The landscape of actionable genomic alterations in lung adenocarcinomas in India.

Lung adenocarcinoma (LUAD), the most prevalent form of non-small cell lung cancer (NSCLC), remains a leading cause of cancer-related death globally, including in India, with a 5-year survival rate below 10%. Despite these grim statistics, recent advances in the use of next-generation sequencing (NGS) for identifying genetic alterations and the emergence of targeted therapies have opened new possibilities for personalized treatment based on distinct molecular signatures. To understand the molecular pattern of NSCLC, a retrospective study was conducted with 53 Indian LUAD patient samples, using a targeted NGS panel of 46 cancer-relevant oncogenes to identify clinically relevant variants. Pathogenic or likely pathogenic variants were detected in 94% of the 53 cases. Non-synonymous mutations, rearrangements, copy number alterations, insertions, and deletions of functional relevance were observed in 31 out of 46 genes. The most frequently mutated genes included TP53 (52.8%) and EGFR (50.9%), followed by RET, PIK3CA and ERBB2; some patients had multiple alterations in the same gene. Gender-based enrichment analysis indicated that ALK and IDH2 alterations were more prevalent in females, while TP53 and PTEN were more common in males. No significant correlation was found between mutations and other clinicopathological attributes, such as age, stage, and subtype. A higher prevalence of EGFR, RET, PIK3CA, ERBB2 and ALK mutations were observed compared to previous LUAD genetic studies coupled with a lower frequency of KRAS mutations. Clinically actionable variants were annotated using OncoKB and categorized into the four therapeutic levels based on their clinical evidence. Seventy-nine percent of cases had at least one clinically actionable alteration. Most patients (39.6%) had the highest level of actionability (Level 1) wherein an FDA-approved drug is available specifically for the observed mutation in lung cancer patients. EGFR Exon19 in-frame deletions and EGFR L858R were the most frequent among targetable variants (20.7%). These findings emphasize the importance of a selective NGS panel in enabling personalized medicine approaches by identifying actionable molecular alterations and informing the choice of targeted therapy for more effective treatment options in Indian NSCLC patients.

A Multi-centric retrospective study into the epidemiological distribution of breast cancer patients in India.

BACKGROUND: A multicentric private hospital-based retrospective study was conducted to understand the epidemiology of breast cancer in terms of demographics and clinical characteristics (staging and hormone receptor status) at the time of diagnosis. METHODS: The data for 5,688 female breast cancer patients were collected from the hospital and clinical records of four study centres. All statistical analysis was performed using Microsoft Excel 2016 and R software. Survival was estimated by the Kaplan-Meier method and compared by the log-rank test. A P value of <.05 was considered statistically significant. RESULTS: The mean and median age of the study population was 52.6 (± 12.4) years and 53.0 (range 51-54 across the four centers) years, respectively. About 68% of patients were in the age category of 41 65 years, 17.6% were <40 years old among whom 23.4% of patients reported a positive family history. Most of the patients (66.3%) were diagnosed at an early stage (Stage I and II). The 3-year OS probability was 100%, 97.5%, 94.1%, and 74.7% for TNM Stages I, II, III, and IV, respectively. The 3-year RFS was 95.7%, 95.5%, 84.5%, and 49% for TNM Stages I, II, III, and IV, respectively. CONCLUSION: The present study highlights the epidemiological distribution of breast cancer patients. It emphasizes the importance of disease awareness among the urban and educated female population as most patients were diagnosed at earlier stages and demonstrated higher OS and RFS than reported in government registries.

Establishment and Characterization of Brain Cancer Primary Cell Cultures From Patients to Enable Phenotypic Screening for New Drugs.

