryzunov and Dobretsov, 1994, 1998; Rolinsky et al., 2007). It is now widely accepted that the dynamics (actual rate of occurrence - not only incidence) of these changes, along with the types of alterations in structure(s) of the immune system cells' lipids/proteins themselves, play a critical role in the maintenance of the immune status of any given organism (Lakowicz, 2000).

As a result of the potential importance of changes in the structural integrity of cells of the immune system, it is important for clinicians to receive information on the biophysical status of these cells via quick, reliable, reproducible methods. In this regard, fluorescent probes have shown to be excellent tools for use in such protocols (Lakowicz, 2000, 2006). The work reported here, which built upon earlier findings reported by our laboratories, investigated the possibility of using the fluorescent probe ABM (an amine derivative of benzanthrone) for the detection of structural/functional alterations in blood plasma albumin and among immunocompetent cells in patients with select types of pathologies, i.e,. cancers. Such an analysis has a great potential for use not only for helping to comprehend mechanisms of immunomodulation associated with the induction/progression of malignancies, but might also have the potential to serve as a very important prognostic indicator of long-term survival among patients with such pathologies.

In the work reported here, ABM fluorescence intensity in blood plasma and following combination with cell suspensions from colorectal cancer patients was examined in the context of the host's immunological parameters and state of cancer progression. For study patients with colorectal cancer were examined: 1) 1 day before and 10 days after their surgical treatment (Stages II-III) (Kalnina et al., 2009); 2) as disease worsened (Stages IIa, IIIb, IV) (Kalnina et al., 2010b); 3) advanced cancer patients, they were divided into two groups in accordance of its survival rate (0-6 months and > 24 months) (Kalnina et al., 2011). Apart from the aforementioned potential benefits from these types of studies to clinicians in general, this type of research is very important in Latvia itself. This is because, in the context

Fluorescent Biomarker in Colorectal Cancer 431

Fig. 2. ABM fluorescence intensity in lymphocytes and in plasma from "Colorectal group" patients. Colorectal cancer patients only (Stage II–III) (*n* = 10). Group number in figure is used to reflect from whom/when samples were isolated [i.e., Group 1: pre-surgery; Group 2: post-surgery; and Group 3: from healthy donors (control group; *n* = 14)]. Solid bar in each set: ABM fluorescence in lymphocytes; hatched bar in each set: ABM fluorescence in plasma.

All intensity values are shown in AU (arbitrary units; mean ± SE). At *P* < 0.05, \*value significantly different from pre-surgical value and/or #significantly different from control

It is of interest to note that while the post-surgery albumin values in these patients samples were ≈18% below control levels, their corresponding fluorescence intensity were now even more depressed relative to that of the controls by ≈ 34%. These results strongly suggest that the noted changes in ABM fluorescence post-surgery in the cancer patients were most likely attributable to some change in the protein(s) themselves rather than due to post-surgery complications (i.e., bleeding or other mechanisms affecting blood volume/composition). To ascertain whether these results (different ABM spectral characteristics) could be explained, in part, by altered structural characteristics of the plasma albumin in the cancer patients, the average binding constant (*K*a) values were determined using the Klotz graphical method. These analyses revealed that the average *K*a values for the pre-surgery samples from the colorectal patient group decreased strongly from the respective constant associated with the control group (i.e., 1.0 X 105 M−1, colorectal cancer group; 1.8 X 105 M−1, healthy control group). These values represent decrements of from 28% to 45% in the binding of ABM to albumin in the plasma of these patients; the reason for the decrement remains to be fully determined. Interestingly, even though the fluorescence intensity values decreased further for the patients following their surgical procedures, the average *K*a values for the samples from the patients increased relative to corresponding preoperative values (i.e., 1.3 X 105

group value.

M−1).

of oncological diseases seen among the Latvian population (as recently as in 2006), colorectal cancers rank third in incidence, only surpassed by lung cancers and urogenital tumors.

#### **2. ABM: Distribution and spectral characteristics in cells and blood plasma**

A new fluorescent probe, a derivative of 3-aminobenzanthrone (ABM) at the Daugavpils University, Daugavpils, Latvia.

Fig. 1.Chemical Structure of probe ABM

Synthesis and properties of probe ABM were described in (Kalnina et al., 2004, 2007, 2009, 2010a; Kirilova et al., 2008).

#### **3. ABM binding with blood plasma albumin**

#### **3.1 ABM binding with blood plasma albumin before and after surgical treatment**

In the colorectal cancer patients, the ABM emission spectra maximum (i.e., at 650 nm)—after combination with the patients' blood plasma—was not altered in comparison to that seen with the plasma from the healthy control volunteers. In contrast, with respect to fluorescence intensity, before their individual surgical treatments, the average ABM intensity in the patients' blood plasma was decreased compared to that seen with the samples from the healthy donors. Specifically, the fluorescence associated with samples from the colorectal cancer group (Figure 2) were decreased by 23.0%. At 10 days after their operations, the average ABM fluorescence intensity in the samples from the colorectal cancer group were decreased further by 13.9%.

