Anupam Sarma

Hybrid Capture 2 Assay Based Evaluation of High-Risk HPV Status in Healthy Women of North-East India

BACKGROUND High risk HPV (HR-HPV) testing has been recommended as an effective tool along with cytology screening in identification of cervical intraepithelial lesions (CINs) and prevention of their progress towards invasive cervical cancer. The aim of this study was to assess the HR-HPV DNA status by Hybrid Capture 2 (HC2) assay in healthy asymptomatic women of North-East India. MATERIALS AND METHODS This study examined cervical cell samples of forty three (n=43) healthy women by HC2 assay. A High Risk HPV DNA kit (Qiagen) was used which can detect 13 high risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. RESULTS The mean relative light units (RLU) for samples was in the range of 141-5, 94, 619. HR-HPV DNA was confirmed in 16% (7/43) of participant women samples. Among demographic and clinical parameters, menstrual irregularity (p=0.039) and infection history (p=0.028) has shown statistically significant differences between the HR-HPV-positive and negative groups. In the HR-HPV positive group, two women were confirmed for CINs after colposcopy and histopathologic examination. CONCLUSIONS We suggest that there may be an association between irregular menstruation and infection history of the urogenital tract with HR-HPV DNA prevalence in North-East Indian asymptomatic women. HC2 assay can be a valuable tool for HR-HPV screening.

Alcohol and Tobacco Increases Risk of High Risk HPV Infection in Head and Neck Cancer Patients: Study from North-East Region of India

Background Human papilloma virus (HPV) associated Head and Neck Cancers (HNCs) have generated significant amount of research interest in recent times. Due to high incidence of HNCs and lack of sufficient data on high-risk HPV (hr-HPV) infection from North -East region of India, this study was conceived to investigate hr-HPV infection, its types and its association with life style habits such as tobacco, alcohol consumption etc. Methods A total of one hundred and six primary HNC tumor biopsy specimens were collected. These samples were analyzed for hr-HPV DNA (13 HPV types) using hybrid capture 2 (HC2) assay and genotyping was done by E6 nested multiplex PCR (NMPCR). Results The presence of hr-HPV was confirmed in 31.13% (n = 33) and 24.52% (n = 26) of the HNC patients by nested multiplex PCR (NMPCR) and HC2 assay respectively. Among hr-HPV positive cases, out of thirteen hr- HPV types analyzed, only two prevalent genotypes, HPV-16 (81.81%) followed by HPV-18 (18.18%) were found. Significant association was observed between hr-HPV infection with alcohol consumption (p <0.001) and tobacco chewing (p = 0.02) in HNC cases. Compared to HPV-18 infection the HPV-16 was found to be significantly associated with tobacco chewing (p = 0.02) habit. Conclusions Our study demonstrated that tobacco chewing and alcohol consumption may act as risk factors for hr-HPV infection in HNCs from the North-East region of India. This was the first study from North-East India which also assessed the clinical applicability of HC2 assay in HNC patient specimens. We suggest that alcohol, tobacco and hr- HPV infection act synergistically or complement each other in the process of HNC development and progression in the present study population.

Expression of aberrant CD markers in acute leukemia: A study of 100 cases with immunophenotyping by multiparameter flowcytometry

BACKGROUND Acute leukemia is a heterogenous disease having diverse phenotypes. Immunophenotyping by flowcytometry is essential for diagnosis of myeloid and lymphoid subtypes. Aberrant phenotype incidence is controversial and dissimilar results have been reported by different groups. OBJECTIVES Purpose of the study was to determine the incidence of aberrant phenotypes in North East Indian patients with acute leukemia. METHODS We analysed a total of 100 cases (AML = 36, ALL = 61, MPAL = 3) by multiparametric flow cytometry using an acute panel of monoclonal antibodies (MoAbs). The MoAbs were selected to identify differentiation-associated antigens of both myeloid and lymphoid lineages. RESULTS Aberrant phenotypes were found in 21 (58.3%) cases of AML, 36 (59.2%) cases of B-ALL and 6 (66.7%) cases of T-ALL. CD7 was the most frequent lymphoid associated antigen found in 33% of AML cases while CD117 was the myeloid antigen most frequently detected in ALL (54%) cases. Aberrant expression of CD 117 is highly significant by Fischers exact test (P< 0.0001). CONCLUSION We conclude that aberrant phenotypes are present in a great majority of acute leukemia patients of North East India. Future studies will be directed to correlate of these markers with prognosis and therapeutic response.

