Molecular epidemiology and clinical characteristics of epidermal growth factor receptor mutations in NSCLC: A single-center experience from India.

Background: The genetic profiling of non-small cell lung cancer (NSCLC) has contributed to the discovery of actionable targetable mutations, which have significantly improved outcomes in disease with poor prognosis. Molecular epidemiological data of driver mutations in Indian populations have not been extensively elaborated compared to western and eastern Asian NSCLC populations. This study assessed the prevalence and clinical outcomes of EGFR (epidermal growth factor receptor) mutations among the Indian NSCLC cohort in South India. Patients and Methods: Retrospective analysis of 2,003 NSCLC patients who had undergone EGFR mutational analysis from 2013 to 2020 was performed. Clinical analysis was performed for 141 patients from 2013 to 2017 using Kaplan-Meier and Chi-square methods. Descriptive and survival statistics were performed using IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp. Results: EGFR-sensitizing mutations were detected in 41.6% (834/2003) in the study cohort with compound mutations detected in 7.55% (63/834) of EGFR-positive cases. A significant relationship with regard to female gender and EGFR mutation status (P <.001) was observed. Exon 18 G719X (8.7%) mutations and exon 20 T790M point mutation (3.1%) were the most frequently isolated uncommon EGFR mutations. In the clinical cohort, EGFR mutations were detected at a significantly higher prevalence in females (P =0.002) and never-smokers (P < 0.001). EGFR mutation demonstrated a significant relationship with regard to brain metastasis (P = 0.011). EGFR mutated individuals had significantly longer median overall survival compared to EGFR wild type (26 months vs. 12 months, P = 0.044). Conclusion: We reports the highest number of EGFR mutation analysis performed from India and mutational analysis indicated a loco-regional variation in India with regard to EGFR mutation frequency and its subtypes.

The role of p53 and ki67 in predicting clinical outcome in breast cancer patients.

Background: ">ki67 may be used as a proliferative index in addition to estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) negative status. p53 gene expression is a well-known biomarker in breast cancer and its role in predicting clinical outcome remains unclear. The current study aimed to determine the relationship between p53 gene mutation and ki67 expression, their clinical characteristics, and overall survival (OS), and to differentiate the significance of p53 and ki67 as the prognostic value in breast cancer patients. Methods: ">In this study, 135 patients were enrolled in the study from December 2015 to May 2017. Medical records for all patients were reviewed prospectively. The inclusion criteria included age more than 18 years with histologically proven breast cancer and willingness to be enrolled in p53 genetic study. Exclusion criteria included dual malignancy, male breast cancer, with a loss to follow-up during the study. Results: ">The mean survival of patients with ki67 ≤20 index was 42.7 months (95% confidence interval [CI] 38.7-46.7) and 129 months (95% CI 101.3-157.2) in patients with ki67 >20. The mean OS was 145 months (95% CI 105.6-185.5) in the p53 wild-type group and 106 months (95% CI 78.0-133.0) in the p53 mutated group, as illustrated. Conclusion: ">Our results indicated that p53 mutational status and high ki67 might have an essential impact on overall survival, with p53 mutated patients having a poorer outcome than p53 wild type patients.

Landscape of clinically actionable mutations in breast cancer 'A cohort study'.

Breast cancer (BC) is a heterogeneous disease. Numerous chemotherapeutic agents are available for early stage or advanced/metastatic breast cancer to provide maximum benefit with minimum side effects. However, the clinical outcome of patients with the same clinical and pathological characteristics and treated with similar treatments may show major differences and a vast majority of patients still develop treatment resistance and eventually succumb to disease. It remains an unmet need to identify specific molecular defects, new biomarkers to enable clinicians to adopt individualized treatment for every patient in terms of endocrine, chemotherapy or targeted therapy which will improve clinical outcomes in BC. Our study aimed to identify frequent hotspot mutation profile in BC by targeted deep sequencing in cancer-related genes using Illumina Truseq amplicon/Swift Accel-Amplicon panel and MiSeq technology in an IRB-approved prospective study in a CLIA compliant laboratory. All the cases had pathology review for stage, histological type, hormonal status and Ki-67. Data was processed using Strand NGS™. Mutations identified in the tumor were assessed for 'actionability' i.e. response to therapy and impact on prognosis.

Molecular landscape of salivary gland cancers.