The average intensity values of the plasma samples from the cancer patients were significantly (*P* < 0.05) different from the control volunteers' average value. Whether these observations tracked actual changes in the levels of plasma albumin were also investigated. The results (data not shown) indicate that plasma albumin concentrations (μg/μL) pre-surgery in the patient group (71.73 ± 1.34 ) were below those in the plasma of the health controls (83.41 ± 1.16). This meant that the pre-surgery values for albumin only indicated levels in colorectal group samples that were ≈ 14% below control, while the samples' fluorescence intensity was correspondingly lower than the control value by 23.0% . The plasma albumin concentrations after surgery seemed to be insignificantly impacted. Specifically, these value was in colorectal cancer patients samples 68.48 ± 1.78 (in μg/μL, mean ± SD).

of oncological diseases seen among the Latvian population (as recently as in 2006), colorectal cancers rank third in incidence, only surpassed by lung cancers and urogenital tumors.

**2. ABM: Distribution and spectral characteristics in cells and blood plasma**  A new fluorescent probe, a derivative of 3-aminobenzanthrone (ABM) at the Daugavpils

Synthesis and properties of probe ABM were described in (Kalnina et al., 2004, 2007, 2009,

The average intensity values of the plasma samples from the cancer patients were significantly (*P* < 0.05) different from the control volunteers' average value. Whether these observations tracked actual changes in the levels of plasma albumin were also investigated. The results (data not shown) indicate that plasma albumin concentrations (μg/μL) pre-surgery in the patient group (71.73 ± 1.34 ) were below those in the plasma of the health controls (83.41 ± 1.16). This meant that the pre-surgery values for albumin only indicated levels in colorectal group samples that were ≈ 14% below control, while the samples' fluorescence intensity was correspondingly lower than the control value by 23.0% . The plasma albumin concentrations after surgery seemed to be insignificantly impacted. Specifically, these value was in colorectal

**3.1 ABM binding with blood plasma albumin before and after surgical treatment**  In the colorectal cancer patients, the ABM emission spectra maximum (i.e., at 650 nm)—after combination with the patients' blood plasma—was not altered in comparison to that seen with the plasma from the healthy control volunteers. In contrast, with respect to fluorescence intensity, before their individual surgical treatments, the average ABM intensity in the patients' blood plasma was decreased compared to that seen with the samples from the healthy donors. Specifically, the fluorescence associated with samples from the colorectal cancer group (Figure 2) were decreased by 23.0%. At 10 days after their operations, the average ABM fluorescence intensity in the samples from the colorectal

University, Daugavpils, Latvia.

Fig. 1.Chemical Structure of probe ABM

**3. ABM binding with blood plasma albumin**

cancer group were decreased further by 13.9%.

cancer patients samples 68.48 ± 1.78 (in μg/μL, mean ± SD).

2010a; Kirilova et al., 2008).

Fig. 2. ABM fluorescence intensity in lymphocytes and in plasma from "Colorectal group" patients. Colorectal cancer patients only (Stage II–III) (*n* = 10). Group number in figure is used to reflect from whom/when samples were isolated [i.e., Group 1: pre-surgery; Group 2: post-surgery; and Group 3: from healthy donors (control group; *n* = 14)]. Solid bar in each set: ABM fluorescence in lymphocytes; hatched bar in each set: ABM fluorescence in plasma. All intensity values are shown in AU (arbitrary units; mean ± SE). At *P* < 0.05, \*value significantly different from pre-surgical value and/or #significantly different from control group value.

It is of interest to note that while the post-surgery albumin values in these patients samples were ≈18% below control levels, their corresponding fluorescence intensity were now even more depressed relative to that of the controls by ≈ 34%. These results strongly suggest that the noted changes in ABM fluorescence post-surgery in the cancer patients were most likely attributable to some change in the protein(s) themselves rather than due to post-surgery complications (i.e., bleeding or other mechanisms affecting blood volume/composition). To ascertain whether these results (different ABM spectral characteristics) could be explained, in part, by altered structural characteristics of the plasma albumin in the cancer patients, the average binding constant (*K*a) values were determined using the Klotz graphical method. These analyses revealed that the average *K*a values for the pre-surgery samples from the colorectal patient group decreased strongly from the respective constant associated with the control group (i.e., 1.0 X 105 M−1, colorectal cancer group; 1.8 X 105 M−1, healthy control group). These values represent decrements of from 28% to 45% in the binding of ABM to albumin in the plasma of these patients; the reason for the decrement remains to be fully determined. Interestingly, even though the fluorescence intensity values decreased further for the patients following their surgical procedures, the average *K*a values for the samples from the patients increased relative to corresponding preoperative values (i.e., 1.3 X 105 M−1).

