Table of Contents

Original ArticlesCorrelations between MHLC Scores and Indicators of Immune Response in Egyptian Women with Breast Cancer .............. .............. 07Eman M. EL-Baiomy, Mohamed L. Salem, Azza El-Amir, Noha A. Sabry, Kenneth A. Wallston, Nehal EL-Mashad

Open label, non-randomized, interventional study to evaluate response rate after induction therapy with docetaxel and cisplatin in locally advanced squamous cell carcinoma of oral cavity ........................................................................................... 12S. H. Manzoor Zaidi, Ahmad Ijaz Masood, Syed Ijaz Hussain Shah, Irfan Hashemy

The Association Between Clinicopathological Features and Molecular Markers in Bahraini Women with Breast Cancer .................. 19Aysha AlZaman, Eman Ali, Bayan Mohamad, Moinul Islam, Entisar AlZaman, Yahya AlZaman

Immunohistochemical Staining for Ras-Related Protein 25 (RAB25) Associates with Luminal B Breast Cancer Subtype .................. 26Amina Belhadj, Lynda Addou-Klouche, Issam Bouakline, Miloud Medjamia, Hamid Jelloul Benammar, Tewfik Sahraoui

Bibliometric and Comparative Analysis of Castration Resistant and Refractory, Hormone Resistant and Refractory Prostate Cancer Publications .......................................................................................................... 34Selahattin Çalışkan, Alkan Çubuk, Abdullah Ilktac

ALK gene rearrangement status in non-squamous non-small cell lung carcinoma in the Middle Eastern population ..................... 38Samah El Naderi, Rosy Abou-Jaoude, Marc Rassy, Hussein Nasreddine, Elie El Rassy, Claude Ghorra

Micronucleus Test for Diagnosing Uncertain Cases (BI-RADS 3) in Breast Cancer Screening: A Review and Preliminary Results ... 45Roberto Menicagli, Ortensio Marotta, Roberta Serra

Preoperative Leukocytosis as a Prognostic Marker in Endometrioid-Type Endometrial Cancer: A Single-Center Experience from Saudi Arabia .................................................................................................................................................................. 51Hany Salem, Ahmed Abu-Zaid, Osama Alomar, Mohammed Abuzaid, Mohammad Alsabban, Tusneem Elhassan, Abdullah Salem,

Yahya Alyamani, Ismail A. Al-Badawi

Review ArticlesA Quick Review of Redox State in Cancer: Focus to Bladder ................................................................................................................... 59Hamid Mazdak, Mehdi Gholampour, Zahra Tolou_Ghamari

Case ReportsAbdominoscrotal Lymphangioma Masquerading as a Communicating Hydrocele: A Case Report ....................................................... 63Ahmed Al Rashed, Zarine Gazali, Vijay Kumar Malladi, Arbinder Kumar Singal

Hurthle Cell Adenoma with Micro-Papillary Carcinoma and Parathyroid Adenoma in a Transplant Recipient with Graft Failure: A Case Report ...................................................................................................................................................................... 66Shameema Sharfudeen, Tasneem Amir, Waddah Eskaf, Mahmoud Elsayed Ghanem, Aysha Al Jassar, Kusum Kapila

Feature ArticleThe State of Cancer Care in the United Arab Emirates in 2020: Challenges and Recommendations, A report by the United Arab Emirates Oncology Task Force .................................................................................................................... 71 Humaid Al-Shamsi, et. al.

Conference Highlights/Scientific Contributions• Highlights of “Management of Breast and Colorectal Cancer: Recent Updates” Kuwait Conference .............................................. 88

• News Notes ........................................................................................................................................................................................... 92

• Scientific events in the GCC and the Arab World for 2020 ................................................................................................................. 96

26

Corresponding author: Amina BELHADJ, Doctor, Biology of development and differentiation laboratory.

