Novel oncogenic transcriptional targets of mutant p53 in esophageal squamous cell carcinoma.

Missense mutations in the DNA binding domain of p53 are observed frequently in esophageal squamous cell carcinoma (ESCC). Recent studies have revealed the potentially oncogenic transcriptional networks regulated by mutant p53 proteins. However, majority of these studies have focused on common "hotspot" p53 mutations while rarer mutations are poorly characterized. In this study, we report the characterization of rare, "non-hotspot" p53 mutations from ESCC. In vitro tumorigenic assays performed following ectopic-expression of certain "non-hotspot" mutant p53 proteins caused enhancement of oncogenic properties in squamous carcinoma cell lines. Genome-wide transcript profiling of ESCC tumor samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumors harboring mutant p53. Of these, ARF6, C1QBP, and TRIM23 were studied further. Reverse transcription-quantitative PCR (RT-qPCR) performed on RNA isolated from ESCC tumors revealed significant correlation of TP53 transcript levels with those of the three target genes. Ectopic expression of wild-type and several mutant p53 forms followed by RT-qPCR, chromatin affinity-purification (ChAP), and promoter-luciferase assays indicated the exclusive recruitment of p53 mutants-P190T and P278L, to the target genes leading to the activation of expression. Several functional assays following knockdown of the target genes revealed a significant suppression of tumorigenicity in squamous carcinoma cell lines. Rescue experiments confirmed the specificity of the knockdown. The tumorigenic effects of the genes were confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of "non-hotspot" mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor-specific p53 mutations.

Short stature and dysmorphic features in Asian Indian siblings with DAAM2-associated steroid-resistant nephrotic syndrome: Expansion of the phenotypic spectrum or a blended phenotype?

Variants in more than 60 different genes, most of which code for podocyte-related proteins, have been found to be associated with monogenic forms of nephrotic syndrome (NS). Biallelic variants in DAAM2, a member of the formin family, were recently identified to cause autosomal recessive (AR) NS type 24 in four unrelated families with steroid-resistant nephrotic syndrome (SRNS). This case report represents only the fifth reported family of DAAM2-associated NS and the first from India, with two sibs who presented with a complex phenotype characterized by steroid-resistant nephrotic syndrome, short stature, dysmorphic facial features, deep-set toenails, myopia, increased thickness of the calvarium of the skull, and sloping ribs. Both sibs were found to have a homozygous likely pathogenic nonsense variant c.196C>T (p.Arg66Ter; NM_001201427.2) in exon 3 of the DAAM2 gene through whole exome sequencing. The dysmorphic features could possibly be part of the DAAM2-related phenotype which has hitherto not been reported or could represent a blended phenotype, with the extrarenal manifestations resulting from a yet to be identified coexisting genetic condition.

Aberrant cytoplasmic localization of ARID1B activates ERK signaling and promotes oncogenesis.

The ARID1B (BAF250b) subunit of the human SWI/SNF chromatin remodeling complex is a canonical nuclear tumor suppressor. We employed in silico prediction, intracellular fluorescence and cellular fractionation-based subcellular localization analyses to identify the ARID1B nuclear localization signal (NLS). A cytoplasm-restricted ARID1B-NLS mutant was significantly compromised in its canonical transcription activation and tumor suppressive functions, as expected. Surprisingly however, cytoplasmic localization appeared to induce a gain of oncogenic function for ARID1B, as evidenced from several cell line- and mouse xenograft-based assays. Mechanistically, cytoplasm-localized ARID1B could bind c-RAF (RAF1) and PPP1CA causing stimulation of RAF-ERK signaling and ß-catenin (CTNNB1) transcription activity. ARID1B harboring NLS mutations derived from tumor samples also exhibited aberrant cytoplasmic localization and acquired a neo-morphic oncogenic function via activation of RAF-ERK signaling. Furthermore, immunohistochemistry on a tissue microarray revealed significant correlation of ARID1B cytoplasmic localization with increased levels of active forms of ERK1 and ERK2 (also known as MAPK3 and MAPK1) and of ß-catenin, as well as with advanced tumor stage and lymph node positivity in human primary pancreatic tumor tissues. ARID1B therefore promotes oncogenesis through cytoplasm-based gain-of-function mechanisms in addition to dysregulation in the nucleus.This article has an associated First Person interview with the first author of the paper.

