Clinico-Pathological Characteristics and Mutational Analysis of Gastrointestinal Stromal Tumors from India: A Single Institution Experience.

BACKGROUND: Gastrointestinal stromal tumor (GIST) is the most common type of mesenchymal neoplasm of gastrointestinal tract. The incidence of GIST in India is not known and its treatment strategy in our country is largely derived from studies in other global populations. Some of the most important features of this type of cancer include its size, site of origin, mitotic index, histology and Immunohistochemistry. In this report we have studied these parameters in the Indian GIST patients presenting at our center. Additionally, we have also studied the mutational spectrum of these GISTs by next generation sequencing. METHODS: Thirty one Indian patients of GIST were enrolled in this study and information regarding age, gender, tumor location and size was collected from their records. Immunohistochemistry studies were performed by the pathologist. Mutational analysis of these samples was performed by next generation sequencing. RESULTS AND DISCUSSION: The most common site of GIST occurrence in our study was stomach. The tumor size for all 31 patients ranged between 0.6 cms to 20 cms. A spindle-cell pattern was present in 24 out of 31 of the cases. 29 out of 31 subjects were positive for CD117 expression. C-KIT was the most highly mutated gene indentified in our patients. Apart from these findings we observed many similarities as well as dissimilarities between the results of our study and literature published previously. CONCLUSIONS: The dissimilarities in the results of our study and published literature could be attributed to the genetic or ethnic differences that exist between the Indian population and other global populations. The results of our study warrant a need to conduct studies of GIST in a much larger population of India. Such large scale studies may also help in better treatment and/or prevention of GIST in developing countries like India.

Identification of Prognostic and Susceptibility Markers in Chronic Myeloid Leukemia Using Next Generation Sequencing.

BACKGROUND: Incidence of Chronic Myeloid Leukemia (CML) is continuously increasing and expected to reach 100,000 patients every year by 2030. Though the discovery of Imatinib Mesylate (IM) has brought a paradigm shift in CML treatment, 20% patients show resistance to this tyrosine kinase inhibiter (TKI). Therefore, it is important to identify markers, which can predict the occurrence and prognosis of CML. Clinical Exome Sequencing, panel of more than 4800 genes, was performed in CML patients to identify prognostic and susceptibility markers in CML. METHODS: Enrolled CML patients (n=18) were segregated as IM responders (n=10) and IM failures (n=8) as per European Leukemia Net (ELN), 2013 guidelines. Healthy controls (n=5) were also enrolled. DNA from blood of subjects was subjected to Next Generation Sequencing. Rare mutations present in one patient group and absent in another group were considered as prognostic markers, whereas mutations present in more than 50% patients were considered as susceptibility markers. RESULT: Mutations in genes associated with cancer related functions were found in different patient groups. Four variants: rs116201358, rs4014596, rs52897880 and rs2274329 in C8A, UNC93B1, APOH and CA6 genes, respectively, were present in IM responders; whereas rs4945 in MFGE8 was present in IM failures. Mutations in HLA-DRB1 (rs17878951), HLA-DRB5 (rs137863146), RPHN2 (rs193179333), CYP2F1 (rs116958555), KCNJ12 (rs76684759) and FUT3 (rs151218854) were present as susceptibility markers. CONCLUSION: The potential genetic markers discovered in this study can help in predicting response to IM as frontline therapy. Susceptibility markers may also be used as panel for individuals prone to have CML.

Association between Altered Expression and Genetic Variations of Transforming Growth Factor ß-Smad Pathway with Chronic Myeloid Leukemia.