Ovarian carcinoma in normal size ovaries with inguinal lymph node metastasis: a case report

It is generally recognized that ovarian cancer tends to remain intra-abdominal even in advanced stages. This case report describes a patient with ovarian carcinoma who had an ulcerated fungating growth in her left groin area. Case history: A 35yrs para2 women presented with a fungating left inguinal node of 5 x 4 cm for last 3 months. Biopsy revealed metastatic papillary adenocarcinoma. Clinical examination showed left sided pedal oedema. Laboratory investigation revealed increased CA125 (412IU/ml).Radiological studies were normal. In view of the raised Ca 125 she was treated in the lines of FIGO stage III cancer ovary with Paclitaxel 260mg and Carboplatin 450mg for three cycles. She responded dramatically with completely healedinguinal nodes. Then laparotomy with total hysterectomy, bilateral salpingo-oophorectomy partial omentectomy and inguinal lymph node dissection were performed. Histology confirmed right ovarian adenocarcinoma consistentwith the earlier histology of the left inguinal lymph node. There were noother sites of involvement. Postoperatively, the patient received three more cycle of chemotherapy. Conclusions: Ovarian cancer with inguinal lymph node metastasis is uncommon and only around 1% of ovarian carcinoma have a normal sized ovary. This case report demonstrates that early distal metastasis, although rare, can occur in patients with ovarian cancer and may be a compelling symptom of ovarian cancer.

Cutaneous metastasis from squamous carcinoma of the base of tongue

Context: Cutaneous metastasis from head and neck cancer is uncommon and it is seen from laryngeal cancer. Cutaneous metastasis from the base of tongue is relatively rare. Case Report: A 55-year-old male, who was a treated case of squamous carcinoma of the base of tongue presented with metastatic nodule on the skin of face and thigh. But, there was complete resolution of the tumor at the primary site. In the present case, clinically obvious cutaneous nodules with metastasis appeared soon after the completion of treatment with concurrent chemo-radiotherapy. The metastasis to the skin of face clinically appeared like an inflammatory lesion. Fine needle aspiration cytology confirmed the diagnosis of metastasis to skin at both the sites. Conclusion: Our case has highlighted that there could be associated occult skin metastasis at the time of diagnosis in squamous carcinoma of the base of tongue.

Abstract 2681: Oral micro biome enhances stemness in oral cancer cells by activating Toll like receptor signaling

