Izhar Bagwan

Expression profiling and significance of VEGF-A, VEGFR2, VEGFR3 and related proteins in endometrial carcinoma.

BACKGROUND Angiogenesis plays a key role in the progression of various tumors, including endometrial carcinomas. Several cytokines and their associated receptors are shown to be involved, particularly VEGF-A with VEGFR1, -2 and -3. METHODS The expressions of VEGF-A, VEGFR2 and VEGFR3 were studied in by immunohistochemistry in 76 endometrial carcinoma specimens. VEGFR2 and VEGFR3 receptor expression were also studied by qRT-PCR in 17 tumors in comparison to normal endometrium. The expression profiles were correlated with tumor type, grade, stage, lymphovascular invasion, disease free survival, and the expressions of other cytokine receptors (EGFR, CXCR1 and CXCR2). RESULTS Immunohistochemically, 63% of endometrial cancers expressed VEGF-A, 55% VEGFR2 and 26% VEGFR3. VEGFR3 was significantly correlated with tumor stage (p=0.02), with a trend towards poorer disease free survival (p=0.09). VEGF-A was significantly correlated with microvessel density (p<0.01). Using qRT-PCR, increased expression of VEGFR2 (17.2-fold) and VEGFR3 (21.9-fold) was seen in endometrial carcinomas compared with normal endometrium, with significant correlations among the expression levels of VEGFR2, VEGFR3, EGFR, CXCR1 and CXCR2. CONCLUSION Our study suggests that evaluation of VEGFR3 expression in tumors may provide prognostic data, and help identify patients who would best benefit from anti-angiogenic therapeutic agents. This is the first report showing correlations between the expressions levels of the different receptors.

Co-expression of HER family members in patients with Dukes' C and D colon cancer and their impacts on patient prognosis and survival.

The human epidermal growth factor receptor (EGFR) is an important therapeutic target in patients with metastatic colorectal cancer and anti-EGFR antibodies cetuximab and panitumumab have been approved for the treatment of such patients. Despite these advances, the duration of response in some patients can be limited. Since, EGFR is capable of forming heterodimers with the other members of the HER (Human epidermal receptor) family, it is important to investigate the co-expression and prognostic significance of all members of the HER family in colorectal cancer patients. The expression of the HER family members were determined in tumour specimens from 86 patients with Dukes’ C and D (metastatic) colon cancer using immunohistochemistry. Sections were scored by the percentage of positive tumour cells and intensity of staining. Their associations with clinicopathological parameters, and overall survival and disease free survival were evaluated using univariate and multivariate analysis. Overall, 43%, 77%, 52% and 92% of the cases were EGFR, HER-2, HER-3 and HER-4 positive respectively. Interestingly, 35%, 24%, 43%, and 18% of the cases had co-expression of EGFR/HER-2, EGFR/HER-3, EGFR/HER-4 and all four members of the HER family respectively. Of these, only the expression of EGFR and co-expression of EGFR/HER-4 were associated with poorer disease-free survival in both univariate and multivariate analysis. Co-expression of all members of the HER family in colon cancer supports the need for further investigations on their predictive value for response to therapy with anti-EGFR mAbs and whether such sub-population of patients may benefit from therapy with the new generation of pan-HER inhibitors.

Incidental Presentation of Gall Bladder MALT Lymphoma

BackgroundPrimary lymphomas of mucosa associated lymphoid tissue (MALT) within the gall bladder are exceedingly rare and may have an incidental presentation.Case ReportWe report a case of gall bladder MALT lymphoma diagnosed after cholecystectomy in a 65-year old woman.ConclusionNormally the gall bladder is devoid of lymphoid tissue; however, it has been suggested that MALT lymphomas may occur secondary to chronic cholecystitis with cholelithiasis or bacterial infection based on similar mechanism as described previously in the stomach and conjunctiva. Surgical resection is considered curative if the disease is localised only to the gall bladder.

Primary hepatic solitary fibrous tumor with histologically benign and malignant areas

Extrapleural solitary fibrous tumor (SFT) is an uncommon mesenchymal neoplasm, presenting most commonly in the intrathoracic sites but which has been reported at numerous extrathoracic locations. The majority of intra-thoracic SFTs are benign, but 10%-15% behave aggressively. We report a case of primary hepatic SFT with histologically benign and malignant areas. A 65-year-old man underwent an abdominal CT scan following a cerebrovascular accident, which demonstrated a sharply demarcated large liver mass with a heterogenous enhancing area and occupying most of the left lobe of the liver. Histological examination following a hemihepatectomy showed an SFT with morphological patterns ranging from benign to malignant areas, including pleomorphism, increased cellularity, herringbone pattern, necrosis and a raised mitotic count. On review of the literature, only an occasional case report with malignant areas in a hepatic SFT was identified. This case highlights that SFT should be included in the differential diagnosis of a hepatic spindle cell lesion, and that on rare occasions, malignant areas can occur in this already uncommon neoplasm.

