Whole exome sequencing identifies novel variants of PIK3CA and validation of hotspot mutation by droplet digital PCR in breast cancer among Indian population.

BACKGROUND: Breast cancer (BC) is the most common malignancy with very high incidence and relatively high mortality in women. The PIK3CA gene plays a pivotal role in the pathogenicity of breast cancer. Despite this, the mutational status of all exons except exons 9 and 20 still remains unknown. METHODS: This study uses the whole exome sequencing (WES) based approach to identify somatic PIK3CA mutations in Indian BC cohorts. The resultant hotspot mutations were validated by droplet digital PCR (ddPCR). Further, molecular dynamics (MD) simulation was applied to elucidate the conformational and functional effects of hotspot position on PIK3CA protein. RESULTS: In our cohort, PIK3CA showed a 44.4% somatic mutation rate and was among the top mutated genes. The mutations of PIK3CA were confined in Exons 5, 9, 11, 18, and 20, whereas the maximum number of mutations lies within exons 9 and 20. A total of 9 variants were found in our study, of which 2 were novel mutations observed on exons 9 (p.H554L) and 11 (p.S629P). However, H1047R was the hotspot mutation at exon 20 (20%). In tumor tissues, there was a considerable difference between copy number of wild-type and H1047R mutant was detected by ddPCR. Significant structural and conformational changes were observed during MD simulation, induced due to point mutation at H1047R/L position. CONCLUSIONS: The current study provides a comprehensive view of novel as well as reported single nucleotide variants (SNVs) in PIK3CA gene associated with Indian breast cancer cases. The mutation status of H1047R/L could serve as a prognostic value in terms of selecting targeted therapy in BC.

Occupational exposure to pesticides in female tea garden workers and adverse birth outcomes.

Pesticides are globally used to eliminate pests from crops and plants. The increased use of pesticides has posed a serious threat to human health. This study evaluates the effects of pesticide exposure on pregnancy outcomes in tea garden workers (TGW). The acetylcholinesterase (AChE) activity was measured in the maternal blood, placenta, and cord blood of TGW and housewives (HWs). The placental structure and expression of hypoxia-inducible factor (HIF)-1α were also analyzed in TGW and HW groups delivering low birth weight (LBW) and normal birth weight (NBW) babies. A significantly decreased AChE activity was observed in maternal blood and cord blood in TGW as compared with HW in the LBW group. However, it did not change significantly in the NBW group (p < .05). The adjusted regression analysis of birth outcomes (birth weight, head circumference, infant's length, and ponderal index) revealed a significant and positive association with the levels of AChE activity in maternal blood, placenta, and cord blood in TGW (p < .05). The histological analysis showed significantly higher placental syncytial knots, chorangiosis, fibrinoid deposition, necrosis, and stromal fibrosis in the LBW group of TGW. Microinfarction, increased fibrinoid deposition, and atypical villi characteristics, such as mushroom-like structures, were observed during scanning electron microscopy along with increased HIF-1α expression in placental tissues of TGW exposed to pesticides. Results suggest that occupational pesticide exposure during pregnancy may decrease AChE activity and cause in utero pathological changes accompanied by an increased HIF-1α expression, which also contributes to placental insufficiency and fetal growth restriction.

Differential expression of Ets-1 in breast cancer among North Indian population.

Breast cancer is a highly aggressive disease contributing to high mortality rate among females across the globe owing to wide geographical variations, change in lifestyle along with rapid tumor growth, drug resistance, and high metastasis rate. To understand the molecular and genetic basis of breast cancer progression; we studied the role of E26 transformation-specific-1 (Ets-1) transcription factor which is implicated to have a role in carcinogenesis like invasion, metastasis, angiogenesis, etc. Our findings revealed an overexpression of Ets-1 gene in 75 breast cancer tumors as compared with their normal adjacent tissues. The findings significantly established a co-relation between Ets-1 expression in breast cancer tissue with hormonal receptor profiles and ductal-lobular histological subtypes in Indian population. In addition, a differential expression pattern of Ets-1 was observed between high, moderate, and low grades of breast cancer patients. The present study demonstrates a crucial role of Ets-1 transcription factor which may serve as a potential biomarker for breast carcinogenesis.