Background: The resistance and progression of cancers after chemotherapy to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Recent studies suggest, microbiomes can induce a cascade of host events to either support or inhibit tumor growth. Specially, in oral cancer, chemotherapy treatment may alter the oral microbial flora, which may favor or inhibit tumor growth. Hence, it is importantl to develop novel experimental approaches to study the role of oral microbial flora in oral cancer stemness (self-renewal and undifferntiated state of cancer stem cells). Importantly, patients in developing area, including Assam, where KaviKrishna laboratory is located, may have distinct oral microbial flora that could favor oral cancer growth. Hence, it is important to include patients from developing countries for such studies. Our previous research showed that chemotherapy ehances stemenss in many cancer cell types, including oral squamous cell carcinoma cell line SSC-25. The stemness switch is characterized by enhanced expression of stemness associated genes including Nanog, Lin28A/B, Oct-4, MYC, HIF-2alpha and inflammation associated genes including Toll like receptor (TLR) 2/4. Here we investigated the role of oral microbiomes in the TLR mediated stemness switch of oral cancer cells. Methods: SCC-25 oral cancer cell line was treated with bacterial product lipopoly saccharide (LPS), and the stemness switch evaluated by isolation of ABCG2+ cells and expression of stemness associated genes by these cells. Capacity of interaction of tumor stromal cells with mesenchymal stem cells was also evaluated. Additionally, we obtained sputum from oral cancer subjects undergoing chemotherapy. The patients were from the Kamrup district of Assam, where KaviKrishna laboratory is located. The sputum was processed and then added to the culture medium of SCC-25 cells. These post-sputum treated SCC-25 cells were subjected to phenotypic stemness switch analysis. Results: We found, LPS and sputum treatment led to the enhanced stemness of ABCG2+ cells, including the high expression of TLR2/4, MYC, Nanog, Sox-2, and HIF-2alpha. Importantly, sputum derived from oral cancer subjects under remission showed inhibitory activity on ABCG2+ cell self-renewal. In contrast, sputum obtained from oral cancer subjects with relapse showed enhanced stemness of ABCG2+ cells, and also increased tumorigenic potential. The post-sputum treated ABCG2+ cells exhibited high expression of TLR2/4 and associated increase of HIF-2alpha and MYC transcriptional activity. The sputum treated with broad spectrum antibiotic ciprofloxacin did not enhance the stemness and TLR2/4 signaling of SCC-25 cells. Conclusion: These results indicate that oral microbiomes may differentially influence the stemness of oral cancer cells. We also conclude that live bacteria present in the sputum may be required to enhance stemness in a TLR2/4 dependent manner. Note: This abstract was not presented at the meeting. Citation Format: Joyeeta Talukdar, Rashmi Bhuyan, Bidisha Pal, Sorra Sandhya, Hong Li, Seema Bhuyan, Sukanya Garhyan, Debabrat Baishya, Anupam Sarma, Jyotirmoy Phukan, Amal Kataki, Bikul Das. Oral micro biome enhances stemness in oral cancer cells by activating Toll like receptor signaling [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 2681. doi:10.1158/1538-7445.AM2017-2681

Abstract 935: Oral cancer cells may hijack stem cell altruism to survive during extreme hypoxia, and exposure to chemotherapeutic drugs

The mechanism of tumor hypoxia induced reprogramming of cancer cells are not understood well. Hypoxia might activate evolutionary preserved cellular defense mechanism that could contribute to tumor progression and metastasis. We recently described that during hypoxia/oxidative stress, human embryonic stem cells (hES) exhibit an altruistic defense mechanism, where a few cells reprogram to a highly undifferentiated state, and secrete glutathione to protect rest of the community of cells from oxidative stress induced DNA damage (Das B et al, Stem Cells, 2012). The altruistic stem cells phenotype exhibited high HIF-2alpha and low p53 activity. After a few weeks, the reprogrammed cells, although highly fit to survives, underwent spontaneous apoptosis/differentiation by re-activating the p53/MDM2 oscillation. Thus, the reprogrammed cells sacrificed its own fitness to enhance the fitness of the rest of the community, an altruistic behavior (Das B et al. Stem Cells, 2012). Here, we investigated the potential hijacking of the altruistic defense mechanism by oral cancer cells during exposure to hypoxia. We exposed the four oral cancer cell lines SCC-25, SCC-15, SCC-9 and SCC-5 to extreme hypoxia followed by re-oxygenation for 72 hours. We found that while majority of cancer cells underwent apoptosis, a few cancer cell lines survived, exhibited side-population (SP) phenotype, high level of HIF-2alpha, Nanog, Sox-2 and MYC. The SP cells exhibited migratory activity, as well as high tumorigenic and metastatic activity in NOD/SCID mice. ChIP assay indicated that HIF-2alhpa interact with MYC and NOTCH1. The conditioned media of SP cells exhibited high level of glutathione, and ability to protect non-SP cells from cisplatin-induced toxicity. These results indicate that HIF-2alpha and MYC may cooperate to reprogram a few oral cancer cells to altruistic stemness phenotype. The altruistic phenotype that exhibited cytoprotective activity against cisplatin mediated toxicity. Thus, similar to bacteria, where altruistic biofilm exhibit novel drug resistance mechanism, stem cell altruism may serve as a novel drug resistance mechanism in oral cancer. Citation Format: Rashmi Bhuyan, Hong Li, Sukanya Gayan, Bidisha Pal, Reza Bayat-Mokhtari, Jyotirmoy Phukan, Debabrata Baishya, Anupam Sarma, Joyeeta Talukdar, Manaf Muhammad Alkurdi, Wael Tasabehji, Seema Bhuyan, Gayatri Gogoi, Ista Pulu, Herman Yeger, Bikul Das. Oral cancer cells may hijack stem cell altruism to survive during extreme hypoxia, and exposure to chemotherapeutic drugs. [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 935.