Fluorescent Biomarker in Colorectal Cancer 433

In the colorectal cancer patients, the ABM emission spectra maximum (i.e., at 650 nm) after combination with the patients' lymphocytes (as with their plasma) was not altered in comparison to that seen with the cells from the healthy control volunteers. Surprisingly, the average ABM fluorescence intensity value noted from colorectal patients group was actually 12.0% greater than the control level (Figure 3); however, even with this increase, the value was not significantly different from the average control value. In contrast to what was observed with the plasma samples, the average ABM fluorescence intensity values noted with the cells from patients at 10 days after their operations were greater than the values seen with the control volunteers' cells by 44%. In comparison to the pre-operative values, these average ABM fluorescence intensity values at 10 days after the patients' operations

**COLORECTAL CANCER PATIENTS (Stage II-III) (n=10)**

b1 ‡15.95±2.18 ‡314.18±39.27 ‡1.27±0.10 ‡3.40±1.20 28.00±1.30 2 \*‡7.93±1.43 \*‡144.75±22.44 \*‡1.50±0.13 \*‡12.70±3.40 \*‡23.50±2.20 3(Controls) 12.50±1.10 389.00±24.11 1.88±0.16 24.60±1.60 28.00±1.30

b Indicates when or from whom samples were isolated: (1) before surgical treatment; (2) after surgical

\* Value significantly different from pre-surgical value (P<0.05); ‡significantly different from control

Table 3. Peripheral blood lymphocyte subpopulation counts in the distinct subsets of the

In general, among the gastrointestinal cancer patients examined here, the ABM emission spectra maximum (i.e., at 650 nm) after combination of the probe with the patients' lymphocytes was not altered in comparison to that seen with the cells from the healthy control volunteers (spectral data not shown). The ABM fluorescence intensity in the samples from colorectal patient group in Stages IIA-IIIB was not significantly different from the average control value (0.28 vs. 0.25 fluorescence units, respectively; Fig.3). In contrast, there was a significant reduction in ths parameter among the cells from the cancer patients in

**4.3 ABM spectral characteristics in lymphocytes of advanced cancer patients**

**T**he ABM fluorescence intensity in the samples from advanced cancer patients (Group 1) was not different from the average value (0.25 vs. 0.25) fluorescence units, respectively: (Table 4). In contrast, there was a significant increase in this parameter among the cells from the cancer patients Group 2 as compared with Group 1 (by 80.8%) and healthy donors (by

a Values shown are in terms of absolute numbers (mean ± SE).

**4.2 ABM binding with lymphocytes as a function of stage**

treatment; and (3) healthy donors (control group; n=14).

Stage IV (a decrease of 44%; (0.14 vs. 0.25 units).

CD16+% aCD16+ CD4+:CD8+ CD38+% Lymphocytes

(%)

**4.1 ABM binding with lymphocytes before and after surgical treatment**

**4. ABM binding with lymphocytes**

had increased by 28.6%.

group value (P<0.05).

study's cancer patients.

112%) (0.25 vs. 0.52 units).

#### **3.2 ABM binding with blood plasma albumin as a function of stage**

The average ABM fluorescence intensity in patient's blood plasma was decreased compared to that seen with healthy donors. Specifically, the fluorescence intensity associated with samples from colorectal cancer patients (Table 1) was decreased by 23% (average value of 1.44 fluorescence units for those at Stage IIA-IIIB) and 42% (average value of 1.09 units for those at Stage IV) relative for the healthy controls(an average value of 1.87 units).

aF (PI) = fluorescence intensity in blood plasma; Values shown are in mean (± SE).

Value (p<0.05) significantly different from that of \* control or # Group 2 patients.

Table 1. Spectral characteristics of ABM in blood plasma of colorectal cancer patients

#### **3.3 ABM spectral characteristics in blood plasma of advanced cancer patients**

The average ABM fluorescence intensity in the patients (Group 1 and Group 2) blood plasma was decreased (i.e., by 37.4% and 24.1%, respectively) as compared to that seen in healthy donors (Table 2). In Group 2 average intensity significantly (p<0.05) differs from Group 1 value and also control group value by 10.6 % and 31.7 %, respectively. Total albumin (TA) concentration in Group 1 and Group 2 patients was decreased (relative to control value) by 23.5% and 16.1%, respectively. Effective albumin (EA) concentration was decreased by 40.0% and 27.7%, respectively. The lowest value EA/TA in these patients plasma reached 0.61 (Group 1), and 0.66 (Group 2); (donor group 0.79-0.81).