Kidney transplant dysfunction in a patient with COVID - 19 infection: role of concurrent Sars-Cov 2 nephropathy, chronic rejection and vitamin C-mediated hyperoxalosis: case report.

BACKGROUND: COVID-19 infection in kidney transplant recipients often lead to allograft dysfunction. The allograft injury has various histopathological manifestations. Our case illustrates the unusual combination of allograft rejection, acute kidney injury secondary to oxalate nephropathy and SARS CoV-2 nephropathy as the cause of irreversible allograft failure. CASE PRESENTATION: A 56 year old renal allograft recipient presented with a history of fever and diarrhoea for the preceding 4 weeks, tested positive for Sars-CoV2 on nasal swab and was found to have severe allograft dysfunction, necessitating haemodialysis. He subsequently underwent an allograft biopsy, which demonstrated antibody mediated rejection along with the presence of extensive oxalate deposition in the tubules. Ultrastructural examination demonstrated spherical spiked particles in the glomerular capillary endothelium and the presence of tubulo-reticular inclusions suggestive of an active COVID-19 infection within the kidney. The intra-tubular oxalate deposition was considered to be the result of high dose, supplemental Vitamin C used as an immune booster in many patients with COVID - 19 infection in India. CONCLUSIONS: This case highlights the complex pathology that may be seen in following COVID-19 disease and the need for kidney biopsies in these patients to better understand the aetiology of disease.

Chronic interstitial nephritis in agricultural communities is a toxin-induced proximal tubular nephropathy.

Almost 30 years after the detection of chronic interstitial nephritis in agricultural communities (CINAC) its etiology remains unknown. To help define this we examined 34 renal biopsies from Sri Lanka, El Salvador, India and France of patients with chronic kidney disease 2-3 and diagnosed with CINAC by light and electron microscopy. In addition to known histopathology, we identified a unique constellation of proximal tubular cell findings including large dysmorphic lysosomes with a light-medium electron-dense matrix containing dispersed dark electron-dense non-membrane bound "aggregates". These aggregates associated with varying degrees of cellular/tubular atrophy, apparent cell fragment shedding and no-weak proximal tubular cell proliferative capacity. Identical lysosomal lesions, identifiable by electron microscopy, were observed in 9% of renal transplant implantation biopsies, but were more prevalent in six month (50%) and 12 month (67%) protocol biopsies and in indication biopsies (76%) of calcineurin inhibitor treated transplant patients. The phenotype was also found associated with nephrotoxic drugs (lomustine, clomiphene, lithium, cocaine) and in some patients with light chain tubulopathy, all conditions that can be directly or indirectly linked to calcineurin pathway inhibition or modulation. One hundred biopsies of normal kidneys, drug/toxin induced nephropathies, and overt proteinuric patients of different etiologies to some extent could demonstrate the light microscopic proximal tubular cell changes, but rarely the electron microscopic lysosomal features. Rats treated with the calcineurin inhibitor cyclosporine for four weeks developed similar proximal tubular cell lysosomal alterations, which were absent in a dehydration group. Overall, the finding of an identical proximal tubular cell (lysosomal) lesion in CINAC and calcineurin inhibitor nephrotoxicity in different geographic regions suggests a common paradigm where CINAC patients undergo a tubulotoxic mechanism similar to calcineurin inhibitor nephrotoxicity.

XPNPEP3 is a novel transcriptional target of canonical Wnt/ß-catenin signaling.

Canonical Wnt/ß-catenin signaling plays important roles in embryonic development and adult tissue regeneration while aberrant Wnt activation is the major driver of sporadic colorectal cancer (CRC). Thus, it is important to characterize the complete ß-catenin target transcriptome. We previously performed microarray-based mRNA profiling of rectal cancer samples stratified for Wnt status. In addition to AXIN2 and EPHB2, XPNPEP3 transcripts were significantly elevated in tumors exhibiting activated Wnt/ß-catenin signaling, validated by Q-PCR. Three different cell lines supported elevated XPNPEP3 transcript levels upon activation of Wnt signaling, confirmed using promoter-luciferase assays. Ectopic expression of XPNPEP3 promoted tumorigenic properties in CRC cells. Immunohistochemistry on a CRC tissue microarray revealed significant correlation between ß-catenin nuclear localization and XPNPEP3 levels. More importantly, XPNPEP3 expression was upregulated compared to normal samples in published expression data sets from several cancers including CRC. Finally, XPNPEP3 expression correlated with poor survival in many cancers. Our results therefore suggest XPNPEP3 to be a transcriptional target of Wnt/ß-catenin pathway with particular significance for CRC.

