**Meet the editor**

Professor Raja Kummoona, Fellow of the Royal College of Surgeons of England (FDSRCS), Emiratus professor of Maxillofacial Surgery of Iraqi Board for Medical Specializations, is one of 40 top scientist in Iraq. He has been a member of the Research Fellow Royal college of Surgeons of England (1975-1977), the President of Iraqi Dental Society (1977-1985). Prof. Kummoona has con-

tributed to the field with a number of publications and also by advocating many surgical procedures. He has conducted research in cancer surgery, flap reconstruction, TMJ surgery and facial injuries caused by missiles.

Contents

**Preface IX**

**A Personal View 1**  Raja Kummoona

**Part 1 History of Neck Dissection 5**

Jeremiah C. Tracy

**Head and Neck Cancer and Neck Dissection -**

**Part 2 Different Techniques of Neck Dissection & Complications 23**

Chapter 1 **A Brief History of Cervical Lymphadenectomy 7**

Chapter 2 **Neck Dissection – Techniques and Complications 25**

Abdul Wadood Mohammed and Anil K. Dash

**Dissection in Multidisciplinary Treatment 49**  Muneyuki Masuda, Ken-ichi Kamizono, Hideoki Uryu,

**Part 3 Advances and Modification of Neck Dissection 69**

Chapter 6 **Advanced Developments in Neck Dissection Technique: Perspectives in Minimally Invasive Surgery 87**

Jaimanti Bakshi, Naresh K. Panda,

Akiko Fujimura and Ryutaro Uchi

Chapter 4 **Complications of Neck Dissection 61**  Nader Saki and Soheila Nikakhlagh

Chapter 5 **Lateral Cervical Flap a Good Access**

Raja Kummoona

**for Radical Neck Dissection 71**

Jandee Lee and Woong Youn Chung

Chapter 3 **Roles of Therapeutic Selective Neck**

### Contents

#### **Preface XI**

**Head and Neck Cancer and Neck Dissection - A Personal View 1**  Raja Kummoona

#### **Part 1 History of Neck Dissection 5**

	- **Part 2 Different Techniques of Neck Dissection & Complications 23**
	- **Part 3 Advances and Modification of Neck Dissection 69**

### Preface

Advances in the management of cervical lymph node deposit required many modalities in surgery, deep X-ray therapy and chemotherapy. In order to achieve maximal effectiveness when treating a patient, it is essential to take considerable criteria factors such as: the pathology of a particular tumor (beside the status of patient's general condition) and the anatomy of the region. It is important that the pathology is assessed by the surgeon and not by the pathologist.

The chosen title of the book is based on different techniques used for lymphadenactomy: radical neck dissection, selective neck dissection and conservative neck dissection, including the preservation of the spinal accessory nerve, internal jugular vein and sterno mastoid muscle, using techniques such as the endoscopic technique with reboot surgery and other types of incisions. Recently the design of the lateral cervical flap proved to be an excellent access for different modalities of lymph adenactomy.

The treatment of the majority of patients with lymph node metastasis required a multidisciplinary approach. The joined efforts of the surgeon, radiotherapist, oncologist and pathologist are necessary for a successful outcome. They should all be involved in the treatment plan and should understand the principle and current applications of chemotherapy and radiotherapy in neck cancer metastasis.

The book comprises 11 chapters (including the Introductory chapter), which fall naturally into three main sections: I) History of neck dissection, II) Different techniques of neck dissection and Complications, III) Advances and modification of neck dissection.

This type of publication required a great support and effort both from the book editor and from the publishing team. I would like to extend my thanks and gratitude to Ms Natalia Reinic, editor relations consultant, and to the publishing process managers Ms Alenka Urbancic, Mr Marko Rebrovic and Ms Silvia Vlase. All these people spent a lot of time into making this book a worthwhile publication and interesting to the readers and top specialist concerned with neck dissection.

**Prof. Raja Kummoona**

Professor Emeritus of Maxillofacial Surgery Acting Chairman of Maxillofacial Surgery Iraqi Board for Medical Specializations Baghdad, Iraq

**Head and Neck Cancer and Neck Dissection -**

Head and neck cancer represent nearly 12% of total malignancies, including the face, the oropharynx, the parotid gland and other salivary glands, the orbit, the jaw , the sinuses and other parts of the face including the skin. These anatomical sites might be affected by other varieties of cancer, such as basal cell carcinoma, squamous-cell carcinoma, fibro sarcoma, osteogenic sarcoma and jaw lymphoma, and non-Hodgkin's lymphoma and Hodgkin's lymphoma. Jaw lymphoma is nominated from other parts of the world and Africa, such as Burkitt lymphoma. Jaw lymphoma is quite different from Burkitt lymphoma in its clinical features, aetiology and even with regard to its treatment. Jaw lymphoma is presented as having a very rapid onset with a fast spread to internal organs and the brain, while Burkitt lymphoma is a slowly growing tumour; it is well known that Burkitt lymphoma can be treated successfully by a few courses of cyclophosphamide (40 mg/square meter) but jaw lymphoma requires a more complicated regimen with combination of many chemotherapeutic agents, such as CHOP( therapeutic regimen of jaw lymphoma consist of eight doses over 24 weeks including 1.5mg/m2 Vincristine,50mg/m2 Adriamycin, 1000mg/m2 Cyclophosphomide, 10mg/m2 Methotroxate and 50mg/m2 prednisolone ) and it rarely deposits its tumour to the lymph nodes. Cancer of the head and neck constitute an important section of the total cancers affecting the body, and oral cancer represents about 4% of this; it is not necessary that all such cancers have nodal deposits in the neck, such jaw lymphoma or Burkitt Lymphoma. Malignant tumours such as squamous cell carcinoma – which form about 95% of oral cancers – and Melanoma – a highly malignant tumour with early metastasis – are rare and aggressive types of tumours and the survival rate is very low. Other malignant tumours, such as adenocarcinoma – which is a slowly growing malignant

Cancer of the paranasal sinuses is considered to be an aggressive type of malignancy with a tendency to invade the orbit and the base of the skull. The most common tumour of the sinuses is squamous cell carcinoma rather than adenocarcinoma, as a result of cellular changes from respiratory columnar type to squamous type due to the recurrence of infection and other irritating agents. These types of tumour metastasise in the cervical lymph nodes. Cancer of the oral cavity represents somewhat less than 4% of total cancer incidence but this might increase to more than 40% – as in India due to dietary causes such as spicy foods and

tumour – have less of a tendency for cervical node metastasis.

**A Personal View**

*Professor Emeritus of Maxillofacial Surgery, Acting Chairman of Maxillofacial Surgery, Iraqi Board for Medical Specializations,*

Raja Kummoona

*Baghdad, Iraq*

## **Head and Neck Cancer and Neck Dissection - A Personal View**

#### Raja Kummoona

*Professor Emeritus of Maxillofacial Surgery, Acting Chairman of Maxillofacial Surgery, Iraqi Board for Medical Specializations, Baghdad, Iraq*

Head and neck cancer represent nearly 12% of total malignancies, including the face, the oropharynx, the parotid gland and other salivary glands, the orbit, the jaw , the sinuses and other parts of the face including the skin. These anatomical sites might be affected by other varieties of cancer, such as basal cell carcinoma, squamous-cell carcinoma, fibro sarcoma, osteogenic sarcoma and jaw lymphoma, and non-Hodgkin's lymphoma and Hodgkin's lymphoma. Jaw lymphoma is nominated from other parts of the world and Africa, such as Burkitt lymphoma. Jaw lymphoma is quite different from Burkitt lymphoma in its clinical features, aetiology and even with regard to its treatment. Jaw lymphoma is presented as having a very rapid onset with a fast spread to internal organs and the brain, while Burkitt lymphoma is a slowly growing tumour; it is well known that Burkitt lymphoma can be treated successfully by a few courses of cyclophosphamide (40 mg/square meter) but jaw lymphoma requires a more complicated regimen with combination of many chemotherapeutic agents, such as CHOP( therapeutic regimen of jaw lymphoma consist of eight doses over 24 weeks including 1.5mg/m2 Vincristine,50mg/m2 Adriamycin, 1000mg/m2 Cyclophosphomide, 10mg/m2 Methotroxate and 50mg/m2 prednisolone ) and it rarely deposits its tumour to the lymph nodes. Cancer of the head and neck constitute an important section of the total cancers affecting the body, and oral cancer represents about 4% of this; it is not necessary that all such cancers have nodal deposits in the neck, such jaw lymphoma or Burkitt Lymphoma. Malignant tumours such as squamous cell carcinoma – which form about 95% of oral cancers – and Melanoma – a highly malignant tumour with early metastasis – are rare and aggressive types of tumours and the survival rate is very low. Other malignant tumours, such as adenocarcinoma – which is a slowly growing malignant tumour – have less of a tendency for cervical node metastasis.