Accurate diagnosis of salivary gland tumors can be challenging because of the many diagnostic entities, the sometimes extensive morphologic overlap, and the rarity of most tumor types. The current understanding of molecular rearrangements in salivary gland tumor pathology, emphasizes the prospects for exploiting molecular alterations in salivary gland tumors for diagnosis and targeted therapy. As new targeted therapies emerge, it will become increasingly vital to incorporate appropriate molecular testing into the pathologic evaluation of salivary gland cancers.

Biomarkers in Non-Small Cell Lung Cancers: Indian Consensus Guidelines for Molecular Testing.

Novel molecular targets and promising targeted therapies have reshaped diagnostics in patients with advanced non-small cell lung cancer (NSCLC). Despite this progress, the implementation of molecular screening to identify predictive biomarkers in Indian clinical and pathology settings has been challenging due to operational and logistical constraints. This consensus guideline brings together medical oncologists, molecular pathologists and pathologists from India to provide a quick and competent reference for biomarker testing in NSCLC. The guideline summarizes the importance of targetable mutations in NSCLC such as epidermal growth factor receptor (EGFR), rearrangements in anaplastic lymphoma kinase and receptor tyrosine kinase encoded by ROS-1 gene, overexpression of programmed cell death ligand-1 and resistant EGFR mutations. It reaffirms recommendations from international working groups, discusses vulnerable pre-analytical procedures and provides a balanced review on the pros and cons of different diagnostic tests (immunohistochemistry, fluorescence in situ hybridization, polymerase chain reaction-based testing and next-generation sequencing). The document also provides an algorithm to aid diagnostic decision-making and a checklist to assess the quality of testing laboratories that will help the medical oncologists make an informed choice. Overall, these recommendations are based on evidence and clinical experience and will aid policymakers, oncologists, health care practitioners and pathologists who strive to implement molecular strategies and make informed decisions for improved care in NSCLC in India.Funding: AstraZeneca Pharma India Limited.

Screening of over 1000 Indian patients with breast and/or ovarian cancer with a multi-gene panel: prevalence of BRCA1/2 and non-BRCA mutations.

PURPOSE: Breast and/or ovarian cancers are among the most common cancers in women across the world. In the Indian population, the healthcare burden of breast and/or ovarian cancers has been steadily rising, thus stressing the need for early detection, surveillance, and disease management measures. However, the burden attributable to inherited mutations is not well characterized. METHODS: We sequenced 1010 unrelated patients and families from across India with an indication of breast and/or ovarian cancers, using the TruSight Cancer panel which includes 14 genes, strongly associated with risk of hereditary breast and/or ovarian cancers. Genetic variations were identified using the StrandNGS software and interpreted using the StrandOmics platform. RESULTS: We were able to detect mutations in 304 (30.1%) cases, of which, 56 mutations were novel. A majority (84.9%) of the mutations were detected in the BRCA1/2 genes as compared to non-BRCA genes (15.1%). When the cases were stratified on the basis of age at diagnosis and family history of cancer, the high rate of 75% of detection of hereditary variants was observed in patients whose age at diagnosis was below 40 years and had first-degree family member(s) affected by breast and/or ovarian cancers. Our findings indicate that in the Indian population, there is a high prevalence of mutations in the high-risk breast cancer genes: BRCA1, BRCA2, TP53, and PALB2. CONCLUSION: In India, socioeconomic inequality limiting access to treatment is a major factor towards increased cancer burden; therefore, incorporation of a cost-effective and comprehensive multi-gene test will be helpful in ensuring widespread implementation of genetic screening in the clinical practice for hereditary breast and/or ovarian cancers.

Detection of high frequency of mutations in a breast and/or ovarian cancer cohort: implications of embracing a multi-gene panel in molecular diagnosis in India.

Breast and/or ovarian cancer (BOC) are among the most frequently diagnosed forms of hereditary cancers and leading cause of death in India. This emphasizes on the need for a cost-effective method for early detection of these cancers. We sequenced 141 unrelated patients and families with BOC using the TruSight Cancer panel, which includes 13 genes strongly associated with risk of inherited BOC. Multi-gene sequencing was done on the Illumina MiSeq platform. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. We were able to detect pathogenic mutations in 51 (36.2%) cases, out of which 19 were novel mutations. When we considered familial breast cancer cases only, the detection rate increased to 52%. When cases were stratified based on age of diagnosis into three categories, ⩽40 years, 40-50 years and >50 years, the detection rates were higher in the first two categories (44.4% and 53.4%, respectively) as compared with the third category, in which it was 26.9%. Our study suggests that next-generation sequencing-based multi-gene panels increase the sensitivity of mutation detection and help in identifying patients with a high risk of developing cancer as compared with sequential tests of individual genes.