Oran1 University. Ahmed Ben Bella, Oran, Algeria. belhadj_ami@yahoo.fr

Abstract

Introduction: Luminal B breast cancer is associated with a poor prognosis and resistance to hormone therapy. Studies have suggested that Ras-related protein 25 (RAB25), a member of Rab small GTPase family, is involved in breast cancer pathogenesis. Our aim in the present study is to analyze the association between RAB25 protein expression and clinical and pathological characteristics, and to investigate whether the expression of RAB25 was associated with a specific molecular subtype of breast cancer.

Materials and Methods: A retrospective study was conducted regarding female patients diagnosed with breast cancer; clinicopathologic data was obtained from medical reports. RAB25 expression was evaluated by immunohistochemistry in 57 primary breast cancer samples. The results were correlated with clinicopathologic variables and different breast cancer molecular subtypes.

Results: RAB25 expression was significantly associated with tumors expressing oestrogen receptor (P=0.03). A high significant difference was observed by analyzing RAB25 expression in various breast cancer subtypes (P=0.01). RAB25 expression was found in 66.7% of Luminal B breast tumors, considered as the most aggressive hormone dependent mammary tumors and was strongly associated with luminal breast cancer subtypes (p=0.004) but not with age, tumor size, SBR grade, axillary lymph node, or tumor stage.

Conclusion: RAB25 deregulated expression is most common in luminal B breast cancer tumors suggesting that RAB25 could be a potential therapeutic target for this molecular subtype.

Keywords: RAB25, Breast cancer, molecular subtypes, Luminal B, Therapeutic target

Original Article

Immunohistochemical Staining for Ras-Related Protein 25 (RAB25) Associates with Luminal B Breast

Cancer SubtypeAmina Belhadj1, Lynda Addou-Klouche2, Issam Bouakline 3, Miloud Medjamia3,

Hamid Jelloul Benammar4, Tewfik Sahraoui1

1 Biology of Development and Differentiation Laboratory. Oran1 University. Ahmed Ben Bella, Oran, Algeria 2 Biotoxicology Laboratory, Department of Biology. Djilali Lyabes University, Sidi Bel Abbes, Algeria

3 Department of Anatomy and Pathology, Regional Military Hospital University. Oran, Algeria. 4 Anatomy and Pathology Laboratory, Sidi Bel Abbes, Algeria.

IntroductionMolecular subtyping of breast cancer has identified

hormone dependent subtypes divided into two categories: luminal A and luminal B subtypes [1]. Luminal B tumors are highly proliferative, resistant to standard therapies [2] and are poorly understood despite identification of some tumor suppressor genes such as L3MBTL4 [3]. Although they express hormone receptors, their metastatic risk and resistance to hormone therapy and to conventional chemotherapy need to identify new therapeutic targets that could lead to the development of appropriate therapies in this aggressive breast cancer molecular subtype.

RAB proteins family is intracellular transport proteins that belong to the Ras superfamily of small GTPases [4]. Some of processes that induct to invasion and metastasis are controlled by members of the deregulated RAB family [5]. Ras-related protein 25 (RAB25) is a recently discovered RAB family member [6]. Overexpression of RAB25 has been reported in some human solid tumors such as: colon cancer [7], liver cancer [8], and bladder cancer

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[9,10]. Recent studies suggested that RAB25 expression can alter the behavior of breast and ovarian cancer cell lines [11]. Cheng et al [12] revealed a high RAB25 expression in ER-positive samples. This suggests that RAB25 is implicated in hormone positive breast cancer.

In this context we proceed to breast cancer molecular subtyping and then assessed the protein level of RAB25 in these molecular subtypes then we investigated whether RAB25 protein expression was specifically associated with luminal B breast cancer subtype. To our knowledge our study is the first to identify a deregulated expression of RAB25 in luminal B breast tumors in Algeria.

Materials and Methods

Breast tumors and general information

A total set of 57 formalin fixed paraffin-embedded tumors were collected from women who underwent radical mastectomy and were diagnosed with invasive breast carcinoma in west Algerian hospital from January 2009 to December 2014. The main clinical characteristics of tumors were recorded by reviewing patient’s medical reports. The patients were female aged from 27-88 years with a median age of 54 years. Each patient gave written informed consent and the study was approved by our institutional review committee.