Cancer of the paranasal sinuses is considered to be an aggressive type of malignancy with a tendency to invade the orbit and the base of the skull. The most common tumour of the sinuses is squamous cell carcinoma rather than adenocarcinoma, as a result of cellular changes from respiratory columnar type to squamous type due to the recurrence of infection and other irritating agents. These types of tumour metastasise in the cervical lymph nodes. Cancer of the oral cavity represents somewhat less than 4% of total cancer incidence but this might increase to more than 40% – as in India due to dietary causes such as spicy foods and

Head and Neck Cancer and Neck Dissection - A Personal View 3

advances in chemotherapy have seen the application of Gemzar (gencitabin) (this drug interferes with the growth and spread of cancer cells by inducing apoptosis and ant metabolite and also been used with Carboplatin) – which is a specific chemotherapy for this type of malignancy and was a promising type of chemotherapy even in cases of fourth-stage of pancreatic adenocarcinoma. In the parotid glands, adenocarcinoma is common and also is mucoepidermoid carcinoma and other malignancies; only rarely is the parotid affected by malignant oncocytoma, this type of tumour metastasises in cervical lymph nodes and

The majority of head and neck tumours require neck dissection at once, affecting the oral cavity and parotid region. However, tumours affecting the middle third of the face – such as the maxilla or the orbit – require radical surgery with flap reconstruction followed by DXT and chemotherapy, rather than radical neck dissection and as there is rarely any metastasis

Melanoma of the orofacial tumour is a highly malignant type of tumour with a high tendency for early cervical metastasis, and the prognosis is not very promising. It requires multiple therapies for controlling its tumours, including chemotherapy and radical surgery, while melanoma of the lower limbs is less aggressive and responds to radical surgery and is

Current cancer research focused now a days on understanding on the response and resistance to treatment and apoptosis. Cancer treatment depend not only on cellular damages as achieved by chemotherapy and DXT but also on the ability of the cell to respond to damages by inducing apoptotic changes and mutation in apoptotic pathway to end with resistance to chemotherapy drugs and radiation. Mitochondria and cell surface receptors

Fig. 1. Jaw lymphoma of the right side of the face of a 2 year old boy with a history of one

requires radical resection of the tumour with chemotherapy and DXT.

in the cervical lymph nodes.

month.

diagnosed with lymphoscintigraphy.

smoking – and these tumours appear as a fissure or exophytic growth or ulcer with white leukoplakia, and the most common site which is affected is the tongue and floor of the mouth. Both of these lesions in early metastasis affect the deep chain of the cervical lymph nodes, and the managements of these cases was based on a combination of three modalities in the form of radical surgery, chemotherapy and deep x-ray therapy. There is no possibility of a single technique for treatment in these cases. A frozen section in theatre is required for any assessment of the complete eradication of tumours

Nowadays, chemotherapy has played an important role in the management of head and neck cancers due to advances in the manufacturing of these drugs and DXT (deep x-ray therapy) which have become more specific and more precise in targeting cancer tumours. One technique of note is the use of the gamma Knife (Cobalt 60) in the management of brain tumours and intraocular malignancies without evisceration of the eye ball (which can be very depressing and inconvenient for patients).

The advancement of surgical management of head and neck tumours was based on advances in flap surgeries, such as a pedicle flaps like the forehead flap, the lateral cervical flap, the deltopectoral flap and the trapezius flap, or else by using free flaps like the forearm flap and the tapes dorsalis flap; these flaps are required for microanastomosis for the reconstruction the surgical defects after radical cancer surgery. We have not forgotten that the traditional use of radical neck dissection as a method of treatment for cervical lymph node metastasis has not often been used as a surgical procedure for the total radical excision of cervical lymph nodes with the radical excision of the sternomastoid muscle, the accessory nerve, deep cervical fascia and internal jugular vein ligation. This procedure has become less popular due to the creation of an obvious vertical band of scars extending all over the neck and dropping off the shoulder with a superficial exposure of the carotid tree just below the skin. This problem was overcome by the advancement of the trapezius flap so as to cover the carotid tree and so avoid any traumatic injuries to carotid content. These complications have been avoided by advances in other techniques, such as selective neck dissection, functional neck dissection and supraomohyoid neck dissection.

The advancements of different diagnostic tools for detection of any cervical lymph node metastasis and assessment of these deposit been used by application of ultra sonography , MRI and CT scan with protocol for management of cervical lymph nodes metastasis is the basis for management of cervical lymph node metastasis.

The most common malignant tumours of the orofacial region is basal cell carcinoma affecting the skin of the face and this is more common among white people who have less melanin pigment in their skin and who have continuous exposure to sun light. This tumour is a slowly growing type with a tendency to invade the underlying structures and it does not metastasis to the cervical lymph nodes. Squamous cell carcinoma represents about 95% of the total oral malignancies mainly affecting the tongue and the floor of the mouth with tendency for cervical lymph node deposits. The management of these tumours requires the application of all modalities of treatment, surgery, DXT and chemotherapy.

Adenocarcinoma is less common in the oral cavity and affects the minor salivary glands – it is more common in the maxilla and it is a slowly growing tumour that rarely metastasises in the cervical lymph nodes and is less aggressive than adenocarcinoma of the gastro-intestinal tract, which is a highly malignant tumour with early metastasis in mesenteric lymph nodes. The eradication of these tumours is rather difficult due to their early metastasis and the complicated anatomy of the area, which makes radical surgery rather difficult. Recent

smoking – and these tumours appear as a fissure or exophytic growth or ulcer with white leukoplakia, and the most common site which is affected is the tongue and floor of the mouth. Both of these lesions in early metastasis affect the deep chain of the cervical lymph nodes, and the managements of these cases was based on a combination of three modalities in the form of radical surgery, chemotherapy and deep x-ray therapy. There is no possibility of a single technique for treatment in these cases. A frozen section in theatre is required for

Nowadays, chemotherapy has played an important role in the management of head and neck cancers due to advances in the manufacturing of these drugs and DXT (deep x-ray therapy) which have become more specific and more precise in targeting cancer tumours. One technique of note is the use of the gamma Knife (Cobalt 60) in the management of brain tumours and intraocular malignancies without evisceration of the eye ball (which can be

The advancement of surgical management of head and neck tumours was based on advances in flap surgeries, such as a pedicle flaps like the forehead flap, the lateral cervical flap, the deltopectoral flap and the trapezius flap, or else by using free flaps like the forearm flap and the tapes dorsalis flap; these flaps are required for microanastomosis for the reconstruction the surgical defects after radical cancer surgery. We have not forgotten that the traditional use of radical neck dissection as a method of treatment for cervical lymph node metastasis has not often been used as a surgical procedure for the total radical excision of cervical lymph nodes with the radical excision of the sternomastoid muscle, the accessory nerve, deep cervical fascia and internal jugular vein ligation. This procedure has become less popular due to the creation of an obvious vertical band of scars extending all over the neck and dropping off the shoulder with a superficial exposure of the carotid tree just below the skin. This problem was overcome by the advancement of the trapezius flap so as to cover the carotid tree and so avoid any traumatic injuries to carotid content. These complications have been avoided by advances in other techniques, such as selective neck dissection,

The advancements of different diagnostic tools for detection of any cervical lymph node metastasis and assessment of these deposit been used by application of ultra sonography , MRI and CT scan with protocol for management of cervical lymph nodes metastasis is the

The most common malignant tumours of the orofacial region is basal cell carcinoma affecting the skin of the face and this is more common among white people who have less melanin pigment in their skin and who have continuous exposure to sun light. This tumour is a slowly growing type with a tendency to invade the underlying structures and it does not metastasis to the cervical lymph nodes. Squamous cell carcinoma represents about 95% of the total oral malignancies mainly affecting the tongue and the floor of the mouth with tendency for cervical lymph node deposits. The management of these tumours requires the

Adenocarcinoma is less common in the oral cavity and affects the minor salivary glands – it is more common in the maxilla and it is a slowly growing tumour that rarely metastasises in the cervical lymph nodes and is less aggressive than adenocarcinoma of the gastro-intestinal tract, which is a highly malignant tumour with early metastasis in mesenteric lymph nodes. The eradication of these tumours is rather difficult due to their early metastasis and the complicated anatomy of the area, which makes radical surgery rather difficult. Recent

application of all modalities of treatment, surgery, DXT and chemotherapy.

any assessment of the complete eradication of tumours

functional neck dissection and supraomohyoid neck dissection.

basis for management of cervical lymph node metastasis.

very depressing and inconvenient for patients).

advances in chemotherapy have seen the application of Gemzar (gencitabin) (this drug interferes with the growth and spread of cancer cells by inducing apoptosis and ant metabolite and also been used with Carboplatin) – which is a specific chemotherapy for this type of malignancy and was a promising type of chemotherapy even in cases of fourth-stage of pancreatic adenocarcinoma. In the parotid glands, adenocarcinoma is common and also is mucoepidermoid carcinoma and other malignancies; only rarely is the parotid affected by malignant oncocytoma, this type of tumour metastasises in cervical lymph nodes and requires radical resection of the tumour with chemotherapy and DXT.

The majority of head and neck tumours require neck dissection at once, affecting the oral cavity and parotid region. However, tumours affecting the middle third of the face – such as the maxilla or the orbit – require radical surgery with flap reconstruction followed by DXT and chemotherapy, rather than radical neck dissection and as there is rarely any metastasis in the cervical lymph nodes.

Melanoma of the orofacial tumour is a highly malignant type of tumour with a high tendency for early cervical metastasis, and the prognosis is not very promising. It requires multiple therapies for controlling its tumours, including chemotherapy and radical surgery, while melanoma of the lower limbs is less aggressive and responds to radical surgery and is diagnosed with lymphoscintigraphy.

Current cancer research focused now a days on understanding on the response and resistance to treatment and apoptosis. Cancer treatment depend not only on cellular damages as achieved by chemotherapy and DXT but also on the ability of the cell to respond to damages by inducing apoptotic changes and mutation in apoptotic pathway to end with resistance to chemotherapy drugs and radiation. Mitochondria and cell surface receptors

Fig. 1. Jaw lymphoma of the right side of the face of a 2 year old boy with a history of one month.