Overexpression of COX2 indicates poor survival in urothelial bladder cancer.

BACKGROUND: COX2 is a cyclo-oxygenase enzyme expressed in the tumor cells, inflammatory cells, stromal and non-epithelial cells. The study was conducted to evaluate the expression of COX2 in Urothelial carcinoma and find the association with progression and recurrence. METHODS: The expression of COX2 was evaluated by real-time PCR and immunohistochemistry. RESULTS: Gene expression of COX2 was found to be upregulated >28-fold in urothelial cancer compared to adjacent normal bladder mucosa. Inflammatory cell expression of COX2 was found in 92% cases whereas only 37% cases showed COX2 overexpression in tumor cells. Tumor cell COX2 overexpression was significantly associated with invasion and recurrence. CONCLUSION: COX2 expression is a marker of invasion, recurrence and poor survival and may have a role in predicting the cases which will benefit from additional treatment with COX2 inhibitors in urothelial carcinoma.

Predictive role of serum and urinary cytokines in invasion and recurrence of bladder cancer.

Urothelial cancer patients are prone to recurrence, and there is no marker to predict which cases become refractory to the immunotherapy given to these patients. Tumour behaviour is decided by the dynamics between the pro- and anti-tumorigenic cytokines. In this study, 27 cytokines were estimated in serum and urine of 72 urothelial cancer patients and 42 healthy volunteer controls. Serum cytokines IL-1RA, IL-4 and RANTES were in significantly higher concentration in serum of patients compared to controls, while IL-2 was significantly less in concentration. Patients were found to have significantly high concentrations of 12 urinary cytokines (IL-2, IL-4, IL-8, IL-10, GM-CSF, IFN-γ, IP-10, MIP-1a, PDGF, MIP-1b, RANTES and VEGF) in comparison to healthy controls. Serum VEGF and urinary IL-1ra, IL-4, IL-10, GM-CSF, IP-10, MIP-1a and MIP-1b concentrations were found significantly higher in concentration in high-grade tumours compared to low-grade tumours. There was no difference in either the serum or urinary cytokines between non-invasive and muscle-invasive cases. Serum IL-1ra, IL-6, IL-10, TNF-α and VEGF and urinary IL-1ra, IL-4, IL-8, IL-10, GM-CSF, IP-10, MIP-1a, PDGF, MIP-1b and VEGF were found to be significantly higher in recurrent patients compared to non-recurrent patients. Of these, high concentrations of urinary IL-1RA, IL-4, IL-10, IP-10, PDGF and VEGF and serum IL-1ra, IL-6, IL-10, VEGF and TNF-α were associated with poor recurrence-free survival. Poor recurrence-free survival was also seen with increasing number of cytokines showing high concentrations. The study shows that the estimation of a combination of these cytokines in minimally or non-invasive samples may act as a prognostic indicator.

High-Grade Urothelial Carcinoma of Bladder Transforming to Micropapillary Variant on Follow-Up.

Micropapillary variant of urothelial carcinoma (UC) of the bladder is an aggressive tumour, comprising 0.6-6% of all UC. It generally presents with high-grade and stage, and has been reported as having a worse prognosis when compared to traditional UC. We report the case of a 58-year-old man who presented with macroscopic haematuria. The patient was diagnosed with high-grade urothelial carcinoma and returned with recurrence after 16 months. Histopathology after transurethral biopsy revealed a non-muscle invasive high-grade bladder tumour at first presentation, whereas tumour recurrence was reported after 1.5years. The histopathology at recurrence revealed a high-grade, muscle invasive, micropapillary variant of urothelial carcinoma with focal adenomatous morphology. Immunohistochemical expression of CK7+/CK20+ in tumour cells and negativity for PSA, AMACR, and CDX2 in paraffin section helped in identifying the tumour as primary in the urinary bladder. Radical cystectomy was performed and the patient has no distant metastases on follow-up. The specific morphology even within the high-grade urothelial cancer cases is important to discern for proper treatment.

Primary Leiomyosarcoma of the Mesentery: A Case Report.