Abstract 4073: The potential role of oral mucosa stem cell altruistic behavior as the initiating event of malignant transformation

Oral cancer presents a major health burden across the globe, specially in developing countries. Kamrup, a district of India, where the KaviKrishna laboratory is located has the highest incidence of oral cancer in the entire world. The mechanism of oral cancer carcinogenesis process is not clearly known. Previous studies indicate the potential role of HPV-16 virus as well as tobacco consumption as major contributing factors of oral cancer. In mouse, the carcinogen agent 4-NQO was found to induce oral cancer having similar phenotype as human oral mucosa cancer. 4-NQO may exert tobacco like oxidative stress toxicity to oral mucosa. Hence, developing both in vitro and in vivo models of HPV-16 and 4-NQO mediated oral carcinogenesis might help to understand the initiating event of oral carcinogenesis. In this study, we propose that oral mucosa stem cells (OMSCs) could be the target of HPV-16, and 4-NQO induced carcinogensis. Thus, in vitro treatment of oral mucosa cells of healthy human volunteers with HPV-16 protein E6, and 4-NQO led to expansion of CD271+ cells, which are enriched in OMSCs. Importantly, the expanded CD271+ cells exhibited high HIF-2alpha, low p53, and high glutathione secretion, a phenotype of stem cell altruism that we recently described in human embryonic stem cells (Das B et al. Stem Cells, 2012). In human ES cells, the altruistic reprogramming served as an initiating event of malignant transformation by altering p53/MDM2 oscillation, in an abnormal state of low p53 and high MDM2. Therefore, we performed a complete evaluation of E6 protein and 4-NQO treated CD271+ oral mucosa cells for altruistic behavior, including low p53 and high MDM2 state. For this purpose, the carcinogen treated oral mucosa cells were subjected to immunomagnetic sorting for CD271+ cells. We found that post-carcinogen treated CD271+ cells exhibited in vitro self-renewal activity, high GSH secretion, and importantly activation of a HIF-2alpha/MYC co-operation. ChiP assay revealed the MYC binding to HIF-2alpha, and Sox-2, an stemness associated transcription factor. Importantly, HIF-2alpha was important for the reversible but prolonged suppression of p53 for more than two weeks. We also found that the high HIF-2alpha and low p53 expressing CD271+ cells could be enriched in a ABCG2+ population. Thus, we were able to enrich a CD271+/ABCG2+ cell population in oral mucosa cells of both human and mouse. Based on these findings we propose to use HPV-16 and 4NQO derived carcinogenesis models to study the potential role of altruistic reprogramming in the malignant transformation of oral mucosa stem cells to oral cancer stem cell like cells. Our study may unravel a novel mechanism of malignant transformation, the failure of altruistic stem cells to sacrifice its fitness (altruism) as a potential initiating event of malignant transformation of stem cells to cancer stem cells. Citation Format: Sukanya Gayan, Hong Li, Rashmi Bhuyan, Sora Sandhya, Joyeeta Talukdar, Bidisha Pal, Jaishree Garhyan, Wael Tasabehji, Manaf Muhammad Alkurdi, Heidar Zohrehei, Seema Bhuyan, Anupam Sarma, Gayatri Gogoi, A.C. Kataki, Rakesh Bhatnagar, Debabrata Baishya, Bikul Das. The potential role of oral mucosa stem cell altruistic behavior as the initiating event of malignant transformation. [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 4073.