Evidence for presence of mismatch repair gene expression positive Lynch syndrome cases in India.

Lynch syndrome (LS), the most common form of familial CRC predisposition that causes tumor onset at a young age, is characterized by the presence of microsatellite instability (MSI) in tumors due to germline inactivation of mismatch repair (MMR) system. Two MMR genes namely MLH1 and MSH2 account for majority of LS cases while MSH6 and PMS2 may account for a minor proportion. In order to identify MMR genes causing LS in India, we analyzed MSI and determined expression status of the four MMR genes in forty eight suspected LS patient colorectal tumor samples. Though a majority exhibited MSI, only 58% exhibited loss of MMR expression, a significantly low proportion compared to reports from other populations. PCR-DNA sequencing and MLPA-based mutation and exonic deletion/duplication screening respectively, revealed genetic lesions in samples with and without MMR gene expression. Interestingly, tumor samples with and without MMR expression exhibited significant differences with respect to histological (mucin content) and molecular (instability exhibited by mononucleotide microsatellites) features. The study has revealed for the first time a significant proportion of LS tumors not exhibiting loss of MMR expression.

Evidence for possible non-canonical pathway(s) driven early-onset colorectal cancer in India.

Two genetic instability pathways viz. chromosomal instability, driven primarily by APC mutation induced deregulated Wnt signaling, and microsatellite instability (MSI) caused by mismatch repair (MMR) inactivation, together account for >90% of late-onset colorectal cancer (CRC). Our understanding of early-onset sporadic CRC is however comparatively limited. In addition, most seminal studies have been performed in the western population and analyses of tumorigenesis pathway(s) causing CRC in developing nations have been rare. We performed a comparative analysis of early and late-onset CRC from India with respect to common genetic aberrations including Wnt, KRAS, and p53 (constituting the classical CRC progression sequence) in addition to MSI. Our results revealed the absence of Wnt and MSI in a significant proportion of early-onset as against late-onset CRC in India. In addition, KRAS mutation frequency was significantly lower in early-onset CRC indicating that a significant proportion of CRC in India may follow tumorigenesis pathways distinct from the classical CRC progression sequence. Our study has therefore revealed the possible existence of non-canonical tumorigenesis pathways in early-onset CRC in India.

P53 nuclear stabilization is associated with FHIT loss and younger age of onset in squamous cell carcinoma of oral tongue.

BACKGROUND: Squamous cell carcinoma of tongue (SCCT) is expected to harbor unique clinico-pathological and molecular genetic features since a significant proportion of patients are young and exhibit no association with tobacco or alcohol. METHODS: We determined P53, epidermal growth factor receptor, microsatellite instability, human papilloma virus infection and loss of heterozygosity status at several tumor suppressor loci in one hundred and twenty one oral SCCT (SSCOT) samples and analyzed their association with clinico-pathological features and patient survival. RESULTS: Our results revealed a significantly higher incidence of p53 nuclear stabilization in early (as against late) onset SCCOT. FHIT loss was significantly associated with p53 nuclear stabilization and the association was stronger in patients with no history of tobacco use. Samples harboring mutation in p53 DNA binding domain or exhibiting p53 nuclear stabilization, were significantly associated with poor survival. CONCLUSION: Our study has therefore identified distinct features in SCCOT tumorigenesis with respect to age and tobacco exposure and revealed possible prognostic utility of p53.

To study the expression of estrogen, progesterone receptor and p53 immunohistochemistry markers in subtyping endometrial carcinoma.