**Part 1**

**History of Neck Dissection**

mediate the pathway of apoptosis and this pathways mediated by Bcl-2 family protein and the final excursion of cell death is performed by caspace cascade which is triggered by release cytochrome C from mitochondria. Most of the activity in the development of apoptosis drugs was concentrated on apoptosis inducers for treatment of malignancies.

The future might be very promising for the control of lymph node deposits by using different methods of accessing neck dissection as well as the recent application of robot surgery (the da Vinci surgical robot system) which is more widely used in prostatic eradication than in any other specialty and which might be used in general surgery. However, this technique is limited in its application in all fields and even in head and neck malignancies. Expanding the role of DXT and chemotherapy as the first line of treatment and as a curative therapy without the need for radical neck dissection, either as an adjuvant with surgery or without as in jaw lymphoma (which is the only line of treatment for such a highly malignant tumour, being a fast spreading and fatal tumour).

Fig. 2. Post-therapy after 2 years of treatment of jaw lymphoma by 6 courses of chemotherapy, with the collaboration with Prof. Selma Al Hadad, Paediatric Oncologist, Medical City Baghdad.


We are IntechOpen, the world's leading publisher of Open Access books Built by scientists, for scientists

5,800+

Open access books available

144,000+

International authors and editors

180M+

Downloads

156 Countries delivered to Our authors are among the

Top 1% most cited scientists

12.2%

Selection of our books indexed in the Book Citation Index (BKCI) in Web of Science Core Collection™

### Interested in publishing with us? Contact<EMAIL_ADDRESS>
Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com

## Meet the editor

Ibrokhim Y. Abdurakhmonov received a BS in Biotechnology from the National University, Uzbekistan, in 1997, an MS in Plant Breeding from Texas A&M University in 2001, and a Ph.D. in Molecular Genetics, DSc in Genetics, and a full professorship in Molecular Genetics and Molecular Biotechnology from the Academy of Sciences of Uzbekistan in 2002, 2009, and 2011, respectively. He founded the Center of Genomics and Bioinfor-

matics of Uzbekistan in 2012. He received the 2010 prize from The World Academy of Sciences (TWAS) and "ICAC Cotton Researcher of the Year 2013" for his outstanding contribution to cotton genomics and biotechnology. He was elected as a fellow to TWAS in 2014 and as a member of the Academy of Sciences of Uzbekistan in 2017. In the same year, he was appointed Minister of Innovative Development of Uzbekistan.

## Contents


*Edited by Hassan Abduljabbar*

Uterine fibroids are benign tumors of the smooth muscle of the uterus. They are the most common pathologic abnormalities of the female genital tract, occurring in 20%–50% of women older than 30 years. Some fibroids present with symptoms, whereas others are asymptomatic. The etiology of uterine fibroids is unclear. As such, this book presents comprehensive information on clinical presentation, diagnosis, and management of myoma. Chapters discuss symptoms such as bleeding, pain, and sexual dysfunction, as well as imaging modalities for diagnosis and options for treatment, including medications and surgery.

ISBN 978-1-83962-995-2

Fibroids

Published in London, UK © 2021 IntechOpen © magicmine / iStock


**Thermo-Hydraulic Performance Analysis on the E**ff**ects of Truncated Twisted Tape Inserts in a Tube Heat Exchanger**

**Mehdi Ghalambaz 1,2, Ramin Mashayekhi 3 , Hossein Arasteh 4 , Hafiz Muhammad Ali 5 , Pouyan Talebizadehsardari 6,7,\* and Wahiba Yaïci 8,\***

Received: 10 September 2020; Accepted: 29 September 2020; Published: 9 October 2020

**Abstract:** This paper investigates the convective heat transfer in a heat exchanger equipped with twisted tape elements to examine effects of the twisted tape truncation percentage, pitch value, position and Reynolds number using 3D numerical simulation. A symmetric heat flux is applied around the tube as the studied heat exchanger. Based on the influences in both heat transfer enhancement and pressure drop, the performance evaluation criterion (PEC) is utilized. Inserting twisted tape elements and reducing the pitch value significantly augment the Nusselt number, friction coefficient and PEC number compared to the plain tube. For the best case with a Reynolds number of 1000, the average Nusselt number increases by almost 151%, which is the case of fully fitted twisted tape at a pitch value of L/4. Moreover, increasing the twisted tape truncation percentage reduces both heat transfer and pressure drop. Furthermore, the highest heat transfer rate is achieved when the truncated twisted tape is located at the entrance of the tube. Finally, it is concluded that for P = L, L/2, L/3 and L/4, the optimum cases from the viewpoint of energy conservation are twisted tapes with truncation percentages of 75, 50, 50 and 0%, in which the related PEC numbers at a Reynolds number of 1000 are almost equal to 1.08, 1.24, 1.4 and 1.76, respectively.

**Keywords:** truncated twisted tape; secondary flow; laminar convective heat transfer; performance evaluation criterion; CFD; heat exchanger


**6. Other immune checkpoints under investigation**

Apart from the above two immune checkpoints, a variety of other immune stimulatory and suppressive checkpoints are currently under investigation as immunotherapy targets that include; TIM-3, LAG-3, TIGIT, VISTA and BTLA-4, these reduce the anti-tumor immune response by regulating T cell activity like CTLA-4 and PD-1. Among them TIM 3, BTLA-4 and LAG-3 are implicated as T cell exhaustion markers in tumors same as that of PD-1. TIM 3 negatively regulates the cytotoxic CD8 T cells and Th1 CD4 T cells, thereby shifting the immune responses. TIGIT is expressed by a number of cells such as: T cells, Treg cells and NK cells

#### **Table 2.**

*Immune checkpoints in immunotherapy.*

#### *Integrating Immunotherapy with Chemotherapy: A New Approach to Drug Repurposing DOI: http://dx.doi.org/10.5772/intechopen.100183*

and is known to bind to poliovirus receptor on APCs or tumor cells. It is supposed to perform both direct and indirect immunosuppressive effects by competitively binding to NK and T cell receptors in place of CD226; it also leads to downstream inhibition of AKT signaling in T cells [14, 44]. In addition, TIGIT increases the suppressive activity and releases inhibitory cytokines by receptor binding of TIGIT on the APCs and Tregs [45]. Moreover, VISTA is expressed by both APCs and T cells play a role in both Treg function and myeloid cell activation [14].

On the contrary, other checkpoints like OX40, ICOS and CD40L are immunostimulatory checkpoints that function in the maintenance and activation of effector T cells. The expression of OX40 is induced at the time of T-cell activation leading to the expression of anti-apoptotic factors such as BCL-2 and BCL-XL that leads to the sustenance of T cells proliferation. It also acts as a co-stimulatory signal in tumor induction and is constitutively expressed on Tregs and OX40 also decreases the Treg function by binding to its receptor on Treg [46–48]. Furthermore, ICOS leads to the activation of second signal in immune activation by binding to B7H/B7RP-1 [49]. Another immunostimulatory checkpoint CD40L interacts with CD40 on APCs induces via NF-ƙB signaling a proinflammatory immune response **Table 2** [14].

The immunostimulatory checkpoints can be inhibited by therapeutic agents targeting recombinant ligand peptides, ligands expressing viral particles or agonistic monoclonal antibody that is in contrast, to the inhibitory checkpoints where monoclonal antibodies inhibit the interaction between the respective ligand and receptors. Therefore, there is an emerging need to fully explore these biomarkers for better prognosis of patients using immunotherapy strategy [14].


*Article* **Different Seasonal Collections of** *Ficus carica* **L. Leaves Diversely Modulate Lipid Metabolism and Adipogenesis in 3T3-L1 Adipocytes**

**Mariachiara Pucci 1,†, Manuela Mandrone 2,†, Ilaria Chiocchio 2, Eileen Mac Sweeney 1, Emanuela Tirelli 1, Daniela Uberti 1, Maurizio Memo 1, Ferruccio Poli 2, Andrea Mastinu 1,\*,† and Giulia Abate 1,†**

**Abstract:** Due to the high prevalence of obesity and type 2 diabetes, adipogenesis dysfunction and metabolic disorders are common features in the elderly population. Thus, the identification of novel compounds with anti-adipogenic and lipolytic effects is highly desirable to reduce diabetes complications. Plants represent an important source of bioactive compounds. To date, the antidiabetic potential of several traditional plants has been reported, among which *Ficus carica* L. is one of the most promising. Considering that plant metabolome changes in response to a number of factors including seasonality, the aim of this study was to evaluate whether *Ficus carica* leaves extracts collected in autumn (FCa) and spring (FCs) differently modulate lipid metabolism and adipogenesis in 3T3-L1 adipocytes. The 1H-NMR profile of the extracts showed that FCs have a higher content of caffeic acid derivatives, glucose, and sucrose than FCa. In contrast, FCa showed a higher concentration of malic acid and furanocoumarins, identified as psoralen and bergapten. In vitro testing showed that only FCa treatments were able to significantly decrease the lipid content (Ctrl vs. FCa 25 μg/mL, 50 μg/mL and 80 μg/mL; *p* < 0.05, *p* < 0.01 and *p* < 0.001, respectively). Furthermore, FCa treatments were able to downregulate the transcriptional pathway of adipogenesis and insulin sensitivity in 3T3-L1 adipocytes. In more detail, FCa 80 μg/mL significantly decreased the gene expression of PPARγ (*p* < 0.05), C/EBPα (*p* < 0.05), Leptin (*p* < 0.0001), adiponectin (*p* < 0.05) and GLUT4 (*p* < 0.01). In conclusion, this study further supports an in-depth investigation of *F. carica* leaves extracts as a promising source of active compounds useful for targeting obesity and diabetes.