The impact of co-expression of wild-type EGFR and its ligands determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer

Anti-EGFR mAbs cetuximab and panitumumab are routinely used for the treatment of patients with KRAS-wild type metastatic colorectal cancer (mCRC). However, in some patients their efficacy remains modest and with no clear association between the EGFR protein expression determined by PharmDx™ kit, and response to anti-EGFR therapies. Therefore, we investigated the relative expression and predictive value of wild-type EGFR (wtEGFR), mutated EGFRvIII and EGFR ligand proteins in mCRC patients treated with cetuximab. The expression levels of wtEGFR, EGFRvIII, and EGFR ligand were determined by immunohistochemistry (IHC) in 60 tumour specimens using specific antibodies. Sections were scored according to the percentage of positive tumour cells, intensity and cellular location of staining, and these were associated with response, overall survival (OS) and progression-free survival (PFS). At cut-off value > 5%, wtEGFR, and EGFRvIII were present in 44%, and 41%, betacellulin (BTC) in 72%, followed by epigen (67%), TGFα (58%), amphiregulin (34%), EGF (31%) of the cases, respectively and 96% of the wtEGFR positive cases had co-expression of at least one ligand. We found a significant association between the expression of wtEGFR and poor response to cetuximab. In addition, the co-expression of wtEGFR with one ligand at a cut-off value of > 5% and > 10% was associated with worse response to cetuximab (P = 0.021, and P = 0.005 respectively). We found a 3-fold and 5-fold increased risk of shorter OS with expression of BTC and epigen. Interestingly, the expression of wtEGFR and its co-expression with one or two ligands was associated with shorter PFS but not with OS. The relative expression of wtEGFR and its competing ligands, which is the target for therapeutic interventions with anti-EGFR antibodies, could serve as a more reliable predictive biomarker of response to therapy with anti-EGFR mAbs in mCRC patients and warrants further investigation in large prospective studies.

Iatrogenic gastric ulceration

Dear Editor, Selective internal radiation therapy (SIRT) with yttrium-90 (Y-90)-emitting microspheres, wherein intra-arterial infusion of Y-90 microspheres is performed, is increasingly recognised as an effective therapy of both primary and secondary hepatic malignancies [1]. This is commonly used as a loco-regional radiation therapy for unresectable hepatic neoplasms. Gastrointestinal complications of this novel therapy are sparse and the incidence varies from 3–24 % [2]. These include radiation ulcers mostly in the gastric antrum, pylorus and duodenum [1, 2]. We present the case of a 39-year-old man who was diagnosed with a T3 N0 M1 adenocarcinoma of the rectosigmoid in January 2010. The tumour was inoperable at the time of diagnosis due to widespread hepatic metastasis. He was KRas mutant and was started on FOXFIRE trial (FOLFOX chemotherapeutic regimen with SIRT). But after his first dose of chemotherapy in February 2010, he developed hypersensitivity reaction to oxaliplatin and hence continued chemotherapy with 5FU alone. After his fifth course of 5FU in May 2010, he developed symptoms of gastritis and was put on proton pump inhibitors. He had no relief of symptoms and an endoscopy done on 8th July 2010 revealed a large pre-pyloric ulcer which was biopsied (Fig. 1). The biopsy showed nonspecialised gastric mucosa admixed with ulcer slough and granulation tissue. The lamina propria showed mixed inflammatory infiltrate, increase in vascularity and scattered round dense eosinophilic radiation microspheres embedded in the submucosa (Fig. 2). Diagnosis of this pre-pyloric ulceration due to SIRT required a high degree of clinical suspicion along with characteristic endoscopic findings and pathognomonic radiation microspheres in the biopsy. The patient showed further disease progression on chemotherapy in later months and finally succumbed to unresponsive metastatic disease in 2011. The current evidence suggests that radio embolizationassociated gastroduodenal ulceration results either as a consequence of indirect irradiation or from direct deposition of Y-90 microspheres and biopsy can help differentiate between the two pathologies. The presence of perfectly round, purple microspheres on histological examination of the biopsy specimen is diagnostic of this type of injury [1, 3]. Awareness and knowledge of this type of injury amongst general histopathologists is very much essential for accurate identification and diagnosis of this condition. The lack of

Development of novel monoclonal antibodies against CD109 overexpressed in human pancreatic cancer