Antibodies and reagents

The following antibodies, were used in this study: rabbit polyclonal anti-Rab25 (Sigma Aldrich), Monoclonal Mouse Anti Human Ki67 Antigen (Dako; Clone MIB-1), Monoclonal Mouse Anti Human Estrogen Receptor α (Dako; clone 1D5), Monoclonal Mouse Anti-Human Progesterone Receptor (Dako; clone PgR 636), Monoclonal Mouse Anti-Human HER2-pY-1248 (Dako, clone PN2A) at a dilution of 1:200; 1:100; 1: 100 , 1: 100 , 1:100 respectively.

Immunohistochemistry staining

The formalin-fixed paraffin-embedded tumor tissue slides were first deparaffinized twice in xylene for 10 minutes each and hydrated twice in alcohols for 10 minutes each, washed once in H2O for 5 minutes, incubated in target retrieval solution Citrate (pH 6.0) in a boiling water-bath for 50 minutes. The slides were cooled for 20 minutes. Following washing in phosphate-buffered saline (PBS), slides were incubated with 3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase activity and washed again in H2O for 5 minutes and then incubated with a blocking serum for 10 minutes.

The slides were incubated in primary antibody for 1 hour, washed in PBS for 5 minutes, then incubated in

secondary antibody (Dako REAL™ EnVision™ Detection System) at room temperature for 30 minutes. The slides were washed and developed in 3.30-diaminobenzidine (Peroxidase DAB, Rabbit Mouse purchased from Dako.) under microscopic observation. The reaction was stopped in tap water and the tissues were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. The images were taken using Leica DFC 280 (Leica DM LB2). The evaluation of the IHC was conducted blindly by H.B and HJ.B.

Immunohistochemistry scoring

The classification of positive immunoreactivity in tumor cells was carried out according to the percentage of immunopositive cells: <10% was classified as negative and > 10% as positive for ER and PR nuclear markers.

Membranous immunoreactivity of HER2 was scored as follows: 0 and 1+ indicates negative; 2+, indeterminate; and 3+, positive for overexpression. Ki67 expression was assessed to distinguish between luminal A and luminal B breast cancer. The cut off Ki67 value was ≥14% as previously reported [13]

For RAB25 an H score was calculated by multiplying the fraction of positively stained tumor (percentage) by staining intensity (0, 1+, 2+, or 3 +). In subsequent statistical analyses, H scores were classified as negative (0 to 9), low (10 to 100), medium (101to 200), or high (201 to 300) as previously describe [14], only cytoplasmatic staining was considered as positive.

Definition of breast cancer molecular subtypes

Breast cancer molecular subtypes were defined by IHC receptor statues as follows: Luminal A (ER+ and/or 0PR+, HER2 +/-, Ki 67<14%); Luminal B (ER+ and/or PR+, HER2+/-, Ki 67 ≥ 14%), Triple negative breast cancer ( ER-/PR-/HER2-) and HER2+ subtype ( ER-/PR-/HER2+) as previously described. [13]

Statistical analysis

All statistical analysis was processed using SPSS 25 (Statistical Package for the Social Sciences, IBM Corporation; Chicago, IL). Results are expressed as means ± standard deviations and percentages. Independent Student’s t-test was used for comparing continuous variables. The Chi-Square test was used to estimate the relation¬ship between RAB25 expression and the main clinicopathological characteristics such as axillary lymph node status (positive vs. negative), pathological tumor size as measured in surgery, SBR grade (I, II, III) and the molecular subtypes (luminal A, luminal B, HER2 subtype, TNBC). Statistical significance was determined with P < .05.

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High RAB25 expression in luminal B breast cancer subtype, Amina Belhadj, et. al.

Results

Molecular classification of breast cancer tumors

We evaluated Ki67 expression on patients with hormone-positive tumors to differentiate between luminal A and luminal B tumors. Of the 57 breast tumors: 21.05 % were luminal A subtype, 36.84% luminal B subtype, 28.07 % were TNBC subtype and 14.03 % were HER2+ subtype.