Abstract 251: Stem cell altruism may serve as a novel drug resistance mechanism in oral cancer

Background: The mechanism of oral squamous cell carcinoma resistance to platinum-based chemotherapy is not clearly known. Previous studies indicated that glutathione (GSH), a cellular antioxidant may detoxify cisplatinum (CDDP), a commonly used chemotherapy agent in oral cancer. Our previous research in human embryonic stem cells (hESCs) indicated the altruistic behavior of ABCG2+ hESCs that secrete high level of GSH to protect other hESCs exposed to oxidative stress (Das B et al. Stem Cells, 2012). Here we investigated if CDDP exposure lead to altruistic stem cell reprogramming of ABCG2+ oral squamous cell carcinoma cells and subsequent GSH-mediated resistance against CDDP. Methods: Two oral squamous cell cancer cell lines SCC-25 and SCC-15 were treated with 3-10 uM CDDP for three-days, and then subjected to flow cytometry and immunomagnetic sorting based evaluation of ABCG2+ cells. The conditioned media (CM) obtained from ABCG2+ cells were examined for GSH content. The CM treated cancer cell lines were examined for resistance against CDDP toxicity. Next, the post-CDDP treated ABCG2+ cells were examined for enhanced stemness phenotype that corresponds to altruistic stem cell phenotype (Das B et al, Stem Cells 2012). Results: We found that CDDP treatment increases the ABCG2+ self-renewal capacity of SCC-25 and SCC-15 cells as measured by serial transplantation assay. The CM of the post-CDDP treated cells exhibited high level of GSH. When the SCC-15 and SCC-25 cells were treated with CM plus CDDP, the cancer cells exhibited 10-15-fold increase in resistance against CDDP toxicity. Next, the post-CDDP treated SCC-25 and SCC-15 cells exhibited enhanced stemness reprogramming phenotype characterized by very high HIF-2alpha, Sox-2 and Nanog transcriptional activity. Furthemore, we found increased expression of EMT (epithelial mesenchymal transition) marker expression including Snail, Twist and vimentin as evaluated by flow cytometry. siRNA HIF-2alpha treatment led to marked loss in the in vivo self-renewal capacity of ABCG2+ SCC-25 and SCC-15 cells. We then noted that post-CDDP ABCG2+ cells exhibited reversible state, as after two weeks of culture, most of the cells either differentiated or underwent apoptosis. Conclusions: These results indicate that oral cancer cells exhibit altruistic defense mechanism to resist the toxicity of CDDP. The altruistic defense mechanism involved high secretion of GSH. Thus, we suggest that similar to bacterial altruism as a mechanism of drug resistance, stem cell altruism may also serve as a novel mechanism of drug resistance in cancer. Citation Format: Bidisha Pal, Reza Bayat-Mokhtari, Hong Li, Rashmi Bhuyan, Joyeeta Talukdar, Sora Sandhya, Anupam Sarma, Wael Tasabehji, Seema Bhuyan, Sukanya Gayan, Amal Ch Kataki, Debabrata Baishya, Herman Yeger, Dean W. Felsher, Bikul Das. Stem cell altruism may serve as a novel drug resistance mechanism in oral 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 251.

Basaloid squamous carcinoma of the supraglottic larynx with neuro-endocrine features

Basaloid squamous carcinoma (BSC) is a rare aggressive variant of squamous cell carcinoma and occurs mainly at the larynx, oropharynx and tongue of the head and neck region. Neuro-endocrine differentiation of BSC is further rare occurrence in laryngeal cancers. We report here a case of BSC of supraglottic larynx with neuro-endocrine differentiation, which was treated by radiotherapy and its response to treatment.