F (Pl) = fluorescence intensity in blood plasma;. Values shown are in mean (± SE).

EA- "healthy" albumin equivalent in patients plasma, g/L

TA- total albumin concentration, g/L

EA/TA- reserve of albumin binding capacity

Table 2. Spectral parameters and binding sites čaracteristics of ABM in blood plasma of advanced cancer patients

The average ABM fluorescence intensity in patient's blood plasma was decreased compared to that seen with healthy donors. Specifically, the fluorescence intensity associated with samples from colorectal cancer patients (Table 1) was decreased by 23% (average value of 1.44 fluorescence units for those at Stage IIA-IIIB) and 42% (average value of 1.09 units for

**3.2 ABM binding with blood plasma albumin as a function of stage**

aF (PI) = fluorescence intensity in blood plasma; Values shown are in mean (± SE).

Value (p<0.05) significantly different from that of \*

**Survival rate**

**(months)**

EA- "healthy" albumin equivalent in patients plasma, g/L

TA- total albumin concentration, g/L EA/TA- reserve of albumin binding capacity

advanced cancer patients

**Group**

P<0.05 between groups

those at Stage IV) relative for the healthy controls(an average value of 1.87 units).

**Group Stage F (PI)a**

Table 1. Spectral characteristics of ABM in blood plasma of colorectal cancer patients

**3.3 ABM spectral characteristics in blood plasma of advanced cancer patients**

plasma reached 0.61 (Group 1), and 0.66 (Group 2); (donor group 0.79-0.81).

F (Pl) = fluorescence intensity in blood plasma;. Values shown are in mean (± SE).

The average ABM fluorescence intensity in the patients (Group 1 and Group 2) blood plasma was decreased (i.e., by 37.4% and 24.1%, respectively) as compared to that seen in healthy donors (Table 2). In Group 2 average intensity significantly (p<0.05) differs from Group 1 value and also control group value by 10.6 % and 31.7 %, respectively. Total albumin (TA) concentration in Group 1 and Group 2 patients was decreased (relative to control value) by 23.5% and 16.1%, respectively. Effective albumin (EA) concentration was decreased by 40.0% and 27.7%, respectively. The lowest value EA/TA in these patients

1 0-6 1.17 ± 0.14 39.0 + 1.1 63,80 + 1.02 0,61 2 > 24 1.42 ± 0.09 47.0 + 1.2 70,80 + 1.14 0,66

Control 1.87 ± 0.13 65.0 + 1.3 83,4 + 1.16 0,78

Table 2. Spectral parameters and binding sites čaracteristics of ABM in blood plasma of

1-2, 1-3, 2-3 1-2, 1-3, 2-3 1-2, 1-3, 2-3

1 IIA-IIIB #\*1.44±0.12 2 IV \*1.09±0.11 3 Controls 1.87±0.13

control or # Group 2 patients.

**F (Pl) EA TA EA/TA**

### **4. ABM binding with lymphocytes**

#### **4.1 ABM binding with lymphocytes before and after surgical treatment**

In the colorectal cancer patients, the ABM emission spectra maximum (i.e., at 650 nm) after combination with the patients' lymphocytes (as with their plasma) was not altered in comparison to that seen with the cells from the healthy control volunteers. Surprisingly, the average ABM fluorescence intensity value noted from colorectal patients group was actually 12.0% greater than the control level (Figure 3); however, even with this increase, the value was not significantly different from the average control value. In contrast to what was observed with the plasma samples, the average ABM fluorescence intensity values noted with the cells from patients at 10 days after their operations were greater than the values seen with the control volunteers' cells by 44%. In comparison to the pre-operative values, these average ABM fluorescence intensity values at 10 days after the patients' operations had increased by 28.6%.

a Values shown are in terms of absolute numbers (mean ± SE).

b Indicates when or from whom samples were isolated: (1) before surgical treatment; (2) after surgical treatment; and (3) healthy donors (control group; n=14).

\* Value significantly different from pre-surgical value (P<0.05); ‡significantly different from control group value (P<0.05).

Table 3. Peripheral blood lymphocyte subpopulation counts in the distinct subsets of the study's cancer patients.

#### **4.2 ABM binding with lymphocytes as a function of stage**

In general, among the gastrointestinal cancer patients examined here, the ABM emission spectra maximum (i.e., at 650 nm) after combination of the probe with the patients' lymphocytes was not altered in comparison to that seen with the cells from the healthy control volunteers (spectral data not shown). The ABM fluorescence intensity in the samples from colorectal patient group in Stages IIA-IIIB was not significantly different from the average control value (0.28 vs. 0.25 fluorescence units, respectively; Fig.3). In contrast, there was a significant reduction in ths parameter among the cells from the cancer patients in Stage IV (a decrease of 44%; (0.14 vs. 0.25 units).