Patients’ characteristics

Our survey has been done over 57 patients, detailed clinical and pathologic information was available in most cases and are shown in Table 1. The mean age was about 48.15 years, ranging from 27 to 88 years old. Patients were relatively young (below 40 years old) with rate of 21.05%. Tumor size varied from 0.4cm to 13cm, 48 tumors were larger than 2cm (84.21%) and 9 tumors were 2cm or less in size (15.79%) cases. We histologically classified breast tumors by using the 2003 WHO classification system. The histologic findings were as follows: 89.47% ductal carcinomas, 5.26% lobular carcinomas and 5.27% of cases represents the other uncommon types. There were 34 early-stage tumors diagnosed in grade 2 (59.65%), and 21 advanced-stage tumors diagnosed in grade 3 (36.84%). At diagnosis the axillary lymph node metastases were present in 37 tumors (64.91%). Immunostaining of steroid receptors (estrogen and progesterone receptors) shows that ER and PR positivity were identified in 31 (55.36%) and 27 (48.21%) tumors respectively, ER and PR negativity were found in 25 (44.64%) and 29 (51.79%) tumors respectively. In addition, we further analyzed if there was any relationship between age at diagnosis and tumors which expressed hormone receptors (Classified as luminal A and luminal B), statistical analysis indicated a highly significant difference among luminal A and luminal B tumors for the risk factor of the age (P=.006) (Table 2).

RAB25 association with clinical and molecular characteristics

Immunohistochemical assessment of the status of RAB25 revealed overexpression of RAB25 in 18 tumors (31.57%), while 39 tumors (68.42%) do not express this protein. We analyzed the correlation between RAB25 status and various clinicopathological parameters.

Overexpression of RAB25 was found to correlate with mammary tumors expressing the estrogen receptor (P = .03), 76.5% of tumors overexpressing RAB25 are associated with the estrogen receptor. Moreover, tumors overexpressing RAB25 are most often associated with high proliferation index (84.6%) rather than a low Ki67 (15.4 %) (P = .06) (Table 3)

Table 1. Clinicopathological characteristics of 57 patients with breast cancer

Age

Pathological tumor size (57)

Histological type (57)

SBR grade (57)

Tumor stage (57)

Pathological axillary

lymph node (57)

Estrogen receptor status (56)

Progesterone receptor

status (56)

HER2 receptor status (56)

Distant metastasis (57)

Breast cancer subtypes

≤ 50 years

> 50 years

≤ 2Cm

>2Cm

Ductal

Lobular

Other

I

II

III

T1-T2

T3-T4

Negative

Positive

Negative

Positive

Negative

Positive

Negative

Positive

M0

M1

Mx

Luminal A

Luminal B

TNBC

HER2+

32 (56.14)

25 (43.86)

09 (15.79)

48 (84.21)

51 (89.48)

03 (5.26)

03 (5.26)

02 (3.51)

34 (59.65)

21 (36.84)

40 (70.17)

17 (29.83)

20 (35.09)

37 (64.91)

25 (44.64)

31 (55.36)

29 (51.79)

27 (48.21)

42 (75)

14 (25)

14 (24.56)

02 (3.51)

41 (71.93)

12 (21.05)

21 (36.84)

16 (28.07)

08 (14.03)

Variables N (%)

Values followed by different letters within a column are significantly different (Student’s t-test: **P < 0.01).

Table 2. Relationship between Luminal breast cancer subtypes and the age at diagnosis

Luminal breast cancer subtypes

Luminal A

Luminal BAge

Means ±SD

55.92±9.26

44.43±11.50

t

-2.94

ddl

31

P

0.006**

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Overexpression of RAB25 in tumors appears to be not associated with the menopausal status of the patients. It was found as many premenopausal women (55.6%) than postmenopausal women (44.4%).

For other prognostic parameters, axillary lymph node infiltration and SBR grade have no significant association with the expression of RAB25. It was noted that most tumors overexpressing RAB25 protein showed axillary lymph node infiltration (66.7%) while only 33.3% of this same group of patients did not show this infiltration.