Pancreatic cancer is one of the most aggressive and lethal types of cancer, and more effective therapeutic agents are urgently needed. Overexpressed cell surface antigens are ideal targets for therapy with monoclonal antibody (mAb)-based drugs, but none have been approved for the treatment of pancreatic cancer. Here, we report development of two novel mouse mAbs, KU42.33C and KU43.13A, against the human pancreatic cancer cell line BxPC-3. Using ELISA, flow cytometry, competitive assay and immunoprecipitation followed by mass spectrometry, we discovered that these two mAbs target two distinct epitopes on the external domain of CD109 that are overexpressed by varying amounts in human pancreatic cancer cell lines. Treatment with these two naked antibodies alone did not affect tumour cell growth or migration in vitro. Of the two mAbs, only KU42.33C was useful in determining the expression of CD109 in tumour cells by Western blot and immunohistochemistry. Interestingly, immunohistochemistry of human pancreatic carcinoma tissue arrays with mAb KU42.33C showed that 94% of the 65 human pancreatic adenocarcinoma cases were CD109 positive, with no expression in normal pancreatic tissues. Our results suggest that these two novel mAbs are excellent tools for determining the expression level of CD109 in the tumour specimens and sera of patients with a wide range of cancers, in particular pancreatic cancer, and for investigating its diagnostic, prognostic and predictive value. Further research is warranted and should aim to unravel the therapeutic potential of the humanised forms or conjugated versions of such antibodies in patients whose tumours overexpress CD109 antigen.

Co-expression and prognostic significance of the HER family members, EGFRvIII, c-MET, CD44 in patients with ovarian cancer

EGFR and HER-2 are important targets but none of the monoclonal antibodies or small molecule tyrosine kinase inhibitors specific for the HER members has been approved for the treatment of patients with ovarian cancers. In some studies, co-expression of other growth factor receptors has been associated with resistance to therapy with the HER inhibitors. The aim of the present study was to determine the relative expression, cellular location, and prognostic significance of HER-family members, the EGFR mutant (EGFRvIII) c-MET, IGF-1R and the cancer stem cell biomarker CD44 in 60 patients with FIGO stage III and IV ovarian cancer. At cut off >5% of tumour cells with positive staining, 62%, 59%, 65% and 45% of the cases were EGFR, HER-2, HER-3 and HER-4 positive, and 3%, 22% and 48.3% of the cases were positive for EGFRvIII, c-MET, and CD44 respectively. Interestingly, 23% co-expressed all four members of the HER family. On univariate analysis, only EGFR staining at >50% of tumour cells (HR = 3.57, p = 0.038) and CD44 staining at 3+ intensity (HR = 7.99, p = 0.004) were associated with a poorer overall survival. EGFR expression (HR = 2.83, p = 0.019) and its co-expression with HER-2, HER-3, HER-2/HER-3, and c-MET were all associated with poorer disease-free survival. Our results suggest co-expression of the HER-family members is common in Stage III and IV ovarian cancer patients. Further studies on the prognostic significance and predictive value of all HER family member proteins for the response to treatment with various forms of the HER inhibitors are warranted.

Abstract 5731: The prognostic significance of EGFR, HER-2, HER-4, EGFRvIII, c-MET, Ki67 and CD44 in patients with FIGO stages III and IV ovarian cancer