**Keywords:** seasonality; *Ficus carica* L.; furanocoumarins; 1H-NMR profile; lipid metabolism; adipogenesis

#### **1. Introduction**

Metabolic syndrome and diabetes pose a significant health burden worldwide and their incidence was found to be highly increased due to recent and alarming changes in food habits and lifestyle [1]. Metabolic syndrome is described as a pre-diabetic condition that includes obesity, dyslipidemia, impaired fasting glucose and/or impaired glucose tolerance, and reduced insulin sensitivity [2]. Diabetes mellitus (DM) is instead a chronic condition characterized by abnormal glucose homeostasis, resulting in elevated plasma glucose levels [3]. When prolonged in life, chronic hyperglycemia is associated with a myriad of long-term DM-related clinical sequelae such as damage to pancreatic β cells and microvascular complications, including retinopathy, nephropathy, and neuropathy [3].

**Citation:** Pucci, M.; Mandrone, M.; Chiocchio, I.; Sweeney, E.M.; Tirelli, E.; Uberti, D.; Memo, M.; Poli, F.; Mastinu, A.; Abate, G. Different Seasonal Collections of *Ficus carica* L. Leaves Diversely Modulate Lipid Metabolism and Adipogenesis in 3T3-L1 Adipocytes. *Nutrients* **2022**, *14*, 2833. https://doi.org/ 10.3390/nu14142833

Academic Editor: Hayato Tada

Received: 28 May 2022 Accepted: 5 July 2022 Published: 10 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The International Diabetes Federation has estimated that 463 million adults lived with diabetes worldwide in 2019, with a projected increase to 700 million by 2045 if effective prevention or strict glycemic control methods are not be adopted in DM patients before disease progression [4].

Several risk factors are known to be involved in the development of diabetes, including genetic factors (i.e., family history), lifestyle, diet, age, ethnicity, high blood glucose during pregnancy, hypertension, dyslipidemia, and obesity [5]. Of note, the increasing literature highlights a contributing role of adipose tissue dysfunction and hyperproliferation in inducing metabolic syndrome and diabetes [6].

Adipose tissue is highly dynamic and is the largest organ in humans involved in lipid storage and mobilization on the basis of energy requirements. It consists of several types of cells, including mature adipocytes that can increase in number and size. Decreasing proliferation and adipogenesis at the early stage of adipocyte differentiation might represent potential targets for preventing or treating obesity and, in turn, diabetes [7].

Besides insulin supplementation, to reduce the cluster of inter-related events of hyperglycemia and hyperlipidemia, a multi-targeted approach controlling both glucose and lipid metabolism is needed. The currently available non-pharmacological options are exercise or dietary modification [8], while pharmacological options include treatment with anti-diabetic, and anti-hyperlipidemic drugs [3].

Unfortunately, these latter therapeutic treatments have undesirable side effects, leading to a growing interest in traditional medicine to treat metabolic disorders, focusing mainly on herbal medicines [9,10]. Noteworthy is the development of metformin, one of the main antihyperglycemic agents, which can be traced back to the traditional use of *Galega officinalis* to treat diabetes [11,12]. With specific regard to the treatment of diabetes, obesity, and blood glucose control, several traditional plants with anti-diabetic and anti-obesity activities have already been reported. Among them, *Ficus carica* L. is considered to be one of the most promising [13,14].

*Ficus carica* is the most popular species of the genus *Ficus* (Moraceae family). It is native to Southwest Asia but has been widely cultivated throughout the Mediterranean basin since ancient times. Overall, the genus *Ficus* is one of the most abundant and varied genera of Angiosperms, including more than 800 species widely distributed in the tropical and subtropical belt of Southeast Asia and South America [15,16]. Traditional systems of medicine such as Ayurveda, Unani, and Siddha use different organs of multiple *Ficus* species such as *F. benghalensis*, *F. religiosa*, *F. glumosa*, *F. deltoidea*, *F. racemosa* and *F. carica* to treat metabolic, respiratory, gastrointestinal, reproductive and infectious disorders [17,18]. In support of traditional use, the bioactivity of *F. carica* extracts was also the object of several studies demonstrating its numerous pharmacological properties, among which include antioxidant, anticancer, anti-inflammatory and, remarkably, antidiabetic and hypolipidemic properties, in both in vitro and in vivo models [19,20]. More specifically, the data in the literature identify *F. carica* leaf extracts as an optimal source of anti-diabetic, anti-hyperglycemic and anti-obesity compounds that can be used as food supplements in diabetes prevention [16,21].

The biological activities of *F. carica* are mainly associated with the presence of heterogeneous phytoconstituents such as phytosterols, anthocyanins, amino acids, organic acids, fatty acids, phenolics, furanocoumarins hydrocarbons and aliphatic alcohols, which have been found in the latex, leaves, fruits and roots [22]. Nevertheless, contradictory results are present, probably due not only to the lack of standardization but also to the high variability in the phytoconstituents. Both these parameters can be influenced by diverse factors such as different preparations of the plant extracts, plant harvesting seasonality, drought stress and soil conditions [23–25].

Therefore, the main objective of this study was to evaluate whether the effects of *F. carica* leaf extracts on glucose and lipid metabolism in fully differentiated 3T3-L1 adipocytes depended on different phytochemical profiles that, in turn, were potentially due to different harvesting seasons. *Ficus carica* leaves were collected in spring and autumn (before and after

fructification), the differences in metabolite content were explored by 1H-NMR profiling and the activity of the extracts as key adipogenic gene modulators with anti-adipogenic and anti-obesity properties was tested in an *in vitro* model.

#### **2. Materials and Methods**

#### *2.1. Plant Material and Extract Preparation*

*Ficus carica* leaves were collected in November (FCa) and May (FCs) 2020 from a tree growing in the botanical garden of Bologna "Orto dei Semplici". Voucher specimen (BOLO0602009) were retained in the Herbarium of Alma Mater Studiorum-University of Bologna. The leaves, deprived of the stalk, were dried in stove for three days at 40 ◦C and consequentially grinded. For 1H-NMR profiling, 30 mg of plant material were extracted with 1 mL of mixture (1:1) of phosphate buffer (90 mM; pH 6.0) in H2O-*d*2 (containing 0.1% TMSP) and MeOH-*d*4 by ultrasonication (TransSonic TP 690, Elma, Germany) for 20 min at 45 ◦C. After this procedure, samples were centrifuged for 10 min (17,000× *g*), then 700 μL of supernatant were transferred into NMR tubes. For bioactivity tests, 120 mg of plant material were extracted in 6 mL of MeOH:H2O (1:1), then sonicated for 30 min and centrifuged for 20 min. The supernatant was then divided into 3 × 2 mL test tubes and dried in a vacuum concentrator (Savant SpeedVac SPD210, Thermo Fisher Scientific, Waltham, MA, USA). Finally, it was resuspended in DMSO and then tested at different concentrations on cells.

#### *2.2. Liquid-Liquid Partition of Ficus Carica Extracts*

In order to elucidate the structure of the furanocoumarins visible in the 1H NMR profile of the extracts, FCa and FCs were subjected to a pre-purification procedure: 2 g of powder material were extracted in 100 mL of MeOH:H2O (1:1), then sonicated for 30 min and centrifuged for 15 min. The supernatant was dried in a rotary evaporator yielding 400 mg of extract, which was resuspended into 30 mL of water to undergo liquid-liquid partition with CHCl3 and EtOAc used in sequence, repeating the procedure three times for each solvent. The three fractions were dried in a rotary evaporator and analyzed by 1H NMR. The chloroform fraction contained the diagnostic signals of the furanocoumarins; therefore, it was subjected to 2D NMR analysis and the two major compounds were identified as psoralen and bergapten

#### *2.3. NMR Analysis*

1H NMR spectra, J-resolved (J-res), 1H-1H homonuclear (COSY) and inverse detected 1H-13C correlation experiments (HMBC, HSQC) were recorded at 25 ◦C on a Varian Inova (Milan, Italy) instrument (equipped with a reverse triple resonance probe). For 1H NMR profiling the instrument was operated at a 1H NMR frequency of 600.13 MHz, and H2O-*d*2 was used as the internal lock. Each 1H NMR spectrum consisted of 256 scans (corresponding to 16 min) with the relaxation delay (RD) of 2 s, acquisition time 0.707 s, and spectral width of 9595.8 Hz (corresponding to δ 16.0). A pre-saturation sequence (PRESAT) was used to suppress the residual water signal at δ 4.83 (power = −6 dB, pre-saturation delay 2 s). The spectra were manually phased and baseline corrected and calibrated to the internal standard trimethyl silyl propionic acid sodium salt (TMSP) at δ 0.0 using Mestrenova software (Mestrelab Research, Spain). The analysis of 1H-NMR profiles of extracts was performed based on an in-house library and in comparison with the literature [26–29].

#### *2.4. Cells Culture and Treatment*

3T3-L1 murine pre-adipocyte cells, purchased from Sigma Aldrich (Sigma-Aldrich, St. Louis, MO, USA), were cultured in a Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS (Sigma Aldrich) at 37 ◦C and 5% CO2 until ~80% cells confluence. Then, cells were seeded at a density of 1 × 104 per well in 24-well plates. After 48 h, ~80% confluence was reached and cells were induced to differentiate (DAY 0) in MDI (Methylisobutylxanthine, Dexamethasone, Insulin) induction medium for 2 days. In particular, MDI induction medium is composed of 0.5 mM 3-isobutil-1-methylxantine, 1 μM dexamethasone and 10 μg/mL insulin in DMEM containing 10% FBS. At differentiation on DAY 3, the MDI medium was switched to a growth medium supplemented with 10 μg/mL insulin (DMEM containing 10% FBS and 10 μg/mL insulin). Medium was replaced every 2 days until fully differentiated adipocytes were obtained (DAY 11) [30,31]. Thus, at DAY 11, >90% of cells showed the characteristic adipocyte phenotype with accumulated lipid droplets. To test the effect of 48 h treatments with FC extracts, 3T3-L1 adipocytes were treated at DAY 9 with different dosages (25, 50, 80 and 100 μg/mL) of FCa and FCs and after 48 h (DAY 11), the MTT test was performed.