RAB25 is highly expressed in luminal B breast cancer tumors

The analysis of the expression of RAB25 in our study reveals that this protein is expressed in breast tumors but

also that RAB25 is weakly expressed in normal mammary gland (Figure 1).

A high significant difference was observed by analyzing the RAB25 expression in various breast cancer subtypes (P = .01) (Table 3), proportions of RAB25 protein expression among molecular subtypes are shown in Figure 2. Findings also revealed that RAB25 expression is highly correlated with the most aggressive hormone dependent mammary tumors : the luminal B subtype compared to the luminal A tumors “good prognosis” (P=.004) (Figure 3); those of HER2-positive phenotype and the triple negative tumors did not show overexpression of the RAB25 protein (Figure 4).

Table 3. Association of RAB25 expression in the tumor cells with clinicopathological variables and molecular subtypes

RAB25- (39)

N (%)

22 (56.4%)

17 (43.6%)

34 (87.2%)

2 (5.1%)

3 (7.7%)

6 (15.3%)

33 (84.7%)

12 (30.77%)

27(69.23 %)

24 (61.5 %)

15 (38.5%)

21(53.8%)

18(46.2%)

23 (59%)

16 (41%)

29(74.4%)

10 (25.6%)

11 (45.8%)

13(54.2%)

10 (25.6%)

9 (23.1%)

7 (17.9%)

13 (33.3%)

4(30.8%)

9 (69.2%)

≤ 50

>50

Ductal

Lobular

Other

< 2CM

≥ 2 CM

N-

N+

II

III

ER-

ER+

PR-

PR+

HER2-

HER2+

KI67 < 14%

KI67≥ 14%

LUMINAL A

LUMINAL B

HER2

TNBC

Yes

No

Age (57)

Histological type (57)

Pathological tumor size (57)

Pathological axillary lymph node (57)

SBR Grade (55)

ER (56)

PR (56)

HER2 (56)

KI67 (37)

MOLECULAR SUBTYPES (57)

Metastatic Relapse (28)

RAB25+ (18)

N (%)

10 (55.6%)

8 (44.4%)

17 (94.4 %)

1 (5.6%)

0 (0%)

2 (11.1%)

16 (88.9%)

6 (33.3 %)

12 (66.7%)

10 (62.5%)

6 (37.5%)

4 (23.5%)

13 (76.5%)

6 (35.3%)

11 (64.7%)

13 (76.5%)

4 (23.5%)

2(15.4%)

11(84.6%)

2 (11.1%)

12 (66.7%)

1 (5.6%)

3 (16.7%)

5 (33.3%)

10 (66.7%)

P –Value

0.95

0.48

0.66

0.84

0.94

0.036

0.10

0.86

0.06

0.01

0.88

Clinicopathological Parameters

30

High RAB25 expression in luminal B breast cancer subtype, Amina Belhadj, et. al.

DiscussionBreast cancer is a complex and heterogeneous

disease, its molecular classification allows to better assess prognosis and therefore better treat patients. The identification of deregulated markers would be of great interest to the most aggressive hormone positive tumors, the luminal B subtype for which no targeted therapies exist yet.

The aberrant expression of small GTPases of the RAB family in solid cancers has been reported in many studies [15-16 -17-18]. Among the RAB family members RAB25 has been implicated in several cancers [19-20-21-8-22-10]. In this context, we assessed the RAB25 protein expression in different molecular subtypes of breast cancer and the association of RAB25 with patient clinicopathological characteristics.

First, the analysis of RAB25 expression in our study demonstrates (as in the literature) that this protein is expressed in breast tumors [12] as well in normal

mammary gland (weak expression) [4], although Cheng et al, using aCGH technique, found that 67% of breast cancers showed at least 1.7-fold increase in RAB25 expression compared to normal breast tissue suggesting that overexpression of RAB25 is an important event in carcinogenesis [22].