Mesenteric masses are infrequent lesions ranging from benign cysts to aggressive malignancies and often present a diagnostic and therapeutic challenge. The mesentery is a frequent recipient of metastases from the gastrointestinal tract, pancreas, and biliary cancers. Primary mesenteric tumors are relatively rare, mostly mesenchymal in origin and benign in nature. Examples include gastrointestinal stromal tumors and smooth muscle tumors. We describe a 50-year-old woman, who presented with a lump in the left hypochondrium along with altered bowel habits of 2 years' duration. Imaging revealed a cystic lesion, raising the differentials of a benign lesion. Exploratory laparotomy revealed a large cystic mass in the mesentery closely abutting the jejunal loops. This was followed by mass resection along with a segment of the jejunum. Histopathological examination revealed features of a leiomyosarcoma. Postoperatively, the patient developed a colocutaneous fistula, which was re-excised. At 6 months' follow-up, the patient is doing well. Our case demonstrates the diagnostic challenge posed by the atypical clinical and imaging features of this tumor at an unusual site.

Determination of sample size for various study designs in medical research: A practical primer.

Formulating a research question and selecting an appropriate study design for answering that question are crucial initial steps in the research process. The population, intervention, control group, and outcomes measures (PICO time and setting [TS]) framework provides a practical guide in this regard, which stands for population, intervention, control, outcome, type of research question, and study design. The various study designs have their own merits and demerits, and implementing the methodology meticulously requires knowledge of all of these. Similarly, different methods of sample size calculation are warranted based on the most appropriate study design and outcome variables of interest. Sometimes, a post hoc power analysis can be performed after the sample size calculation, to check whether the study was adequately powered or not. There are multiple validated free software tools for sample size calculation, including Open-Epi, R, StatCalc, etc. The practice by most researchers of reporting significant P values is to be replaced by reporting effect sizes, as the latter is a much better estimate of the strength of association. This review provides a comprehensive, ready reckoner for busy family physicians to quickly identify the appropriate study design for answering any applied research questions in their minds and estimating the sample size required for the same.

Bioimpedance: A Tool for Screening Oral Cancer - A Systematic Review.

Objective: The successful management of cancer depends on proper screening and treatment methods. Bioimpedance spectroscopy (BIS) is an established technique in detecting breast cancer, cervical cancer, and prostate cancer. This systematic review sought to investigate the current evidence regarding the clinical application of bioimpedance in the detection of oral squamous cell carcinoma and oral potentially malignant disorders. Study Design: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed to perform this review. Electronic databases such as PubMed, MEDLINE, Embase, EBSCOhost, and Google Scholar were searched till March 2022. Articles published in the English medical literature on human participants report on the application of BIS in the screening of precancerous and cancerous lesions. The primary endpoint was defined as the ability to differentiate between normal and cancerous tissue. Results: A total of 6754 articles were identified; of which 481 were eligible for inclusion. Only five articles met the eligibility criteria and were included in the study. Qualitative analysis for each study was done to assess the data provided. All the studies demonstrated a significant divergence in BIS metrics between cancerous and normal tissue at 20 Hz and 50 KHz. Conclusion: Bioimpedance appears to be a promising novel tool for the detection of various malignancies which can be used in community screening due to its noninvasiveness and portability.

Protective Effects of Rifampicin and Its Analog Rifampicin Quinone in a Mouse Model of Obesity-Induced Type 2 Diabetes.