#### *2.5. Cell Viability*

The 3T3-L1 preadipocytes were seeded at a density of 1 × 104 cells per well in 96-well plates and incubated in culture medium. The cells were then treated with different dosages (25–100 μg/mL) of FCa and FCs. After 48 h, cells were incubated with 500 mg/mL of MTT (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 1h at 37 ◦C. The supernatants were then removed and DMSO was added to each well. Plates were agitated to dissolve the formazan crystal products. The absorbance at 595 nm was measured using a Bio-Rad 3350 microplate reader (Bio Rad Laboratories, Richmond, CA, USA). The percentage of viable cells was calculated by defining the viability of untreated cells as 100%.

#### *2.6. Red O Staining*

3T3-L1 adipocytes were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. After fixation, cells were washed two times with PBS and one time with 60% isopropanol. Then, cells were stained with filtered Oil-Red-O working solution (working solution: 60% Oil-Red-O stock solution and 40% distilled water) for 1 h at room temperature, washed 4 times with distilled water, and plates were dried and scanned for images. Oil-Red-O dye was then extracted using 100% isopropanol alcohol and measured spectrophotometrically at 490 nm.

#### *2.7. Quantitative Real-Time PCR*

Total RNA was extracted from 1 × 104 cells following TRIzol® reagent protocol (Invitrogen Corporation, Carlsbad, CA, USA). Then, 2μg of total RNA was retrotranscribed with M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA), using oligo-dT (15–18) as a primer in a final volume of 40μL reaction mix containing 1X RT buffer with MgCl2 5 mM, DDT 10 mM, oligo-dT 5 mM, dNTPs 1 mM, RNase inhibitor 1U/mL and Reverse Transcriptase (M-MLV Reverse Transcriptase, Invitrogen) 10 U/mL. The reaction was incubated at 70 ◦C for 10 min and then at 4 ◦C for 2 min, followed by 37 ◦C for 60 min as reported in [32]. SYBR Green-based Real-Time PCR was used to determine cDNA levels. PPAR-γ, C/EBP-α, Adiponectin, Leptin, GLUT4, FAS and β-actin primers reported in Table 1 were provided by Metabion (Metabion International AG, Planegg, Germany). β-actin was used as the endogenous reference. Quantitative RT-PCR was performed with the ViiA7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the iQ™SYBR Green Supermix method (Bio-Rad Laboratories, Richmond, CA, USA) according to the manufacturer's instructions. Samples were run in triplicate in a 25 μL of reaction mix containing 12.5 μL x SYBR Green Master Mix (Bio-Rad), 12.5 pmol of each forward and reverse primer and 2 μL of diluted cDNA. The PCR mixtures were incubated at 95 ◦C for 10 min, followed by 40 cycles at 95 ◦C for 15 s and 60 ◦C for 60 s. A subsequent dissociation curve analysis verified the Ct for the target gene minus the mean of the Ct for the internal control gene. The Ct represented the mean difference between the Ct of the test minus the Ct of the calibrator. The N-fold differential expression in the target gene of the test compared with the calibrator (β-actin) was expressed as 2−ΔΔCt. Data analysis and graphics were performed using GraphPad Prism 9 software (GraphPad, San Diego, CA, USA).

#### **Table 1.** Primers used for q-PCR.

#### *2.8. Statistical Analysis*

Statistical differences were determined by the analysis of variance (one-way ANOVA) followed, when significant, by an appropriate post hoc test; value of *p* ≤ 0.05 was considered statistically significant. The results are reported as mean ± standard error mean (SEM) of at least three independent experiments.

#### **3. Results**

#### *3.1. NMR Profiling and Compounds Identification*

This work represents the first report of 1H-NMR profiling of *Ficus carica* leaves. As reported in Figure 1, the main metabolites identified in the samples were both primary such as: sucrose, α-glucose, β-glucose, malic acid, alanine and valine, and secondary such as: bergapten, psoralen and a caffeic acid derivative.

The structure of the furanocoumarins (bergapten and psoralen) was confirmed by further 2D NMR experiments (Tables 2 and 3 and Supplementary Figures S1–S4) performed on a pre-purified chloroform fraction obtained by liquid/liquid partition. The leaves collected in May (FCs) showed higher content of the caffeic acid derivative, glucose and sucrose compared to the sample collected in November (FCa). Conversely, the concentration of malic acid and furanocoumarins is higher in FCa than FCs. The quantity of psoralen was always greater than that of bergapten.

**Table 3.** NMR spectral references of bergapten.

**Figure 1.** Comparison of 1H-NMR profiling of FCs and FCa: on top, entire spectrum; at the bottom, region from δ 8.6 to 6.2 (orange), region from δ 5.5 to 3.8 (brown), region from δ 3.0 to 0.4 (red) where a = bergapten, b = psoralen, c = caffeic acid derivative, d = sucrose, e = α-glucose, f = β-glucose, g = malic acid, h = alanine, i = valine.

#### *3.2. In Vitro Evaluation of Cell Viability and Biocompatibility of FCa and FCs Treatments*

From the phytochemical characterization of *Ficus carica* leaf extracts collected in autumn and spring, important differences in their composition were highlighted. Therefore, it was important to evaluate whether these variations in phytochemical composition consequently reflect different biological activities. Hence, at first, the possible effects of FC treatments on cell viability, biocompatibility and cell metabolic activity were assessed with MTT assay (Figure 2). MTT assay measures the activity of a mitochondrial enzyme and the colorimetric signal generated is an index of the number of viable cells and their metabolic activity [33].

**Figure 2.** Effect of *Ficus carica* phytoextract collected (A) in autumn (FCa) and (B) in spring (FCs) on 3T3-L1 cell viability evaluated with MTT test. Data are shown as mean ± SEM; \* *p*< 0.05 vs. control group (untreated).

Specifically, 3T3-L1 adipocytes were treated at different concentrations of FCa ranging from 25 μg/mL to 100 μg/mL for 48 h. MTT assay results showed that FCa treatment does not induce any cytotoxic effect on 3T3L-1 up to a concentration of 80 μg/mL, whereas dosages higher than 80 μg/mL resulted in cell suffering with a statistically significant decrease in cell viability (*p* < 0.05) (Figure 2A). On the contrary, FCs treatment showed good cell viability for all the concentrations tested (25, 50, 80 and 100 μg/mL) (Figure 2B) and interestingly, the lowest FCs dosage used (25 μg/mL) significantly increased cell metabolic activity (*p* < 0.05). Therefore, FC dosages at which no toxicity and good biocompatibility and cell viability was observed (25, 50 and 80 μg/mL) were selected for the following experiments.

#### *3.3. FCa and FCs Differently Modulate Lipid Accumulation in Mature Adipocytes*

To further investigate the potential of *Ficus carica* phytoextracts in modulating lipid accumulation, 3T3-L1 preadipocyte were induced to differentiate by using MDI induction medium which contained pro-adipogenic factors such as insulin, dexamethasone, and isobutylmethyl xanthine. Different concentrations of FCa and FCs (25, 50, 80 μg/mL) were added to the medium at DAY 9 to observe the effects of 48 h treatments on mature adipocytes formation and lipid droplets accumulation. Thus, adipocytes treated and untreated with FC phytoextracts were stained by Oil Red-O at DAY 11.

Figure 3 shows that FCa treatments, but not FCs, were able to induce a dose-dependent decreasing trend of adipocytes stained with Oil Red-O that specifically binds to fatty acids and lipid depots [34]. In order to semi-quantitatively assess lipid accumulation in cultured adipocytes, Oil Red-O dye was extracted with isopropanol and then evaluated spectrophotometrically. As shown in Figure 3A, FCa treatment was able to significantly reduce lipid accumulation by 20% at the lowest dosages (25 μg/mL; *p* < 0.05), up to a reduction of nearly 40% at the highest dosage used (80 μg/mL; *p* < 0.001). On the contrary, no significant differences were observed for FCs treatment for any of the dosages used (Figure 3B).

**Figure 3.** Effect of *Ficus carica* phytoextract collected (**A**) in autumn (FCa,) and (**B**) in spring (FCs) on 3T3-L1 lipid accumulation evaluated with Red O Staining. Data are shown as mean ± SEM; \* *p*< 0.05; \*\* *p*< 0.01; \*\*\* *p*< 0.001 vs. control group (untreated).

#### *3.4. FCa and FCs Modulate Genes Involved in Adipogenesis and Adipocytes Maturation*

Considering the results obtained from cell cytotoxicity and lipid accumulation, FC phytoextracts treatments at both 50 and 80 μg/mL were chosen to further investigate whether FCa and FCs were able to modulate genes involved in adipogenesis, adipocyte differentiation and lipid accumulation. In this regard, the gene expression of key adipogenesis activators such as Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) were investigated [35]. They mutually induce the expression of each other and have been reported to cooperate in the activation of a few adipogenic marker genes as Leptin, Adiponectin and GLUT4 [36]. In particular, our results demonstrated that FCa significantly decreased the gene expression of PPARγ (*p* < 0.05, Figure 4A) at both dosages used.