#### **4.3 ABM spectral characteristics in lymphocytes of advanced cancer patients**

**T**he ABM fluorescence intensity in the samples from advanced cancer patients (Group 1) was not different from the average value (0.25 vs. 0.25) fluorescence units, respectively: (Table 4). In contrast, there was a significant increase in this parameter among the cells from the cancer patients Group 2 as compared with Group 1 (by 80.8%) and healthy donors (by 112%) (0.25 vs. 0.52 units).

Fluorescent Biomarker in Colorectal Cancer 435

progressed (actual values: 28.6%, Stage IIA-IIIB; 23.7%, Stage IV; and, 38.4%, controls). Patients with Stage IV colorectal cancer yielded any statistically significant shift from control

The levels of lymphocytes (both total and the CD4+ and CD8+ sub-populations) in the blood samples of the cancer patients and healthy volunteers were also assessed here. The results show that among the patients in both cancer groups, the relative percentages of lymphocytes were moreover not significantly altered relative to the control levels irrespective of disease stage (Figures 4 and 5). Of all the patients, only those in Stage IV had lower blood lymphocyte levels that approached or reached statistical significance. Among the Stage IV colorectal cancer patients, levels were decreased 18.5% (a shift from 28.0%

subject levels of CD8+ cells (an increase of ≈38%, i.e., shift from 19.5% to 26.9%).

Fig. 4. Lymphocyte counts (as percentage %) and T-lymphocyte subpopulation Levels (as % of all lymphocytes present) in cancer patients as a function of stage. 1) Stage IIA-IIIB (black fill); 2) Stage IV (clear fill); 3) healthy donors (control group;

53.2%) at Stages IIA-IIIB and IV, respectively, from the 1.88 value for the controls.

Because shifts in CD4+:CD8+ ratios are often used as indices of altered host immune status, these values were also calculated from the patients' blood samples. The results show that among the colorectal cancer patients (Fig 5.), the CD4+:CD8+ ratios were all significantly lower than those for the healthy control subjects and became significantly further lower as the stage worsened. Specifically, the ratios dropped to 1.27 and 0.88 (shifts of 32.4% and

[control] down to ≈22.8%);

striped fill).

Fig. 3. ABM fluorescence intensity in lymphocytes from colorectal cancer patients as a function of stage. 1) Stage IIA-IIIB (black fill); 20 Stage IV (clear fill); 3) healthy donors (control group; stripped fill). All values are shown as mean (-+SE). At p< 0.05, values significantly different from that of "control patients, Group 2 patients.

#### **5. Lymphocyte count and subpopulations**

#### **5.1 Lymphocytes count and subpopulations before and after surgical treatment**

The absolute number of CD3+ cells and CD16+ natural killer cells; the relative percentages of all lymphocytes, CD16+ and CD38+ cells; as well as the CD4+:CD8+ ratio in the blood samples of the healthy volunteers and of the cancer patients, before and after each underwent their operations, were determined. The results among the patients in the "colorectal" group (Table 3) indicate that, before surgery, the numbers of CD16+ cells, the relative percentage of CD38+ cells, and the CD4+:CD8+ ratio were each significantly decreased (i.e., by 19.2%, 86.2%, and 32.4%, respectively) as compared to corresponding control subject values. Somewhat unexpectedly, relative percentages of CD16+ cells in this group were actually significantly greater (by 27.6%)—and the relative percentage of lymphocytes no different—than in the blood of the control volunteers. Within this same group, after surgery, the number and percentages of CD16+ cells, as well as the percentages of all lymphocytes, were each significantly reduced (i.e., by 53.9%, 50.3%, and 16.1%, respectively) relative to corresponding pre-surgery levels. Again, the relative percentage of CD38+ cells and the CD4+:CD8+ ratio increased (by 27.4% and 18.1%, respectively) compared to pre-surgical values, but again did not reach control levels (i.e., still were 48.4 and 20.2% lower, respectively).

#### **5.2 Lymphocytes count and subpopulations as a function of stage**

On the other hand, there were significant changes in the relative percentages of CD4+ cells at almost every stage in both cancer groups (Fig. 4). Among colorectal cancer patients, the percentages of CD4+ cells decreased 25.5 and 38.3% from control levels as the stages

Fig. 3. ABM fluorescence intensity in lymphocytes from colorectal cancer patients as a function of stage. 1) Stage IIA-IIIB (black fill); 20 Stage IV (clear fill); 3) healthy donors (control group; stripped fill). All values are shown as mean (-+SE). At p< 0.05,