Background: Chronic myeloid leukemia (CML) is a hematological disorder caused by fusion of BCR and ABL genes. BCR-ABL dependent and independent pathways play equally important role in CML. TGFß-Smad pathway, an important BCR -ABL independent pathway, has scarce data in CML. Present study investigate the association between TGFß-Smad pathway and CML. Materials and Methods: Sixty-four CML patients and age matched healthy controls (n=63) were enrolled in this study. Patients were segregated into responder and resistant groups depending on their response to Imatinib mesylate (IM). TGFß1 serum levels were evaluated by ELISA and transcript levels of TGFß1 receptors, SMAD4 and SMAD7 were evaluated by Real-Time PCR. Sequencing of exons and exon-intron boundaries of study genes was performed using Next Generation Sequencing (NGS) in 20 CML patients. Statistical analysis was performed using SPSS version 16.0. Results:TGFß1 serum levels were significantly elevated (p = 0.02) and TGFßR2 and SMAD4 were significantly down-regulated (p = 0.012 and p = 0.043 respectively) in the patients. c.69A>G in TGFß1, c.1024+24G>A in TGFßR1 and g.46474746C>T in SMAD7 were the most important genetic variants observed with their presence in 10/20, 8/20 and 7/20 patients respectively. In addition, TGFßR1 transcript levels were reduced in CML patients with c.69A>G mutation. None of the genes differed significantly in terms of expression or genetic variants between responder and resistant patient groups. Conclusion: Our findings demonstrate the role of differential expression and genetic variants of TGFß-Smad pathway in CML. Decreased TGFßR2 and SMAD4 levels observed in the present study may be responsible for reduced tumor suppressive effects of this pathway in CML.

Prevalence and coexistence of KRAS, BRAF, PIK3CA, NRAS, TP53, and APC mutations in Indian colorectal cancer patients: Next-generation sequencing-based cohort study.

Colorectal cancer incidences are on a rise in India. In this study, we have analyzed the mutation frequencies of six potential biomarkers, their coexistence, association with clinicopathological characteristics, and tumor location in Indian colorectal cancer patients. Next-generation sequencing was performed to identify mutations in the six potential biomarker genes using formalin-fixed paraffin-embedded tissue blocks of 112 colorectal cancer patients. The mutation frequency observed in KRAS, BRAF, PIK3CA, NRAS, TP53, and APC was 35.7%, 7.1%, 16.1%, 6.3%, 39.3%, and 29.5%, respectively. The significant associations of mutations were KRAS with age less than 60 years (p = 0.041), PIK3CA with males (p = 0.032), tumor stage I-II (p = 0.013), lack of metastasis in lymph nodes (p = 0.040), NRAS with rectum (p = 0.002), and APC with T2 stage of tumor growth (p = 0.013). No single patient harbored mutations in these six genes or any five genes simultaneously. Significance was noted in coexistence of KRAS with APC (p = 0.024) and mutual exclusion of KRAS with BRAF (p = 0.029). PIK3CA exon 9 was observed to be more frequently associated with KRAS mutations than PIK3CA exon 20 (p = 0.072). NRAS mutations were mutually exclusive with BRAF and PIK3CA mutations. As per our knowledge, this is the first next-generation sequencing-based biomarker study in Indian colorectal cancer patients. Frequent coexistence of gene mutations in pairs and triplets suggests that synergistic effect of overlapping mutations might further trigger the disease. In addition, infrequent coexistence of multiple gene mutations hints toward different signaling pathways for colorectal cancer tumorigenesis.

Targeted molecular profiling of rare genetic alterations in colorectal cancer using next-generation sequencing.