Advanced glycation end-products (AGEs) form when glucose reacts non-enzymatically with proteins, leading to abnormal protein function, oxidative stress, and inflammation. AGEs are associated with aging and age-related diseases; their formation is aggravated during diabetes. Therefore, drugs preventing AGE formation can potentially treat diabetic complications, positively affecting health. Earlier, we demonstrated that rifampicin and its analogs have potent anti-glycating activities and increase the life span of Caenorhabditis elegans. This study aimed to investigate the effects of rifampicin during hyperglycemia in C. elegans and in a mouse model of obesity-induced type 2 diabetes. The effects of rifampicin were assessed by determining the life span of C. elegans cultured in the presence of glucose and by measuring HbA1c, AGE levels, and glucose excursions in the diabetic mouse model. Our results show that rifampicin protects C. elegans from glucose-induced toxicity and increases life span. In mice, rifampicin reduces HbA1c and AGEs, improves insulin sensitivity, and reduces indications of diabetic nephropathy without inducing hepatotoxicity. Rifampicin quinone, an analog with lower anti-microbial activity, also reduces HbA1c levels, improves glucose homeostasis and insulin sensitivity, and lowers indications of diabetic nephropathy, without adversely affecting the liver of the diabetic mice. Altogether, our results indicate that rifampicin and its analog have protective roles during diabetes without inflicting hepatic damage and may potentially be considered for repositioning to treat hyperglycemia-related complications in patients.

Insights from establishing a high throughput viral diagnostic laboratory for SARS-CoV-2 RT-PCR testing facility: challenges and experiences.

Background: The World Health Organization declared the coronavirus disease 2019 (COVID-19) a global pandemic on 11 March 2020. Identifying the infected people and isolating them was the only measure that was available to control the viral spread, as there were no standardized treatment interventions available. Various public health measures, including vaccination, have been implemented to control the spread of the virus worldwide. India, being a densely populated country, required laboratories in different zones of the country with the capacity to test a large number of samples and report test results at the earliest. The Indian Council of Medical Research (ICMR) took the lead role in developing policies, generating advisories, formulating guidelines, and establishing and approving testing centers for COVID-19 testing. With advisories of ICMR, the National Institute of Cancer Prevention and Research (NICPR) established a high-throughput viral diagnostic laboratory (HTVDL) for RT-PCR-based diagnosis of SARS-CoV-2 in April 2020. HTVDL was established during the first lockdown to serve the nation in developing and adopting rapid testing procedures and to expand the testing capacity using "Real-Time PCR." The HTVDL provided its testing support to the national capital territory of Delhi and western Uttar Pradesh, with a testing capacity of 6000 tests per day. The experience of establishing a high-throughput laboratory with all standard operating procedures against varied challenges in a developing country such as India is explained in the current manuscript which will be useful globally to enhance the knowledge on establishing an HTVDL in pandemic or non-pandemic times.

Inhibition of protein kinase C isozymes causes immune profile alteration and possibly decreased tumorigenesis in bladder cancer.

Protein kinase C (PRKC) isozymes activate many signaling pathways and promote tumorigenesis, which can be confirmed by masking the kinase activity. In the present study, the kinase activity of PRKC ε and ζ isozymes was masked by siRNA in bladder cancer, and the consequent gene profile was evaluated. Here, we show that the commonly dysregulated genes affected by both the isozymes were the chemokines (CXCL8 & CXCL10), adhesion molecules (ICAM1, SPP1, MMP3, VEGFA) and mutated isoform of TP53. As these same genes were upregulated in bladder cancer patients, the activity of the kinase in downregulating them is confirmed. These genes are associated with regulating the tumor microenvironment, proliferation and differentiation of cancer cells and poor prognosis. The effect of kinase masking in downregulating these genes in bladder cancer indicates the benefits PRKC inhibitors may have in managing these patients.

Community-Minimal Invasive Tissue Sampling (cMITS) using a modified ambulance for ascertaining the cause of death: A novel approach piloted in a remote inaccessible rural area in India.

BACKGROUND: Melghat in India is a hilly, forested, difficult to access, impoverished rural area in northeast part of Maharashtra (Central India) with difficult healthcare access. Melghat has very high Mortality rates, because of grossly inadequate medical facilities. (1) Home deaths contribute to 67% of deaths,(2) which are difficult to track and where cause of death is mostly unknown. METHODS: A feasibility study was carried out in 93 rural villages and 5 hospitals to assess feasibility of tracking real-time community mortality and to ascertain cause of death in 0-60 months and 16-60 years age group using Minimal Invasive Tissue Sampling (MITS) in purpose-modified ambulance. We used the network of village health workers (VHW)s, to establish real-time community mortality tracking. Upon receipt of reports of home death, we performed MITS within 4 h of death in the vicinity of the village. RESULTS: We conducted 16 MITS. Nine, in MITS ambulance in community and seven at MAHAN hospital. The acceptance rate of MITS was 59.26%. Standard operating procedure (SOP) of conducting community MITS in an ambulance, is established. Major challenges were, Covid19 lockdown, reluctance of tribal parents for consent for MITS due to illiteracy, superstitions and fear of organ removal. Ambulance was an easy to reach transport means in remote area, provided a well-designed and discrete facility to perform MITS in community, winning the confidence of bereaved family. This has reduced time interval between time of death and performing MITS. CONCLUSIONS: MITS in purpose-modified Ambulance can be used worldwide for community MITS especially in areas which are remote and lack healthcare access. This solution needs to be assessed in different cultural settings to document culture specific issues.