Considering the gene expression of C/EBPα, only a reducing trend was observed for 50 μg/mL of FCa treatment but became significant when 80 μg/mL of FCa was used (*p <* 0.01, Figure 4A). Similarly, a decreasing trend in Adiponectin gene expression was found for the 50μg/mL dosage of FCa, which became significant when FCa 80 μg/mL was tested (*p* < 0.05). FCa treatment was also able to highly reduce the gene expression of Leptin and GLUT4 at both dosages used. In details, FCa at 50 μg/mL significantly reduced the gene expression of Leptin (*p* < 0.01) and GLUT4 (*p* < 0.05) and their reduction is even more evident at higher dosage (*p* < 0.0001 and *p* < 0.01, respectively). Interestingly, FCa phytoextract was unable for any of the concentrations used to modulate the gene expression of FAS, an adipogenic enzyme involved in the de novo lipogenesis.

**Figure 4.** mRNA expressions of pro-adipogenic genes in 3T3-L1 adipocytes treated with *Ficus carica* phytoextract collected (**A**) in autumn (FCa,) and (**B**) in spring (FCs). mRNA expression of adipogenic transcription factors such as PPARγ and C/EBPα and of their related target genes as Leptin, Adiponectin and GLUT4. Additionally, the gene expression of the adipogenic enzyme FAS has been analyzed. Data are shown as mean ± SEM; \* *p*< 0.05; \*\* *p*< 0.01; \*\*\*\* *p*< 0.0001 vs. control group (untreated).

Surprisingly, FCs phytoextracts showed an opposite effect when compared to FCa (Figure 4B). In fact, cells treated with FCs at 50 μg/mL showed a significant increase in PPARγ gene expression, while for C/EBPα, an increasing trend was found. These FCs effects were even more evident with increasing dosage of treatment. FCs at 80 μg/mL was able to induce a significant increase in the gene expression of PPARγ (*p* < 0.05) and C/EBPα (*p* < 0.05) and of their related target genes as Leptin (*p* < 0.05), Adiponectin (*p* < 0.05) and GLUT 4 (*p* < 0.01). Finally, not even FC phytoextract collected in spring were able to modulate FAS gene expression. Overall, these data suggest FCa phytoextract can exert an anti-adipogenic effect while FCs have shown an opposite pro-adipogenic-like effect on adipocyte differentiation and maturation.

#### **4. Discussion**

Targeting adipose tissue represents a valuable therapeutic strategy in obesity treatment and for diabetes prevention [37]. In fact, even though many therapies already exist for the treatment of adipose tissue-related disorders, nowadays nutritional interventions are recommended as first-line treatments and, among them, botanicals are considered promising coadjutants [18,38]. Despite the contradictory findings found in the literature [39], several studies have reported that *Ficus carica* L. extracts are endowed with potential health benefits against obesity-associated metabolic disorders and diabetes, but their mechanism of action is still debated [14,19] and the compounds responsible for the bioactivities have not yet been identified. Moreover, the plant metabolome is strongly affected by a number of biotic and abiotic factors, including seasonal variation [27]. The fluctuation in plant metabolome can be consequently reflected in their biological activities. In case of *F. carica* leaves, variations in its metabolites across the seasons were already reported. In particular, Innocenti et al. analyzed samples harvested from June to August, finding an increase in total coumarins content in *F. carica* leaves harvested in August [40]. Conversely, Marrelli et al. found that samples collected in June were endowed with a higher concentration of psoralen and bergapten than the ones collected in September [41].

This work was not designed to generically monitor the seasonal variation in *F. carica* metabolome, in fact, only two time points were chosen, therefore it is not possible here to draw general conclusions on seasonal variations. However, the 1H-NMR analysis suggested that coumarin content in the autumn extracts is higher than in the spring, while in the spring extracts there is a higher content of a caffeic acid derivative. The two samples not only showed differences in terms of phytochemical profile but also in their biological activity. In fact, in this study, only *F. carica* leaves harvested in autumn (FCa) were able to reduce lipid accumulation inside mature adipocytes. Moreover, FCa extract was able to modulate the expression of many genes involved in adipocyte differentiation, lipid accumulation and metabolism, whereas, notably, this occurs with opposite effects for *F. carica* leaves harvested in spring (FCs). In particular, in this work it has been demonstrated that FCa decreased the gene expression of PPAR-γ, the master regulator of adipogenesis, while FCs increased its expression. PPAR-γ plays a very important role, not only in adipocytes' differentiation but also in lipid and glucose homeostasis [42]. It is well documented that compounds able to reduce PPAR-γ expression have anti-obesity effects [43]. In mature adipocytes, the reduced gene expression of PPAR-γ may contribute to decreased lipid accumulation, as also confirmed by our results in Red O Staining.

FC extracts were also found able to modulate the gene expression of C/EBP-α, another key adipogenic gene that acts as a marker of adipocytes differentiation and remains elevated during cell life [42]. Interestingly, C/EBP-α has a role in promoting lipid accumulation, thus the inhibition of C/EBP-α can aid in reducing lipid content. Our data showed that C/EBPα expression was oppositely and significantly modulated according to FC's seasonality, but only at the highest concentration (80 μg/mL) of both treatments. In detail, after 48 h treatment, a reduction in C/EBP-α gene expression was observed when FCa was used, while a decreasing tendency can also be noticed at the lower dosage. Differently, when leaves were harvested in spring, C/EBP-α gene expression was found to increase after 48 h of treatments.

Adipose tissue is also endowed with secretory properties as a huge number of proteins and other factors with biological activities can be secreted from adipocytes such as circulating hormones, pro-inflammatory and anti-inflammatory proteins [44]. Among these secreted factors, adipokines were found to play a role in the development of a metabolic syndrome and in adipose tissue expansion [45].

Among adipokines, we explored the potentiality of FC extracts in Adiponectin and Leptin gene expression modulation. Regarding Adiponectin, FCa caused a significant reduction in its expression only when the 80 μg/mL dosage of FCa was used, while at lower dosage (50 μg/mL) just a decreasing trend was observed. On the other thand, Adiponectin gene expression was increased after treating cells with FC harvested in spring at the highest dosage used. Adiponectin is also a pro-adipogenic marker [46], thus its reduction suggests an anti-adipogenic effect, while an opposite pro-adipogenic like effect can be attributed to its increased expression. These data supported that FC-dependent modulation of Adiponectin is upstream mediated by PPAR-γ modulation. In fact, it is well reported in the literature that adiponectin gene expression is under the control of PPAR-γ and C/EBP-α [46,47]. Of note, the seasonal opposite adipogenic effect of FC was also confirmed in the Leptin and GLUT4 gene expression data. Leptin is one of the adipokines responsible for regulating energy intake and expenditure [48]. The most wellknown effect of Leptin is to regulate body weight, the feeding behavior of the animal, and energy balance [49], but it also has fundamental roles in tissue remodeling, inflammation, and glucose and lipid homeostasis [50,51]. GLUT-4 is the glucose transporter that is responsible for glucose uptake and whose function is disturbed in some conditions such as insulin resistance and type 2 diabetes [52]. During adipocytes differentiation, the gene expression of GLUT-4 favors glucose uptake and, in turn, glucose metabolism [53]. Thus, pharmacological drugs or natural compounds inhibiting both Leptin and GLUT-4 are highly desired for the treatment of obesity and diabetes. Interestingly, *F. carica* extracts did not modulate fatty acid synthase (FAS) levels, either when harvested in spring or in

autumn. FAS is involved in the de novo lipogenesis [54] and, interestingly, it is not under the control of C/EBP-α-PPAR-γ axis [55,56], corroborating the hypothesis that PPAR-γ expression is a specific target gene of FC.

Overall, according to our results, FCa showed effective anti-adipogenic activities, by reducing lipid accumulation when 3T3-L1 mature adipocytes were treated for 48 h with FCa. These effects can be related to the FC phytochemicals content as demonstrated by the NMR spectra. In fact, most characterizing components, such as furanocoumarins were present only in FCa, which showed anti-obesity potentiality, while their absence was associated with reduced effectiveness: an almost opposite effect was observed. Furthermore, FCs NMR spectrum also revealed a higher presence of sucrose and glucose, two well-known energy sources involved in increasing the expression of pro-adipogenic regulators and fat storage [53,57], suggesting a highly relevant contributing role in exerting FCs effects on 3T3-L1 cells.

The data obtained in this work showed the promising activity of *F. carica* harvested in autumn as a potential source of anti-obesity and anti-adipogenic compounds, pointing out that this activity is strongly dependent on its phytochemical profile. These data lay the foundation for future investigations to fully explain seasonal variation in F. carica leaves metabolome, and how it could be reflected in the modulation of adipogenesis. Further biological experiments could also be conducted in vivo to investigate the possible adverse effects due to furanocoumarins content. In fact, different in vitro and in vivo studies have also demonstrated that furanocoumarins possess positive biological activities such as anti-inflammatory and anti-oxidative activities and bone health promoting effects [58,59]. However, they can also cause undesirable effects due to interactions with certain medications or by inducing phototoxicity [60,61].

In conclusion, this study further supports an in-depth investigation of *F. carica* leaves extract as a promising source of active compounds useful for targeting obesity and diabetes. However, whether the compounds contribute individually or accumulatively to the traditionally believed "medicinal" attributes of the plant is still unclear and under research.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nu14142833/s1. Figure S1: HSQC spectrum of *Ficus carica* chloroform fraction where 1H-13C correlations of bergapten (blue) and psoralen (green) are highlighted. Figure S2: HMBC spectrum of *Ficus carica* chloroform fraction where 1H-13C correlations of bergapten are highlighted. Figure S3: HMBC spectrum of *Ficus carica* chloroform fraction where 1H-13C correlations of psoralen are highlighted. Figure S4: COSY spectrum of *Ficus carica* chloroform fraction where 1H-1H correlations of bergapten (blue) and psoralen (green) are highlighted.