Mutation frequencies of common genetic alterations in colorectal cancer have been in the spotlight for many years. This study highlights few rare somatic mutations, which possess the attributes of a potential CRC biomarker yet are often neglected. Next-generation sequencing was performed over 112 tumor samples to detect genetic alterations in 31 rare genes in colorectal cancer. Mutations were detected in 26/31 (83.9 %) uncommon genes, which together contributed toward 149 gene mutations in 67/112 (59.8 %) colorectal cancer patients. The most frequent mutations include KDR (19.6 %), PTEN (17 %), FBXW7 (10.7 %), SMAD4 (10.7 %), VHL (8 %), KIT (8 %), MET (7.1 %), ATM (6.3 %), CTNNB1 (4.5 %) and CDKN2A (4.5 %). RB1, ERBB4 and ERBB2 mutations were persistent in 3.6 % patients. GNAS, FGFR2 and FGFR3 mutations were persistent in 1.8 % patients. Ten genes (EGFR, NOTCH1, SMARCB1, ABL1, STK11, SMO, RET, GNAQ, CSF1R and FLT3) were found mutated in 0.9 % patients. Lastly, no mutations were observed in AKT, HRAS, MAP2K1, PDGFR and JAK2. Significant associations were observed between VHL with tumor site, ERBB4 and SMARCB1 with tumor invasion, CTNNB1 with lack of lymph node involvement and CTNNB1, FGFR2 and FGFR3 with TNM stage. Significantly coinciding mutation pairs include PTEN and SMAD4, PTEN and KDR, EGFR and RET, EGFR and RB1, FBXW7 and CTNNB1, KDR and FGFR2, FLT3 and CTNNB1, RET and RB1, ATM and SMAD4, ATM and CDKN2A, ERBB4 and SMARCB1. This study elucidates few potential colorectal cancer biomarkers, specifically KDR, PTEN, FBXW7 and SMAD4, which are found mutated in more than 10 % patients.

Lack of any association of the CTLA-4 +49 G/A polymorphism with breast cancer risk in a North Indian population.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is an important protein involved in the regulation of the immune system. The +49 G/A polymorphism is the only genetic variation in the CTLA-4 gene that causes an amino acid change in the resulting protein. It is therefore the most extensively studied polymorphism among all CTLA-4 genetic variants and contributions to increasing the likelihood of developing cancer are well known in various populations, especially Asians. However, there have hiterto been no data with respect to the effect of this polymorphism on breast cancer susceptibility in our North Indian population. We therefore assayed genomic DNA of 250 breast cancer subjects and an equal number of age-, sex- and ethnicity-matched healthy controls for the CTLA-4 +49 G/A polymorphism but no significant differences in either the gene or allele frequency were found. Thus the CTLA-4 +49 G/A polymorphism may be associated with breast cancer in other Asians, but it appears to have no such effect in North Indians. The study also highlights the importance of conducting genetic association studies in different ethnic populations.

Feasibility study of docetaxel, oxaliplatin and capecitabine combination regimen in advanced gastric or gastroesophageal adenocarcinoma.

BACKGROUND AND OBJECTIVES: At present, there is no standard regimen for the treatment of gastroesophageal cancer. Docetaxel, cisplatin and fluorouracil (DCF) has been shown to be an effective regimen; however, toxicity is an area of concern in the palliative case setting. Capecitabine and oxaliplatin have been shown to be as effective as fluorouracil and cisplatin, respectively. To reduce the toxicity of DCF while maintaining efficacy, we conducted this study to evaluate the efficacy of docetaxel, oxaliplatin and capecitabine (DOX) combination in advanced gastroesophageal cancer. METHODS: Patients with histologically confirmed metastatic or locally advanced adenocarcinoma of the stomach or gastroesophageal junction received docetaxel 25 mg/m2 and oxaliplatin 50 mg/m2 on days 1 and 8 with capecitabine 625 mg/m2 twice daily from day 1-14, in 21-day cycles. The primary endpoint was overall response rate (ORR). RESULTS: Of 21 patients, there were 16 males and 5 females with a median age of 57 years, range 37-80 years. The primary tumor was located at the gastroesophageal junction in 7 patients and in other parts of the stomach in the remaining 14 patients. One patient had locally advanced tumor without distant metastases and 20 patients presented with metastatic disease. Grade 3/4 toxicities included diarrhea (24%), hand-foot syndrome (5%) and febrile neutropenia (5%). The ORR was 29%. The median survival was 8.4 months. At the time of analysis, 5 of the 21 patients (24%) were alive. CONCLUSIONS: The DOX combination is tolerable, active and a promising day-care regimen for advanced gastroesophageal cancer.