A localized hydrogel-mediated chemotherapy causes immunogenic cell death via activation of ceramide-mediated unfolded protein response.

Treatment of triple-negative breast cancer (TNBC) is challenging because of its "COLD" tumor immunosuppressive microenvironment (TIME). Here, we present a hydrogel-mediated localized delivery of a combination of docetaxel (DTX) and carboplatin (CPT) (called DTX-CPT-Gel therapy) that ensured enhanced anticancer effect and tumor regression on multiple murine syngeneic and xenograft tumor models. DTX-CPT-Gel therapy modulated the TIME by an increase of antitumorigenic M1 macrophages, attenuation of myeloid-derived suppressor cells, and increase of granzyme B+CD8+ T cells. DTX-CPT-Gel therapy elevated ceramide levels in tumor tissues that activated the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-mediated unfolded protein response (UPR). This UPR-mediated activation of apoptotic cell death led to release of damage-associated molecular patterns, thereby activating the immunogenic cell death that could even clear the metastatic tumors. This study provides a promising hydrogel-mediated platform for DTX-CPT therapy that induces tumor regression and effective immune modulation and, therefore, can be explored further for treatment of TNBC.

Expression of androgen receptor in breast cancer & its correlation with other steroid receptors & growth factors.

BACKGROUND & OBJECTIVES: Breast cancer is the second most common malignancy in Indian women. Among the members of the steroid receptor superfamily the role of estrogen and progesterone receptors (ER and PR) is well established in breast cancer in predicting the prognosis and management of therapy, however, little is known about the clinical significance of androgen receptor (AR) in breast carcinogenesis. The present study was aimed to evaluate the expression of AR in breast cancer and to elucidate its clinical significance by correlating it with clinicopathological parameters, other steroid receptors (ER and PR) and growth factors receptors (EGFR and CD105). METHODS: Expression of AR, ER, PR, epidermal growth factor receptor (EGFR) and endoglin (CD105) was studied in 100 cases of breast cancer by immunohistochemistry (IHC). Risk ratio (RR) along with 95% confidence interval (CI) was estimated to assess the strength of association between the markers and clinicopathological characteristics. Categorical principal component analysis (CATPCA) was applied to obtain new sets of linearly combined expression, for their further evaluation with clinicopathological characteristics (n=100). RESULTS: In 31 cases presenting with locally advanced breast cancer (LABC), the expression of AR, ER, PR, EGFR and CD105 was associated with response to neoadjuvant chemotherapy (NACT). The results indicated the association of AR+ (P=0.001) and AR+/EGFR- (P=0.001) with the therapeutic response to NACT in LABC patients. The AR expression exhibited maximum sensitivity, specificity and likelihood ratio of positive and negative test. The present results showed the benefit of adding AR, EGFR and CD105 to the existing panel of markers to be able to predict response to therapy. INTERPRETATION & CONCLUSIONS: More studies on the expression profiles of AR+, AR+/CD105+ and AR+/EGFR- in larger set of breast cancer patients may possibly help in confirming their predictive role for therapeutic response in LABC patients.

Peroxiredoxins - Urinary Surveillance Biomarkers in Urothelial Cancer.