**Author Contributions:** Conceptualization, M.P.; G.A.; A.M. and M.M. (Manuela Mandrone); investigation, M.P.; G.A.; I.C. and M.M. (Manuela Mandrone); formal analysis, M.P.; G.A. and A.M.; writing—original draft preparation, M.P.; G.A.; A.M. and M.M. (Manuela Mandrone); writing review and editing, E.M.S.; E.T.; I.C.; D.U.; M.M. (Maurizio Memo) and F.P.; supervision, A.M.; project administration, G.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** Giulia Abate benefits from funding FSE-REACT-EU, within the program PON "Research and Innovation" 2014–2020 (PON R&I), DM1062, Action IV.6 "Contratti di ricerca su tematiche Green".

**Data Availability Statement:** Data are available upon request to the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**2. The concept of EDCs**

Originally articulated in the early 1990s by Colbron [2], the theory of *endocrine disruption* referes to exogenous chemicals present in the environment and/or diet that interfere and disrupt physiological hormonal systems, inducing adverse effects on human and wildlife health. The term "endocrine disruptor" was first used at the Wingspread Conference in Wisconsin, USA in 1991 for those EDCs, which may lead to an adverse health effect [3]. Initially the term focused on chemicals with estrogenic activity that would alter reproductive function, commonly referred to as environmental estrogens or xenoestrogens; it has expanded to compounds with androgenic activity, as well as thyroid-active chemicals [4]. Consequently, different variable terms appeared, e.g. endocrine disrupter or endocrine disruptor, hormone mimics, hormone inhibitors or endocrine modulators [5]. Today, these compounds are commonly referred to as EDCs.

Various attempts to set up a scientific definition of an EDC have been made. Today is generally wide acceptance of using the WHO definition [6], which is similar with the EU definition [7]: "*An endcorine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations".*

EDCs interfere and disrupt physiological hormonal balance inducing adverse effects on human health through different mechanisms including direct interaction with hormone receptors, competition on binding and transport proteins or interference with hormone metabolism (blocking or inducing the synthesis of the hormones). Starting from this definition there are clearly two requirements for a substance to be defined as an EDCs, namely the demonstration of an adverse effect and an endocrine disruption mode-of-action. Additionally, the definition implies proof of causality between the observed adverse effect and the endocrine disruption mode-of-action.

It is important to underline that the definition of EDC includes the term "adverse" which was considered as a key criterion to differentiate a genuine EDC from a mere endocrine modulator (that elicits an adaptative reversible response in endocrine homeostasis). According to WHO the term "adversity" means: "*a change in morphology, physiology, growth, reproduction, develop‐ ment or lifespan of an organism which results in impairment of functional capacity or impairment of capacity to compensate for additional stress or increased susceptibility to the harmful effects of other environmental influences*." [8].

This definition is not covering the potential or indicates EDC. A potential (or suspected) EDC may alter function(s) of the endocrine system and consequently may cause adverse health effects, while a substance with indication of endocrine disrupting properties (called indicated EDC) might be expected to lead to endocrine disruption in an intact organism, or its progeny, or (sub)populations.

The need for an expansion of the general term to potential EDCs is reflected on the new Guidance document on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption (OECD 2011) [9]: "*A possible endocrine disrupter is a chemical that is able to alter the* *functioning of the endocrine system but for which information about possible adverse consequences of that alteration in an intact organism is uncertain*".

**2. The concept of EDCs**

220 Treatment of Type 2 Diabetes

EDCs.

*(sub)populations".*

disruption mode-of-action.

*environmental influences*." [8].

or (sub)populations.

Originally articulated in the early 1990s by Colbron [2], the theory of *endocrine disruption* referes to exogenous chemicals present in the environment and/or diet that interfere and disrupt physiological hormonal systems, inducing adverse effects on human and wildlife health. The term "endocrine disruptor" was first used at the Wingspread Conference in Wisconsin, USA in 1991 for those EDCs, which may lead to an adverse health effect [3]. Initially the term focused on chemicals with estrogenic activity that would alter reproductive function, commonly referred to as environmental estrogens or xenoestrogens; it has expanded to compounds with androgenic activity, as well as thyroid-active chemicals [4]. Consequently, different variable terms appeared, e.g. endocrine disrupter or endocrine disruptor, hormone mimics, hormone inhibitors or endocrine modulators [5]. Today, these compounds are commonly referred to as

Various attempts to set up a scientific definition of an EDC have been made. Today is generally wide acceptance of using the WHO definition [6], which is similar with the EU definition [7]: "*An endcorine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or*

EDCs interfere and disrupt physiological hormonal balance inducing adverse effects on human health through different mechanisms including direct interaction with hormone receptors, competition on binding and transport proteins or interference with hormone metabolism (blocking or inducing the synthesis of the hormones). Starting from this definition there are clearly two requirements for a substance to be defined as an EDCs, namely the demonstration of an adverse effect and an endocrine disruption mode-of-action. Additionally, the definition implies proof of causality between the observed adverse effect and the endocrine

It is important to underline that the definition of EDC includes the term "adverse" which was considered as a key criterion to differentiate a genuine EDC from a mere endocrine modulator (that elicits an adaptative reversible response in endocrine homeostasis). According to WHO the term "adversity" means: "*a change in morphology, physiology, growth, reproduction, develop‐ ment or lifespan of an organism which results in impairment of functional capacity or impairment of capacity to compensate for additional stress or increased susceptibility to the harmful effects of other*

This definition is not covering the potential or indicates EDC. A potential (or suspected) EDC may alter function(s) of the endocrine system and consequently may cause adverse health effects, while a substance with indication of endocrine disrupting properties (called indicated EDC) might be expected to lead to endocrine disruption in an intact organism, or its progeny,

The need for an expansion of the general term to potential EDCs is reflected on the new Guidance document on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption (OECD 2011) [9]: "*A possible endocrine disrupter is a chemical that is able to alter the* Recently, the Endocrine Society published a statement of principles on endocrine disruptors [10] in which another definition of an EDC has been proposed: *"An EDC is an exogenous chemical, or mixture of chemicals, that interferes with any aspect of hormone action."* This definition empha‐ sizes that the ability of a chemical to interfere with hormone action is of itself a reliable predictor for adverse outcomes.

A wide variety of chemicals act as EDCs. The Endocrine Disruption Exchange List (TEDX) to date lists almost 1000 endocrine disruptors [11]. The group of EDCs is highly heterogeneous and includes synthetic chemicals used as industrial solvents/lubricants and their by-products (persistent organic pollutants – POPs, dioxins like 2,3,7,8-tetrachlorodibenzo-p-dioxin – TCDD), plastics (bisphenol A – BPA), pesticides (as dichlorodiphenyltrichloroethane – DDT), plasticizers (phthalates like diethylhexyl phthalate – DEHP), heavy metals (arsenic, cadmium), tributyl tin (TBT), fungicides (vinclozolin) and pharmaceutical agents (diethylstilbestrol – DES). POPs comprise a broad class of organohalides, including polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs) and organochlorine pesticides.

However, one should keep in mind that there is also a large number of EDCs of natural origin occurring in plants consumed as food and also some secondary metabolites from fungi that may contaminate food. Examples of EDCs with oestrogenic activity are present in soy (e.g. genistein and daidzein), mycotoxins in cereals (e.g. zearalenone), goitrogens in cabbage (with the potential to inhibit iodine uptake) and glycirrhizine in liquorice (with the potential to disturb the mineralocorticoid system) [12].

EDCs are widely dispersed in the environment. Some are persistent, having long half-lives [13], while others are rapidly degraded in the environment or human body or may be present for only short periods of time but at critical periods of development, considered windows of susceptibility.

The effects of EDCs are observed especially on sensitive groups (foetus and child), based on their susceptibility to hormonal effects [14]. Therefore, for these groups the adverse effects may occur at concentrations that are far below levels that would be considered harmful in the adult [15]

Of a special concern are EDCs such as phthalates [16], PBBs [17] and BPA, detected in pregnant women, fetuses and newborns, taking into account that exposure occuring early in pregnancy can have short-term health effects, while exposure later or during early childhood may induce cognitive and developmental deficiencies [18].

Published studies illustrate that *in utero* developmental period is a critically sensitive window of vulnerability. Disruptions during this time-frame can lead to subtle functional changes that may not emerge until later in life [19] or even later to the next generation.

Actually, is considered that *in utero* and early postnatal exposures play an important role in the development of reproductive defects, obesity and metabolic syndrome as a result of inheritable chemical-induced epigenetic changes [20, 21, 22]. As an example of epigenetic change involved in reproductive defects one can note the developmental exposure to vinclo‐ zolin causing an increase in spermatogenic cell apoptosis in the adult rats [23]. This sperma‐ togenic defect was found to be transgenerational at least to F4 generation due to a permanent altered DNA methylation of the male germ-line [21].

**Table 1.** Classification of EDCs based on Danish criteria

Many other chemicals have also been implicated in promoting toxicity for multiple genera‐ tions, including BPA [24] or pesticides [25], but in these cases the multigenerational effects involved direct exposures, therefore are not considered transgenerational because they are not transmitted solely through the germ cells. Only effects appearing in the F3 generation are considered to be truly transgenerational [26].