**5.1 Lymphocytes count and subpopulations before and after surgical treatment**  The absolute number of CD3+ cells and CD16+ natural killer cells; the relative percentages of all lymphocytes, CD16+ and CD38+ cells; as well as the CD4+:CD8+ ratio in the blood samples of the healthy volunteers and of the cancer patients, before and after each underwent their operations, were determined. The results among the patients in the "colorectal" group (Table 3) indicate that, before surgery, the numbers of CD16+ cells, the relative percentage of CD38+ cells, and the CD4+:CD8+ ratio were each significantly decreased (i.e., by 19.2%, 86.2%, and 32.4%, respectively) as compared to corresponding control subject values. Somewhat unexpectedly, relative percentages of CD16+ cells in this group were actually significantly greater (by 27.6%)—and the relative percentage of lymphocytes no different—than in the blood of the control volunteers. Within this same group, after surgery, the number and percentages of CD16+ cells, as well as the percentages of all lymphocytes, were each significantly reduced (i.e., by 53.9%, 50.3%, and 16.1%, respectively) relative to corresponding pre-surgery levels. Again, the relative percentage of CD38+ cells and the CD4+:CD8+ ratio increased (by 27.4% and 18.1%, respectively) compared to pre-surgical values, but again did not reach control levels (i.e., still were 48.4

values significantly different from that of "control patients, Group 2 patients.

**5.2 Lymphocytes count and subpopulations as a function of stage**

On the other hand, there were significant changes in the relative percentages of CD4+ cells at almost every stage in both cancer groups (Fig. 4). Among colorectal cancer patients, the percentages of CD4+ cells decreased 25.5 and 38.3% from control levels as the stages

**5. Lymphocyte count and subpopulations**

and 20.2% lower, respectively).

progressed (actual values: 28.6%, Stage IIA-IIIB; 23.7%, Stage IV; and, 38.4%, controls). Patients with Stage IV colorectal cancer yielded any statistically significant shift from control subject levels of CD8+ cells (an increase of ≈38%, i.e., shift from 19.5% to 26.9%).

The levels of lymphocytes (both total and the CD4+ and CD8+ sub-populations) in the blood samples of the cancer patients and healthy volunteers were also assessed here. The results show that among the patients in both cancer groups, the relative percentages of lymphocytes were moreover not significantly altered relative to the control levels irrespective of disease stage (Figures 4 and 5). Of all the patients, only those in Stage IV had lower blood lymphocyte levels that approached or reached statistical significance. Among the Stage IV colorectal cancer patients, levels were decreased 18.5% (a shift from 28.0% [control] down to ≈22.8%);

Fig. 4. Lymphocyte counts (as percentage %) and T-lymphocyte subpopulation Levels (as % of all lymphocytes present) in cancer patients as a function of stage. 1) Stage IIA-IIIB (black fill); 2) Stage IV (clear fill); 3) healthy donors (control group; striped fill).

Because shifts in CD4+:CD8+ ratios are often used as indices of altered host immune status, these values were also calculated from the patients' blood samples. The results show that among the colorectal cancer patients (Fig 5.), the CD4+:CD8+ ratios were all significantly lower than those for the healthy control subjects and became significantly further lower as the stage worsened. Specifically, the ratios dropped to 1.27 and 0.88 (shifts of 32.4% and 53.2%) at Stages IIA-IIIB and IV, respectively, from the 1.88 value for the controls.

Fluorescent Biomarker in Colorectal Cancer 437

1 0-6 0,25±0,03 13,31±1,16 20,93±1,13 27,00±1,39 0,78±0,09

2 >24 0,53±0,11 16,95±1,18 26,14±1,32 26,70±1,31 0,98±0,08

Control 0,25±0,03 28,00±1,30 38,40±2,10 19,50±1,20 1,88±0,16

Table 4. ABM fluorescence intensity in lymphocytes. Peripheral blood lymphocytes

**6. Examination of relationship between ABM fluorescence and blood**

Pre-operation, in the colorectal patient group, the ABM fluorescence intensity was found to correlate with the relative number of CD38+ cells (*r* = +0.956). After the operations, both the CD4+:CD8+ ratio and relative number of CD38+ cells in patients blood was observed to be

In colorectal cancer group, the ABM fluorescence intensity in blood plasma and the lymphocytes was found to correlate with CD4+:CD8+ ratios (in all stages of cancer) and the percentage (%) of lymphocytes/ subtypes in their blood. The degree of any relationship between cancer progression/staging, lymphocyte levels, and fluorescence among the lymphocytes was less obvious. Specifically, in terms of disease progression, cell levels and fluorescence intensity weakly tracked together (i.e., r = +0.512) for the colorectal patients. . With respect to associations between the plasma albumin fluorescence measurements and each of the individual lymphocyte-associated endpoints measured (except for cell fluorescence itself), many of the same patterns as noted above were evident. Once again, in terms of disease progression, although cell levels and albumin fluorescence intensity weakly