Overexpression and activation of HER (human epidermal growth factor receptor) family members have been reported in a wide range of epithelial tumours. Although several monoclonal antibodies (mAbs) and small molecule tyrosine kinase inhibitors (TKIs) specific for the HER members have been approved for the treatment of patients with a wide range of tumours, none has yet been approved for the treatment of patients with ovarian cancer. In some studies, the co-expression of other growth factor receptors (e.g. c-MET), the presence of cancer stem cells (CSCs) have been associated with resistance to therapy with the HER inhibitors. The aim of the present study was to determine the expression level and prognostic significance of the EGFR, HER-2, HER-4 and EGFRvIII, as well as c-MET, CD44 and cell proliferation marker Ki67 in 60 patients with FIGO stage III and IV ovarian cancer. The expression levels of these markers were determined, at different cut off values, using immunohistochemistry (IHC), and their associations with the overall survival and disease free survival were evaluated using Chi-squared and Kaplan-Meier survival curves and log-rank test as well as the univariate Cox-regression analysis. At cut off values of >5% of tumour cells with positive immunostaining, 62%, 93%, 45%, 3%, 21%, 48%, and 95%, of the cases were positive for EGFR, HER-2, HER-4, EGFRvIII, c-MET, CD44, Ki67 respectively and 28% were positive for the co-expression of EGFR/HER-2/HER-4. The cellular location of immunostaining was membranous for EGFR, HER-2, c-MET and CD44 and was present in 33%, 10%, 3% and 50% of the cases examined respectively. In univariate analysis, the expression of EGFR at cut-off values of >50% (HR, 3.57; 95% CI, 1.07 to 11.85; P= 5 % (HR, 8.20; 95% CI, 2.02 to 33.2; P= 5% of tumour cells, was associated with better overall survival (HR, 0.13; 95% CI, 0.02 to 0.73; P= 0.021 respectively). In addition, at cut-off value of >5% of tumour cells with positive immunostaining, EGFR expression (HR, 2.83; 95% CI 1.18 to 6.77; P = 0.019) and >10% (HR, 2.40; 95% CI 1.07 to 5.37; P= 0.032 ), as well as the co-expression of EGFR/HER-2 (HR, 2.83; 95% CI 1.18 to 6.77; P= 0.019), EGFR/c-MET (HR, 3.05; 95% CI 1.2 to 7.75; P= 0.019) and EGFR/Ki67 (HR, 2.83; 95% CI 1.18 to 6.77; P= 0.019) were all associated with poorer disease free survival in these patients. Our results suggest that co-expression of EGFR\HER-2\HER4 is common in patients with Stages III and IV ovarian cancers and support the need for investigations on the therapeutic potential of various forms of pan-HER inhibitors in such patients. Citation Format: Soozana Puvanenthiran, Sharadah Essapen, Ben Haagsma, Izhar Bagwan, Alan Seddon, Helmout Modjtahedi. The prognostic significance of EGFR, HER-2, HER-4, EGFRvIII, c-MET, Ki67 and CD44 in patients with FIGO stages III and IV ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5731. doi:10.1158/1538-7445.AM2017-5731

Abstract 5014: Predictive value of wild-type EGFR determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer

The aberrant expression of the epidermal growth factor receptor (EGFR) has been reported in patients with colorectal cancer, and EGFR is an important therapeutic target in such patients. To date, of the anti-EGFR monoclonal antibodies (mAbs), only cetuximab and panitumumab have been approved for the treatment of patients with metastatic colorectal cancer (mCRC). While treatment with a combination of antibodies and cytotoxic drugs improves response rate and median time to progression in some colorectal cancer patients, the duration of response is often limited. In addition, no clear association has been found between the expression level of the EGFR protein in the tumours, determined by the FDA-approved EGFR PharmDx kit, or other standard anti-EGFR antibodies, and the response to the EGFR inhibitors. We have previously shown that kits such as the EGFR PharmDx™ kit, used to determine the expression of EGFR, does not discriminate between the wild-type EGFR (wtEGFR) and the type III deletion mutant EGFR (EGFRvIII), and as such could have contributed to the lack of association between the expression of EGFR and response to anti-EGFR antibodies. Therefore, the aim of this study was to determine the predictive value of wtEGFR for response to therapy with cetuximab. The expression levels of wtEGFR, mutated EGFRvIII, and phosphorylated EGFR (pEGFR Tyr1068 & Tyr1173) were determined using immunohistochemistry and specific antibodies in 61 FFPE tumour specimens from patients with mCRC treated with cetuximab. Sections were scored by the percentage of positive tumour cells, location and intensity of staining. Their associations with response and progression free survival were evaluated using Chi-squared and Kaplan-Meier survival curves and log-rank test respectively. Overall 11% and 46% of the cases were found to express membranous and cytoplasmic EGFR, using mAb specific for wtEGFR (DAK-H1-WT). On the other hand using anti EGFR.113 clone antibody, which does not discriminate between the wtEGFR and EGFRvIII, 11.5% and 18% of the cases were found to express the cytoplasmic and membranous EGFR respectively. While all cases were negative for the pEGFR, the expression of EGFRvIII was mainly cytoplasmic, occurring in 41% of the cases examined. Of these, only the cytoplasmic expression of wtEGFR was found to be significantly associated with poor response to treatment (P = 0.002) and progression free survival (95% CI, 0-3 months vs 4.5-17 months, P = 0.001). Taken together, our results suggest that cytoplasmic expression of wtEGFR is associated with a significantly shorter progression free survival, and is a predictive marker for poor response to treatment with the anti-EGFR mAb cetuximab. These findings underlie the need for further investigations, in a larger population of patients, to validate the predictive value of wtEGFR for response to therapy with anti-EGFR antibodies in patients with mCRC. Citation Format: Said Abdullah Khelwatty, Sharadah Essapen, Izhar Bagwan, Margaret Green, Alan Seddon, Helmout Modjtahedi. Predictive value of wild-type EGFR determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5014.