There are multiple classifications of EDCs, based on their mechanisms of action (on enzymes, transport proteins or receptors), the pathways modulated (e.g., xenoestrogens/antiestrogens, xenoandrogens/antiandrogens), or the biological outcomes (simulation or inhibition) [27]. However, the best classification is the Danish criteria, based on available data (*in vitro, in vivo* and epidemiological studies), which is scientifically legitimated by Hass et al. [28]. Using this approach, EDCs are divided into 3 categories: *category 1,* for substances known to produce endocrine disrupting adverse effects in humans or animal species, *category 2a* for suspected EDCs, and *category 2b* for indicated EDC. Details regarding classification criteria are included in Table 1.


**Gene-Wide Analysis of Aquaporin Gene Family in** *Malus domestica* **and Heterologous Expression of the Gene** *MpPIP2;1* **Confers Drought and Salinity Tolerance in** *Arabidposis thaliana*

#### **Haili Liu 1,**† **, Leilei Yang 1,**† **, Miaomiao Xin 1 , Fengwang Ma 2 and Jingying Liu 1,\***

Received: 24 June 2019; Accepted: 26 July 2019; Published: 29 July 2019

**Abstract:** The aquaporins (AQPs) are a family of integral membrane proteins involved in the transcellular membrane transport of water and other small molecules. A scan of the apple (*Malus domestica*) genome revealed the presence of 42 genes encoding putative AQPs. Based on a phylogenetic analysis of the deduced peptide sequences of the AQPs generated by *Arabidopsis thaliana*, poplar (*Populus trichocarpa*), and rubber (*Hevea brasiliensis*), the apple AQPs were each assigned membership of the five established AQP subfamilies, namely the PIPs (eleven members), the TIPs (thirteen members), the NIPs (eleven members), the SIPs (five members), and the XIPs (two members). The apple AQPs included asparagine-proline-alanine (NPA) motifs, an aromatic/arginine (ar/R) selectivity filter, and the Froger's positions. The heterologous expression of *MpPIP2;1* in *A. thaliana* was shown to enhance the level of tolerance exhibited against both drought and salinity.

**Keywords:** apple; aquaporin; functional analysis; stress tolerance


**6.2. IDH1/2 mutations**

IDHs catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate with production of NADH/NADPH and they are involved in the Krebs cycle.

Recurrent somatic point mutations affect the arginine (Arg) residue at codon 132 in the IDH1 gene on chromosome 2q33.3. Less frequently, they occur at the homolog Arg (R) residue at codon 172 in the IDH2 gene on chromosome 15q26.1. The IDH1/2 mutation rate is in the range of 70– 80% in OII and OIII [50–53] and less in OAII and OAIII, with a higher frequency in 1p/ 19q co-deleted tumors [37]. IDH1 mutations prevail in astrocytic tumors whereas IDH2 mutations are more common in oligodendroglial tumors [51, 54].

In low grade gliomas, they are prognostic favorable factors [53, 55, 56].

The c.395G>A (p.R132H) mutation can be easily detected by a anti-IDH1R132H mutation-specific antibody by immunohistochemical techniques [53, 57]. Tumor cells show IDH1R132H immuno‐ positivity in their cytoplasms, whereas reactive astrocytes and normal glia cells are negative. This is particularly evident in the picture of cortical perineuronal satellitosis where positive (tumor) and negative (normal) satellites can be found, at the beginning of invasion.

## *6.2.1. ATRX mutations*

The ATRX gene is located on chromosome Xq21.1, contains 35 exons, and encodes a 2,492 amino acid protein. ATRX belongs to the H3.3-ATRX-DAXX chromatin remodeling path‐ way, involved in chromatin stabilization [58]. ATRX and its binding factor death-associated protein 6 (DAXX) incorporates the histone protein H3.3 into the nucleosome at telomeres and pericentric heterochromatin [59, 60]. Alterations of this function lead to loss of structural integrity at telomeres leading to tumorigenesis. In fact, ATRX or DAXX protein loss is associated to the alternative lengthening of telomeres (ALT), a telomerase-independent mechanism of telomere lengthening [61–66].

Germline ATRX mutations give rise to a syndrome characterized by severe mental retarda‐ tion [67] and to α-thalassemia.

Somatic ATRX mutations occur in gliomas of different types and histologic grades [38, 61–65, 68–70]. They are more frequent in grade II (67%) and in grade III (73%) astrocytic tumors and in secondary GBMs (57%), as well as in mixed gliomas (25% in grade II and 27–53.8% in grade III tumors) [64, 65, 69, 70]. In contrast, they are rare in primary GBMs (4%), pediatric GBMs (20%) and in pure oligodendroglial tumors (<10%) [63, 64, 68]. Very importantly, ATRX mutations do not affect pilocytic astrocytomas [64].

ATRX mutations occur in 70% of IDH mutant and intact 1p/19q low grade gliomas [62, 64, 65]. Restricted to IDH mutant tumors, they are significantly associated to TP53 mutations and nuclear p53 overexpression and to astrocytic differentiation; they are mutually exclusive with 1p/19q co-deletion [71, 72]. ATRX and IDH1/2 mutations occur in association and they may represent early genetic alterations in the development of gliomas affecting progenitors before their differentiation along the two lineages.

In pediatric gliomas, all ATRX mutations cluster nearthe C-terminal helicase domains; in adult tumors, they are evenly distributed across the gene, mainly as frameshift mutations leading to truncated proteins [63, 64, 69].

The relatively large size of the ATRX gene makes the mutation analysis difficult to be applied in the routine diagnostic procedures. The immunohistochemical evaluation of the ATRX protein expression could represent an alternative method to assess the ATRX status. Although studies reported concordant results between the mutation analysis and IHC [38, 72], tumor heterogeneity in the ATRX expression and concurrent normal non-tumor cells with constitu‐ tive ATRX expression may explain possible discrepancy. As a matter of fact, ATRX muta‐ tions/ATRX protein loss characterizes astrocytic gliomas, whereas retained ATRX immunoreactivity characterizes oligodendroglial gliomas. Referred to OA, the former is typical of the astrocytic component while the latter of the oligodendroglial one [71].

## *6.2.2. ATRX and prognosis*

**6.2. IDH1/2 mutations**

346 Neurooncology - Newer Developments

*6.2.1. ATRX mutations*

mechanism of telomere lengthening [61–66].

mutations do not affect pilocytic astrocytomas [64].

before their differentiation along the two lineages.

tion [67] and to α-thalassemia.

IDHs catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate with production

Recurrent somatic point mutations affect the arginine (Arg) residue at codon 132 in the IDH1 gene on chromosome 2q33.3. Less frequently, they occur at the homolog Arg (R) residue at codon 172 in the IDH2 gene on chromosome 15q26.1. The IDH1/2 mutation rate is in the range of 70– 80% in OII and OIII [50–53] and less in OAII and OAIII, with a higher frequency in 1p/ 19q co-deleted tumors [37]. IDH1 mutations prevail in astrocytic tumors whereas IDH2

The c.395G>A (p.R132H) mutation can be easily detected by a anti-IDH1R132H mutation-specific antibody by immunohistochemical techniques [53, 57]. Tumor cells show IDH1R132H immuno‐ positivity in their cytoplasms, whereas reactive astrocytes and normal glia cells are negative. This is particularly evident in the picture of cortical perineuronal satellitosis where positive

The ATRX gene is located on chromosome Xq21.1, contains 35 exons, and encodes a 2,492 amino acid protein. ATRX belongs to the H3.3-ATRX-DAXX chromatin remodeling path‐ way, involved in chromatin stabilization [58]. ATRX and its binding factor death-associated protein 6 (DAXX) incorporates the histone protein H3.3 into the nucleosome at telomeres and pericentric heterochromatin [59, 60]. Alterations of this function lead to loss of structural integrity at telomeres leading to tumorigenesis. In fact, ATRX or DAXX protein loss is associated to the alternative lengthening of telomeres (ALT), a telomerase-independent

Germline ATRX mutations give rise to a syndrome characterized by severe mental retarda‐

Somatic ATRX mutations occur in gliomas of different types and histologic grades [38, 61–65, 68–70]. They are more frequent in grade II (67%) and in grade III (73%) astrocytic tumors and in secondary GBMs (57%), as well as in mixed gliomas (25% in grade II and 27–53.8% in grade III tumors) [64, 65, 69, 70]. In contrast, they are rare in primary GBMs (4%), pediatric GBMs (20%) and in pure oligodendroglial tumors (<10%) [63, 64, 68]. Very importantly, ATRX

ATRX mutations occur in 70% of IDH mutant and intact 1p/19q low grade gliomas [62, 64, 65]. Restricted to IDH mutant tumors, they are significantly associated to TP53 mutations and nuclear p53 overexpression and to astrocytic differentiation; they are mutually exclusive with 1p/19q co-deletion [71, 72]. ATRX and IDH1/2 mutations occur in association and they may represent early genetic alterations in the development of gliomas affecting progenitors

(tumor) and negative (normal) satellites can be found, at the beginning of invasion.

of NADH/NADPH and they are involved in the Krebs cycle.

mutations are more common in oligodendroglial tumors [51, 54].

In low grade gliomas, they are prognostic favorable factors [53, 55, 56].

Patients harboring ATRX mutations would show a better outcome [65]. ATRX has important prognostic implications in anaplastic gliomas [65]. ATRX loss is a prognostic factor in IDH mutant and non 1p/19q co-deleted low grade gliomas [62, 65]. In GBMs, ATRX loss affects younger patients [72].