In both groups of advanced cancer the ABM fluorescence intensity in blood plasma and lymphocytes was found to correlate to with CD4+/CD8+ ratios. In advanced cancer groups (in contrast to other groups) there is direct (not inverse) correlation between lymphocytes count and ABM fluorescence intensity. There seemed to be a good relationship between total lymphocyte (and subpopulation) levels and ABM fluorescence in both groups of patients. There is also good associations between the plasma albumin fluorescence measurements and each of the individual lymphocyte/albumin associated endpoints measured "effective" and total albumin concentration, reserve of albumin binding

1-2;2-3 1-2;1-3;2-3 1-2;1-3;2-3 1-3;2-3 1-2;1-3;2-3

F(Ly), a.u. Ly (%) CD4+(%) CD8+(%) CD4+/CD8+

Group

3

p<0.05, between groups

increased.

capacity.

Survival rate, months

F(Ly) - fluorescence intensity in lymphocytes

subpopulation counts in advanced cancer patients

tracked together (i.e., r = +0.513) among the colorectal patients.

Values shown are in mean (±SE)

**lymphocyte profiles**

Fig. 5. T-lymphocyte subpopulation ratios in cancer patients as a function of stage. 1) Stage IIA- IIIB (black fill); 2) Stage IV (clear fill); 3) healthy donors (control group; striped fill).

#### **5.3 Lymphocytes count and subpopulations in advanced cancer patients**

Among the patients in both advanced cancer groups, the relative number of lymphocytes were significantly decreased (i.e.by 52.6% and 39.6%, respectively) as compared to corresponding control values. It is necessary to note that relative number of lymphocytes in Group 2 is significantly higher than in the Group 1 patients, but lower than the control value (see Table 4). In patients with advanced cancer and metastases there is reduction in both numbers of lymphocytes and proportions of CD4+/CD8+ T-lymphocytes which are thought to play an important role in cell-mediated immunity. The results indicate that in Group 1 and Group 2 patients the ratio CD4+/CD8+ was significantly reduced (i.e. by 58.5% and 47.9%, respectively) relative to corresponding control level. Actual values of Group 1 and Group 2 – 0.78, 0.98, respectively from the 1.88 control value. It is of interest to note that in Group 2 this parameter stay significantly higher as compared with results in Group 1, but did not reached control value.

Fig. 5. T-lymphocyte subpopulation ratios in cancer patients as a function of stage.

**5.3 Lymphocytes count and subpopulations in advanced cancer patients**

fill).

did not reached control value.

1) Stage IIA- IIIB (black fill); 2) Stage IV (clear fill); 3) healthy donors (control group; striped

Among the patients in both advanced cancer groups, the relative number of lymphocytes were significantly decreased (i.e.by 52.6% and 39.6%, respectively) as compared to corresponding control values. It is necessary to note that relative number of lymphocytes in Group 2 is significantly higher than in the Group 1 patients, but lower than the control value (see Table 4). In patients with advanced cancer and metastases there is reduction in both numbers of lymphocytes and proportions of CD4+/CD8+ T-lymphocytes which are thought to play an important role in cell-mediated immunity. The results indicate that in Group 1 and Group 2 patients the ratio CD4+/CD8+ was significantly reduced (i.e. by 58.5% and 47.9%, respectively) relative to corresponding control level. Actual values of Group 1 and Group 2 – 0.78, 0.98, respectively from the 1.88 control value. It is of interest to note that in Group 2 this parameter stay significantly higher as compared with results in Group 1, but

F(Ly) - fluorescence intensity in lymphocytes

Values shown are in mean (±SE)

Table 4. ABM fluorescence intensity in lymphocytes. Peripheral blood lymphocytes subpopulation counts in advanced cancer patients

#### **6. Examination of relationship between ABM fluorescence and blood lymphocyte profiles**

Pre-operation, in the colorectal patient group, the ABM fluorescence intensity was found to correlate with the relative number of CD38+ cells (*r* = +0.956). After the operations, both the CD4+:CD8+ ratio and relative number of CD38+ cells in patients blood was observed to be increased.

In colorectal cancer group, the ABM fluorescence intensity in blood plasma and the lymphocytes was found to correlate with CD4+:CD8+ ratios (in all stages of cancer) and the percentage (%) of lymphocytes/ subtypes in their blood. The degree of any relationship between cancer progression/staging, lymphocyte levels, and fluorescence among the lymphocytes was less obvious. Specifically, in terms of disease progression, cell levels and fluorescence intensity weakly tracked together (i.e., r = +0.512) for the colorectal patients. . With respect to associations between the plasma albumin fluorescence measurements and each of the individual lymphocyte-associated endpoints measured (except for cell fluorescence itself), many of the same patterns as noted above were evident. Once again, in terms of disease progression, although cell levels and albumin fluorescence intensity weakly tracked together (i.e., r = +0.513) among the colorectal patients.