Aim: Desmoplastic infantile ganglioglioma (DIG), is a rare tumor arising mainly during the first 2 years of life. Molecular characterization of these benign yet rapidly proliferating tumors has been limited to evaluating a few mutations in few genes. Our aim was to establish a live cell culture to enable the understanding of the cellular processes driving the non-malignant growth of these tumors. Methods: Tumor tissue from a rare non-infantile 8-year-old female DIG patient was dissociated and digested using collagenase to establish live cultures. Both 2D monolayer and 3D neurospheres were successfully cultured and characterized for proliferative potential, intrinsic plasticity, presence of cancer stem cells and the expression of stem cell markers. Cells cultured as 3D were embedded as tissue blocks. Immunohistochemistry was performed in both tissue and 3D sections for markers including synaptophysin, vimentin, neurofilament and MIB-1. Mutation analysis by NGS was performed using a-100 gene panel. Results: Using immunohistochemistry, the 3D cultures were shown to express markers as in the original DIG tumor tissue indicating that the spheroid cultures were able to maintain the heterogeneity found in the original tumor. Cells continued proliferating past passage 10 indicative of immortalization. Enrichment of cancer stem cells was observed in neurospheres by FACS using CD133 antibody and RT-PCR. Mutation analysis indicated the presence of germline mutations in three genes and somatic mutations in two other genes. Conclusion: A spontaneous cell line-like cell culture with high percentage of stem cells has been established from a DIG tumor for the first time.

A study on the impact of CYP2C19 genotype and platelet reactivity assay on patients undergoing PCI.

BACKGROUND: A thorough understanding of the patient's genotype and their functional response to a medication is necessary for improving event free survival. Several outcome studies support this view particularly if the patient is to be started on clopidogrel due to the prevalence of clopidogrel resistance. Such guided therapy has reduced the incidence of Major Adverse Cardiac Events (MACE) after stent implantation. METHODS: Between August 2013 and August 2014, 200 patients with coronary artery disease undergoing percutaneous coronary intervention (PCI) were prescribed any one of the anti-platelet medications such as clopidogrel, prasugrel or ticagrelor and offered testing to detect CYP2C19 gene mutations along with a platelet reactivity assay (PRA). Intended outcome was modification of anti-platelet therapy defined as either dose escalation of clopidogrel or replacement of clopidogrel with prasugrel or ticagrelor for the patients in clopidogrel arm, and replacement of ticagrelor or prasugrel with clopidogrel if those patients were non-carrier of mutant genes and also if they demonstrated bleeding tendencies in the ticagrelor and prasugrel arms. CONCLUSION: Clopidogrel resistance was observed to be 16.5% in our study population. PRA was useful in monitoring the efficacy of thienopyridines. By having this test, one can be safely maintained on clopidogrel in non-carriers, or with increased dose of clopidogrel in intermediate metabolizers or with newer drugs such as ticagrelor or prasugrel in poor metabolizers. Patients on ticagrelor and prasugrel identified as non-carriers of gene mutations for clopidogrel metabolism could be offered clopidogrel resulting in economic benefits to the patients. Patients at high risk of bleeding were also identified by the PRA.

The complex etiology of multiple sclerosis.

Multiple sclerosis is a demyelinating disease which is presumed to be a consequence of infiltrating lymphocytes autoreactive to myelin proteins. This is substantiated by several lines of clinical evidence and supported by correlative studies in preclinical models. The development of new therapeutics for MS has been guided by this perspective; however, the pathogenesis of MS has proven to be quite complex as observations exist which question the role of autoreactive lymphocytes in the etiology of MS. In addition the current immunomodulatory therapeutics do not prevent most patients from progressing into more serious forms of the disease. The development of truly transformational therapeutics for MS will likely require a broad assault that expands beyond the concept of MS being an autoimmune disease.