Introduction: Urinary bladder cancer ranks the fourth most common cancer in men worldwide. Peroxiredoxins (PRDXs) are antioxidant enzymes that play an important role in cell proliferation and apoptosis. In the present study, we investigated whether PRDX 1 and 2 can be used as a urinary biomarker for surveillance of recurrence in urothelial cancer. Materials and Methods: PRDX1 and PRDX2 expression levels were examined in 119 bladder tumor specimens by immunohistochemistry and in 150 urine samples (case: 100; healthy controls: 50) using ELISA and their association with recurrence and survival of patients was evaluated. Results: Immunohistochemistry on FFPE tissue showed that both PRDX1 and PRDX2 were positive in bladder tumors and expressed in the cytoplasm and membrane of tumor cells. A significant elevation of urinary PRDX1 and PRDX2 concentration was found in bladder cancer patients and recurrent cases compared to the urine of healthy controls and primary bladder cancer patients (p<0.001 & p<0.01) respectively. However, the concentration of both proteins was not found associated with survival. Conclusion: Elevated urinary PRDX1 and PRDX2 in bladder cancer patients was found to be associated with recurrence and the estimation of urinary PRDX1 and PRDX2 during follow-up may help to extend the period between cystoscopies in patient follow-up.

Progesterone limits the tumor-promoting effects of the beta-subunit of human chorionic gonadotropin via non-nuclear receptors.

The post-menopausal state in women is associated with increased cancer incidence, the reasons for which remain obscure. Curiously, increased circulating levels of beta-hCG (human chorionic gonadotropin) (a hormonal subunit linked with tumors of several lineages) are also often observed post-menopause. This study describes a previously unidentified interplay between beta-hCG and progesterone in tumorigenesis. Progesterone mediated apoptosis in beta-hCG responsive tumor cells via non-nuclear receptors. The transgenic expression of beta-hCG, particularly in the absence of the ovaries (a mimic of the post-menopausal state) constituted a potent pro-tumorigenic signal. Significantly, the administration of progesterone had significant anti-tumor effects. RNA-seq profiling identified molecular signatures associated with these processes. TCGA analysis revealed correlates between the expression of several newly identified genes and poor prognosis in post-menopausal patients of lung adenocarcinoma, colon adenocarcinoma, and glioblastoma. Specifically in these women, the detection of intra-tumoral/extra-tumoral beta-hCG may serve as a useful prognostic indicator, and treatment with progesterone on its detection may prove beneficial.

Effect of in utero exposure to SARS-CoV-2 infection on pregnancy outcomes and growth and development of infants: protocol for a multicentre ambispective cohort study in India.

INTRODUCTION: Poor pregnancy and neonatal outcomes in infants born to COVID-19 positive mothers have been reported, but there is insufficient evidence regarding subsequent growth and development of these children. Our study aims to explore the effect of in-utero exposure to SARS-CoV-2 on pregnancy outcomes and growth and development of infants. METHODS AND ANALYSIS: A multicentric ambispective cohort study with comparison group (1:1) will be conducted at six sites. A total of 2400 participants (exposure cohort, n=1200; comparison cohort, n=1200), ie, 400 participants from each site (200 retrospectively; 200 prospectively) will be included. Exposure cohort will be infants born to women with documented COVID-19 infection anytime during pregnancy and comparison cohort will be infants born to women who did not test positive for SARS-CoV-2 anytime during pregnancy. All infants will be followed up till 1 year of age. Anthropometric measurement, age of attainment of developmental milestones and clinical examination findings will be recorded at each follow-up. Data regarding possible cofactors affecting the outcomes will be collected from both groups and adjusted for during analysis. The two groups will be compared for prevalence of every variable considered in the study. Relative risk, attributable and population attributable risks will be calculated. All risk factors with p<0.1 on bivariate analysis will be subjected to multiple logistic regression analysis. A final multivariable model will be developed by including the statistically significant risk factors. ETHICS AND DISSEMINATION: The study has been approved by the Institutional Review Board of IIHMR Delhi (IRB/2021-2022/006) and will be required to be approved at all participating study sites. The study is scheduled from September 2021 to August 2023. Data from retrospective cohort will be reported by August 2022. All participants will provide written informed consent. We plan to publish our results in a peer-reviewed journal and present findings at academic conferences.