In this study, as in the study of Cheng et al [4], found a significant relationship between RAB25 overexpression and ER+/HER2- tumors; we also noted that the Ki67 expression level is much higher in tumors overexpressing RAB25. In relation to our results, Ki67 is known to be a poor prognosis marker in patients with breast cancer [23] and RAB25 protein is involved in cell survival and growth by activating the PI3 kinase/AKT/PTEN signaling pathway [24]. Our findings strongly demonstrate the highly proliferative nature and aggressiveness of hormone positive tumors overexpressing RAB25 nonetheless known to be associated with a good prognosis. To gain further insight it was essential to better classify ER+ tumors to provide better treatment for hormone positive

Figure 1. Representative RAB25 Immunohistochemical staining

(a) Positive RAB25 immunostaining. RAB25 immuno-reactivity was detected in the cytoplasm of the tumor cells. (b) RAB25 immunostaining in normal mammary gland Magnification X40

Figure 2. Distribution of molecular subtypesdepending on the status of expression of RAB25

Figure 3. Box plot of RAB25 expression in luminal breast cancer subtypes tumors

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patients that express a highly level expression of Ki67 proliferation index.

The molecular distribution of breast cancer in our series of 57 tumors, luminal B tumors came in first place followed by the TNBC subtype and luminal A and in the last position the HER2+ tumors. We found that triple negative tumors ( TNBC ) are characterized by a lack of expression of RAB25, this result is in agreement with those of previous studies that suggest that RAB25 may have a tumor suppressor role in triple negative tumors [4- 25], studies on colon cancer and colorectal cancer were cited [26-27]. Our results showed that tumors belonging to HER2+ and luminal A subtypes are not associated with overexpression of RAB25 as reported in another study [12]. In a more recent study, it was found that FIP1C an effector

of RAB25 would act as a tumor suppressor in breast tumors subtype HER2 + [28].

Our results showed that breast cancer tumors overexpressing RAB25 were significantly associated with the expression of estrogen receptor (ER+ tumors) rather than ER- negative tumors, these results corroborate those of Yin et al [11]. Transcriptomic studies have classified hormone-positive breast cancers into two groups: luminal A and luminal B subtypes[1], since luminal B tumors are highly proliferative and resistant to hormone therapy [2]

, furthermore there is no targeted regimen for the time being, it became crucial to identify a therapeutic target for the luminal B subtype tumors.

Interestingly, our results showed that the expression of RAB25 was different in tumors expressing hormone

Figure 4. Immunostaining for RAB25 in different breast cancer molecular subtypes

(a) Lack of expression of RAB25 in luminal A tumors (X40). (b) RAB25 overexpression in Luminal B tumors (X40). (c) Negative RAB25 immunostaining in HER2+ tumors (X40). (d) Negative RAB25 immunostaining in TNBC tumors (X40).

a b

c d

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High RAB25 expression in luminal B breast cancer subtype, Amina Belhadj, et. al.

receptors (ER/PR positive): luminal A and luminal B, suggesting that these two subtypes have different tumor biology and required different treatment approaches. Overexpression of RAB25 was found in 66.7% of luminal B tumors subtype, while only 11% in luminal A subtype tumors. We have showed that overexpression of RAB25 was specifically and significantly correlated (P = .01) with luminal B subtype tumors, suggesting its potential involvement in the genesis of this particularly aggressive form of breast cancer, our findings are supported by those of Mitra et al[24], indeed tumor cells which overexpress the RAB25 protein are more aggressive and are associated with a worse clinical response; this protein might represent a novel therapeutic target or marker of tumor progression in luminal B breast cancer subtype.

Conclusion In summary we investigated the potential involvement

of the RAB25 protein in breast cancers. We demonstrated for the first time that the expression of RAB25 was significantly elevated in luminal B breast cancer subtype. We have shown that overexpression of RAB25 was associated with highly aggressive and proliferative tumors. RAB25 could serve as a prognosis marker in breast cancers and might be an interesting therapeutic target for Luminal B breast cancer subtype since most of these tumors showed positive immunostaining for RAB25 protein and are resistant to hormone therapy.

Acknowledgments We are grateful to the team of anatomy pathology

unit at the military hospital of Oran for its help for the extraction of the data.

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