In both groups of advanced cancer the ABM fluorescence intensity in blood plasma and lymphocytes was found to correlate to with CD4+/CD8+ ratios. In advanced cancer groups (in contrast to other groups) there is direct (not inverse) correlation between lymphocytes count and ABM fluorescence intensity. There seemed to be a good relationship between total lymphocyte (and subpopulation) levels and ABM fluorescence in both groups of patients. There is also good associations between the plasma albumin fluorescence measurements and each of the individual lymphocyte/albumin associated endpoints measured "effective" and total albumin concentration, reserve of albumin binding capacity.

Fluorescent Biomarker in Colorectal Cancer 439

that patients in the groups in the present study had ingested painkillers (both prescribed and retail) during the course of their disease; thus, a presence of these drugs/ metabolites on their albumin could have contributed to the noted shifts in ABM fluorescence /*K*a values. Our future studies will endeavor to recruit non-cancer patients with a "similar" history of painkiller intake in order to ascertain whether this was a main reason underlying our observations (regarding the albumin outcomes) or if there is something more inherently unique to the patient's cancer-bearing status that influenced the measured endpoints. This second standpoint is not without foundation. In oncopathology, the blood plasma content of two important unsaturated fatty acids (i.e., oleic acid and arachidonic acid) is increased, and these natural constituents also increasingly occupy binding sites on albumin (Gryzunov and Dobretsov, 1994, 1998). Both are observed to occupy binding sites distributed across the protein that happen to also be bound by medium or long-chain saturated fatty acids. The resulting restrictions imparted on the binding configurations of the protein would then account for shifts in the binding affinities at the primary sites between polyunsaturated fatty acids and their saturated or mono-unsaturated counterparts (Petitpas et al., 2001). It remains to be determined whether these alterations in fatty acid composition/binding also result in conformational changes in the albumin that impact upon ABM binding to its major (high selectivity) binding sites. As noted earlier, changes in fluorescence parameters of the cancer patients' lymphocytes could be reflective of changes in one/more inherent characteristics of their cells. In these studies, at least two, that is, proliferative activity and phenotypical character, could readily, albeit indirectly, be evaluated by examining changes in lymphocyte populations (i.e., their numbers) themselves. While the flow cytometry studies did indicate significant changes in lymphocyte (and subpopulation) levels among the cancer patients, unfortunately, the studies failed to yield overall lymphocyte (or subtype) population patterns that paralleled the concurrent changes in ABM fluorescence (i.e., Table 1 vs. Figure 2, example of this "lack of comparativeness"). Among all the subpopulation endpoints reported, only those of "CD38+%" and the "CD4+:CD8+ ratios" approached reflecting trends seen with the patients' fluorescence measurements. Specifically, the pre-surgery levels of each of these cytometric values were "maximally" reduced relative to the control subjects' values; post-surgery, these two values were increased, but in contrast to the fluorescence levels, these values did not reattain (or surpass) counterpart control levels. In light of the cancer patients' post-surgical (1) persistent lower numbers of lymphocytes (both total and within subclasses) and (2) fluorescence values that were uniformly significantly greater than in control subjects' cells, we surmise some factor(s) about these patients' lymphocytes (i.e., some undefined phenotypical characteristics) can cause amplification of the ABM fluorescent response. The fact that this "disconnect" between these two parameters is most predominant during the post-operative period strongly suggests that these as yetundefined modifying factors in the cancer patients might be related to their general immune response to the surgical procedure. Our future studies will need to recruit non-cancer patients with a "similar" history of surgical intervention/protocols (such as among patients suffering enterocolitis, undergoing local biopsies for non-cancer disorders, etc.) to ascertain whether the surgical procedure itself was a main reason for our observations (regarding the "disconnect") or whether, as with the albumin findings, there is something more inherently unique to a cancer-bearing status that influenced the measured endpoints. As expected, the CD4+:CD8+ ratios were seen to be increased in the cancer patients after they had undergone their respective operation. This would be expected as it is well accepted that CD4+ helper

#### **7. Discussion**

The novel fluorescent probe ABM (an amino derivative of benzanthrone) localizes deep within the phospholipids bilayer of lymphocytes membrane. Thus, in studies with lymphocytes, it can be concluded that changes in the spectral parameters of ABM (i.e., shifts in magnitude of fluorescence or actual wavelength associated with normal maximal fluorescence [i.e., *F*max]) could reflect modifications in one/ more interdependent (i.e., inter-related) properties of the cells. These could include the lymphocytes' (1) outer membrane physicochemical state, (2) membrane microviscosity,