Drug-sensitive FGFR3 mutations in lung adenocarcinoma.

Background: Lung cancer is the leading cause of cancer-related deaths across the world. In this study, we present therapeutically relevant genetic alterations in lung adenocarcinoma of Indian origin. Materials and methods: Forty-five primary lung adenocarcinoma tumors were sequenced for 676 amplicons using RainDance cancer panel at an average coverage of 1500 × (reads per million mapped reads). To validate the findings, 49 mutations across 23 genes were genotyped in an additional set of 363 primary lung adenocarcinoma tumors using mass spectrometry. NIH/3T3 cells over expressing mutant and wild-type FGFR3 constructs were characterized for anchorage independent growth, constitutive activation, tumor formation and sensitivity to FGFR inhibitors using in vitro and xenograft mouse models. Results: We present the first spectrum of actionable alterations in lung adenocarcinoma tumors of Indian origin, and shows that mutations of FGFR3 are present in 20 of 363 (5.5%) patients. These FGFR3 mutations are constitutively active and oncogenic when ectopically expressed in NIH/3T3 cells and using a xenograft model in NOD/SCID mice. Inhibition of FGFR3 kinase activity inhibits transformation of NIH/3T3 overexpressing FGFR3 constructs and growth of tumors driven by FGFR3 in the xenograft models. The reduction in tumor size in the mouse is paralleled by a reduction in the amounts of phospho-ERK, validating the in vitro findings. Interestingly, the FGFR3 mutations are significantly higher in a proportion of younger patients and show a trend toward better overall survival, compared with patients lacking actionable alterations or those harboring KRAS mutations. Conclusion: We present the first actionable mutation spectrum in Indian lung cancer genome. These findings implicate FGFR3 as a novel therapeutic in lung adenocarcinoma.

Epidermal growth factor receptor mutation subtypes and geographical distribution among Indian non-small cell lung cancer patients.

BACKGROUND: The Medical Oncology Department at Tata Memorial Hospital, the single largest tertiary cancer care center in Asia, receives in-house registered and referral patient samples from all parts of the country. Our recent studies establish 23% EGFR mutation frequency among Indian population. Here, we extend our study and report further analysis of distribution of different types of EGFR mutations in 1018 non small cell lung cancer patient, and its co-relation with clinical parameters and geographical variations across the country. MATERIAL AND METHODS: This study is a retrospective analysis on all the patients who were referred for EFGR testing as a routine service over a 1.5 year period. This was part of standard care. EGFR kinase domain mutations in exon 18-21 were probed by TaqMan probe-based assays in 1018 NSCLC patients. RESULTS AND DISCUSSION: While EGFR exon 19 mutations, the most frequent EGFR mutation, were found be higher among non smokers females, we find surprisingly higher incidence of exon 21 mutations among EGFR mutation positive male smokers of Indian ethnicity. Furthermore, as Indian population is known to be composed of a gradient admixture of Ancestral North Indian (with genetic influence from Middle Easterners, Central Asians, and Europeans harboring variant EGFR mutation frequency) and Ancestral South Indians, as a paradox our study indicates comparable EGFR mutation frequency across different geographical locations within India CONCLUSION: Geographically there is uniform distribution in the EGFR mutation frequency within India. Further more, while exon 19 mutations are predominant among non smokers, higher incidence of exon 21 mutations exists among EGFR mutation positive male smokers of Indian ethnicity.

Role of RT-PCR and FISH in diagnosis and monitoring of acute promyelocytic leukemia.

BACKGROUND: Patients with a presence of Promyelocytic Leukemia-Retinoic Acid Receptor Alpha (PML-RARA) genes rearrangement predict a favorable response to all-trans retinoic acid (ATRA), and a significant improvement in survival. Therefore, establishing the presence of PML-RARA rearrangement is important for optimal patient management. AIM: The objective of this study is to compare and assess the role of fluorescent in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR) in the diagnosis and long-term monitoring of Acute Promyelocytic Leukemia (APL). MATERIALS AND METHODS: We compared 145 samples received at different interval of times to analyze the sensitivity of RT-PCR and FISH. RESULTS: The failure rate for RT-PCR was 4% at baseline, 13% at induction, and 0% at the end of consolidation. And for FISH it was 8% at baseline, 38% at induction, and 66% at the end of consolidation. The predictive values of relapse in the patients who were positive and negative by RT-PCR, at the end of induction, were 60% and 3%, respectively, and at end of consolidation it was 67% and 4%, respectively. On the other hand the predictive values of relapse in patients who were positive and negative by FISH at end of induction were 57% and 6%, respectively; while at end of consolidation it was 14% who were negative by FISH. CONCLUSION: Both RT-PCR and FISH are important for the diagnosis of APL cases, as both techniques complement each other in the absence or failure of any one of them. However, RT-PCR is more sensitive than FISH for the detection of minimal residual disease in the long-term monitoring of these patients. The present study shows that the predictive value of relapse is more associated with minimal residual disease (MRD) results by RT-PCR than that by FISH.

Identification of PML/RARalpha fusion gene transcripts that showed no t(15;17) with conventional karyotyping and fluorescent in situ hybridization.

Acute promyelocytic leukemia (APL) is characterized by a reciprocal translocation, t(15;17)(q22;q11-21), resulting in the fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARalpha) genes. Using conventional cytogenetic methods, these translocations are normally detected in about 70-90% of patients; most negative results are due to technical problems or cryptic variants. These masked PML/RARalpha fusions can be identified by molecular analyses, such as reverse transcriptase-polymerase chain reaction (RT-PCR) or fluorescence in situ hybridization (FISH). Approximately 5 to 10% of all APL cases reported do not show PML/RARalpha fusion transcripts, even with dual-colored FISH. We report three of 40 diagnosed APL cases that showed morphological, cytochemical, and immunophenotypic features of hypergranular APL, but did not show a PML/RARalpha fusion signal or any of its variants, on FISH. All cases were identified by RT-PCR, which was further confirmed by cDNA sequencing. Conventional karyotyping showed other clonal aberrations in these cases, but failed to show t(15;17) or any other variants or complex translocations.

Analysis and comparison of clinicohematological parameters and molecular and cytogenetic response of two Bcr/Abl fusion transcripts.

Different forms of p210 are produced by alternative splicing, namely b2a2 and b3a2. There have been many contrasting data establishing a relationship between the two Bcr/Abl transcripts and platelet counts and also response to treatment. However, the data published to date have been on a small group of patients. The aim of the present study was to determine whether there was any difference between clinical and hematological parameters at diagnosis between the two Bcr/Abl fusion transcripts in our population, and whether the two transcripts responded differently or similarly to imatinib treatment. RT-PCR was performed in 202 cases for detection of Bcr/Abl transcripts in newly diagnosed chronic myelogenous leukemia cases in one year. The two transcripts were compared and correlated with clinical, hematological and FISH data and with response to treatment. A total of 138 cases were of b3a2 and 64 were of b2a2 transcript. There was no correlation between the hematological parameters and the type of transcript. There was a significant association of blast crisis with b2a2, especially with myeloid blast crisis. When compared to FISH results, 10% of b3a2 were found to have a significant association with 5'Abl deletion as compared to 3% of b2a2. On analyzing the therapeutic response, we did not find any difference between the two transcripts. In conclusion, our findings confirm that the b3a2 type transcript is not significantly associated with thrombocytosis, that the short transcript, b2a2, occurs with acute phase, i.e., blast crisis, and that there is no difference in treatment response between the two transcripts. However, further studies are required to understand the molecular pathways involved in the Bcr/Abl mechanism.