MdmX inhibits ARF mediated Mdm2 sumoylation.

Mdm2, by virtue of an intrinsic E3 ubiquitin ligase activity, is capable of autoubiquitination and the ubiquitination of the p53 tumor suppressor protein. Additionally, Hdm2 has been reported to undergo a p14ARF-dependent sumoylation with concurrent Hdm2 stabilization. In this present work, we report that MdmX can undergo ARF-mediated sumoylation similar to that reported for Mdm2. When coexpressed, MdmX overexpression results in a dose-dependent inhibition of Mdm2 sumoylation and a concurrent increase in Mdm2 ubiquitination. This switch from Mdm2 sumoylation to Mdm2 ubiquitination may explain the destablization of Mdm2 previously observed in cells overexpressing both ARF and MdmX. Given that MdmX can heterodimerize with Mdm2 and separately associate with ARF we employed a series of MdmX mutants to examine how MdmX blocks Mdm2 sumoylation. A MdmX miniprotein capable of binding to ARF, but not p53 or Mdm2 was able to competitively inhibit Mdm2 sumoylation and reverse ARF mediated activation of p53 transactivation. Taken together, these results demonstrate that MdmX can affect post-translational modification and stability of Mdm2 and p53 activity through interaction with ARF.

MdmX represses E2F1 transactivation.

Based on knockout mouse studies, Mdm2 and MdmX have been identified as critical regulators of the p53 tumor suppressor protein, at least during early development. While many of the functions attributed to Mdm2 and MdmX involve p53 and overexpression of each gene appears to have oncogenic activities, a number of studies have suggested that each protein also possesses p53-independent functions. While examining the effect of Mdm2 overexpression on E2F1 transactivation we uncovered a novel MdmX function, the ability to inhibit E2F1 transactivation in a p53 and Mdm2 independent manner. Using a series of MdmX deletion mutants the central region of MdmX, amino acids 128-444 appears to possess the repressive domain. While an in vivo association of MdmX with either E2F1 or DP1 was not observed, a slight reduction in DP1 and an increased cytoplasmic localization of E2F1 were seen in cells overexpressing MdmX. These results suggest that elevated MdmX expression may repress E2F1-regulated genes like p14ARF and thus represent another regulatory mechanism in the Rb-p53 signaling pathway.

Overexpression of Mdm2 and MdmX fusion proteins alters p53 mediated transactivation, ubiquitination, and degradation.

Mdm2 and MdmX function as cellular regulators of the p53 tumor suppressor protein. Mdm2, a p53 inducible protein, negatively regulates p53 by inhibiting p53 transcriptional activity and promoting ubiquitin mediated proteasome degradation. The Mdm2 ring finger domain has been shown to possess E3 ligase activity and to be a necessary domain for targeting p53 degradation. MdmX, a p53 binding protein sharing a high degree of structural homology with Mdm2, has emerged as another negative regulator of the p53 tumor suppressor. MdmX has also been shown to block p53 transactivation but unlike Mdm2 cannot induce p53 degradation. Since MdmX also possesses a ring finger domain that allows MdmX to associate with Mdm2, this study focused on elucidating how the ring and zinc fingers of these two proteins affected p53 function. We have generated a series of fusion proteins between Mdm2 and MdmX by swapping the ring finger domains with or without the zinc finger domains and examined how these fusions regulated p53 induced transactivation, ubiquitination, and degradation. All fusions inhibited the transcriptional activity of p53. In the absence of Mdm2, none of the fusion proteins could trigger p53 ubiquitination or degradation. However, in a cell line with endogenous Hdm2, Mdm2:X fusions containing the ring finger domain with or without the zinc finger domain demonstrated p53 ubiquitination presumably through stabilization of Hdm2. Additionally, an Mdm2:XZFRF fusion also degraded p53 when endogenous Hdm2 was present. Results from immunofluorescence studies suggest that p53 is colocalized to the cytoplasm when coexpressed with a Mdm2:X fusion (Mdm2:XZFRF) and that this fusion is capable of stabilizing endogenous Hdm2. Since none of the fusions triggered p53 ubiquitination in cells lacking Mdm2, these results indicate that the E3 ligase domain within the ring finger of Mdm2 when part of MdmX and the MdmX ring finger fused to Mdm2 were not sufficient to trigger p53 ubiquitination, in vivo.