Novel inosine monophosphate dehydrogenase inhibitor VX-944 induces apoptosis in multiple myeloma cells primarily via caspase-independent AIF/Endo G pathway.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) is a small-molecule, selective, noncompetitive inhibitor directed against human IMPDH. In this report, we show that VX-944 inhibits in vitro growth of human multiple myeloma (MM) cell lines via induction of apoptosis. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs) do not protect against VX-944-induced MM cell growth inhibition. VX-944 induced apoptosis in MM cell lines with only modest activation of caspases 3, 8, and 9. Furthermore, the pan-caspase inhibitor z-VAD-fmk did not inhibit VX-944-induced apoptosis and cell death. During VX-944-induced apoptosis, expressions of Bax and Bak were enhanced, and both apoptosis-inducing factor (AIF) and endonuclease G (Endo G) were released from the mitochondria to cytosol, suggesting that VX-944 triggers apoptosis in MM cells primarily via a caspase-independent, Bax/AIF/Endo G pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin and melphalan even in the presence of BMSCs. Taken together, our data demonstrate a primarily non-caspase-dependent apoptotic pathway triggered by VX-944, thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional agents which trigger caspase activation.

Regulation of inosine monophosphate dehydrogenase type I and type II isoforms in human lymphocytes.

The enzyme inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo biosynthesis of guanine nucleotides. Inhibition of IMPDH leads to immunosuppression by decreasing guanine nucleotides that are required for the proliferation of lymphocytes. IMPDH activity is mediated by two highly conserved isoforms, type I and type II. We have characterized the mRNA and protein expression of the two isoforms in a variety of human tissues, peripheral blood mononuclear cells (PBMCs), and selected cell lines to investigate their regulation. Type I mRNA was expressed in most tissues with high expression in PBMCs and low expression in thymus. IMPDH type II transcript was also detected in most tissues with low expression in spleen and PBMCs. In PBMCs, induction of both type I and type II mRNAs was observed within 12 hr of mitogenic stimulation. Using type-selective IMPDH antibodies, an increase in the levels of type I and type II proteins was observed after mitogenic stimulation. The effect of two IMPDH inhibitors, MPA and VX-497, was investigated on the expression of type I and type II isoforms. VX-497 is an orally bioavailable, potent and reversible inhibitor of IMPDH, with broad applicability in many viral and immune system-mediated diseases. MPA and VX-497 inhibit both isoforms of IMPDH in vitro. Prolonged treatment of lymphocytes with either VX-497 or MPA did not lead to an increase in type I or type II IMPDH protein levels. These results are discussed in the context of IMPDH being a target for immunosuppressive, anti-viral and anti-cancer therapy.

Targeted disruption of the inosine 5'-monophosphate dehydrogenase type I gene in mice.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I(-/-) HPRT(-/0) splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.

Characterization of pharmacological efficacy of VX-148, a new, potent immunosuppressive inosine 5'-monophosphate dehydrogenase inhibitor.

Inosine 5'-monophosphate dehydrogenase (IMPDH) enzyme catalyzes the rate-limiting step in the de novo biosynthesis of guanine nucleotides. Proliferation of lymphocytes is critically dependent on this de novo nucleotide synthesis pathway. Hence, IMPDH is an attractive target for the development of immunosuppressive drugs. VX-148 is a novel, uncompetitive IMPDH inhibitor with a K(i) value of 6 nM against IMPDH type II enzyme. VX-148 is slightly more potent than mycophenolic acid and VX-497 in inhibiting the proliferation of mitogen-stimulated primary human lymphocytes (IC(50) value of ~80 nM). The inhibitory activity of VX-148 is alleviated in the presence of exogenous guanosine. VX-148 does not inhibit proliferation of nonlymphoid cell types such as fibroblasts, indicating selectivity for inhibition of IMPDH activity. VX-148 is orally bioavailable in rats and mice; oral administration of VX-148 inhibits primary antibody response in mice in a dose-dependent manner with an ED(50) value of 38 mg/kg b.i.d. VX-148 significantly prolongs skin graft survival at 100 mg/kg b.i.d. in mice. These results demonstrate that VX-148 is a potent and specific IMPDH inhibitor with a favorable pharmacokinetic profile and good pharmacological activity in mice, and thus support development of VX-148 as an immunosuppressive drug.