Background: Endometrial cancer is one of the most commonly diagnosed cancers in women worldwide. Aim and Objectives: To study the expression of estrogen receptor (ER), progesterone receptor (PR) and p53 immunohistochemistry (IHC) markers in subtyping endometrial carcinoma. Materials and Methods: A total of 100 cases of carcinoma endometrium submitted during January 2016 to October 2018 were included in our study. The ER, PR and p53 expressions were scored as per the adopted scoring system. Agreement between ER, PR and p53 IHC expression and the consensus HE diagnosis, FIGO grading and tumour staging were assessed using Chi square tests. Results: There was a statistical association between ER, PR and p53 status and tumour histologic type with a P value < 0.01. There was no statistical significance observed between ER and PR expressions and different FIGO grades. Statistical significance (P = 0.036) between p53 and different FIGO grades seen. No statistical significance was observed between ER, PR and p53 expressions and different tumour stages and tumour invasiveness. There was a statistical association between ER and PR status and lymph node metastasis. p53 did not show a statistical significance. Conclusion: Combination of ER, PR and p53 IHC markers can be used to distinguish type 1 and type 2 endometrial cancers. PR expression is more specific than ER in endometrioid carcinomas. p53 expression is more specific in serous carcinoma, however, p53 IHC alone cannot be used to distinguish different grades of endometrioid carcinomas as there is variability of staining in endometrioid carcinomas.

Distinct genetic aberrations in oesophageal adeno and squamous carcinoma.

BACKGROUND: The two main oesophageal cancer subtypes namely adenocarcinoma and squamous cell carcinoma exhibit interesting clinical, pathological and geographical variations with the former being more common in the West and the latter in Asia. MATERIALS AND METHODS: We evaluated status of p53, EGFR, Wnt and HPV in addition to microsatellite instability and loss of heterozygosity of several chromosomal loci in the two oesophageal cancer subtypes from India. The comparative analysis was extended to two oesophageal adenosquamous mixed cancer samples. RESULTS: Our results reveal a high frequency of EGFR overexpression in ESCC as against EAC, while Wnt activation was a significantly more common event in EAC as against ESCC. Frequencies of p53 perturbations were not significantly different in the two subtypes. Interestingly, the EGFR and Wnt status in adenocarcinoma and squamous components of the two oesophageal adenosquamous cancer samples were identical to primary tumours. In addition, no common molecular aberration (including instability and loss of heterozygosity) in several microsatellites was detected in DNA isolated from the two components in both adenosquamous cancer samples. CONCLUSIONS: Our results reveal the presence of distinct aberrations in oesophageal adenocarcinoma and squamous cell carcinoma which are replicated in the respective components of adenosquamous cancers. The study therefore suggests perhaps an independent origin of the two components of oesophageal adenosquamous mixed cancer.

Successful Renal Transplant in Castleman Disease - First Case Report from India.

Castleman disease (CD) comprises a rare group of heterogenous benign lymphoproliferative disorders with pathologic similarities. However, they present with diverse clinical manifestations. Renal involvement is rare in CD and is mainly reported with plasma cell type of multicentric disease. Various glomerular pathologies, interstitial involvement, or thrombotic microangiopathies have all been reported, some of which progress to end-stage renal disease (ESRD). Progression of CD to ESRD is well documented; however, a patient on dialysis developing CD is rare. Moreover, kidney transplantations have seldom been performed on patients with CD. We report a patient with ESRD of unknown etiology who developed multicentric CD while on dialysis. He was treated with four doses of rituximab and later underwent a living kidney transplant with his wife as a donor. He has been clinically well ever since. We believe that this is possibly the first successful case of renal transplantation in CD with ESRD being reported from India.

A Study of Focal and Segmental Glomerulosclerosis according to the Columbia Classification and Its Correlation with the Clinical Outcome.

Introduction Focal and segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome in both adults and children. The "Columbia classification of FSGS" includes five variants; not otherwise specified (NOS), tip, perihilar, cellular, and collapsing variants that may have different prognostic and therapeutic implications. Materials and Methods This is a retrospective study and was carried out in the Department of Histopathology, Apollo Hospitals, Hyderabad. Of a total of 11,691 kidney biopsies over a 7-year period, from 2006 to 2012, 824 cases were diagnosed as FSGS, of which 610 cases in which detailed clinical findings were available were included in this study. FSGS was then categorized according to the Columbia classification. Results FSGS, NOS was the predominant histomorphological variant. Serum creatinine was significantly high in the collapsing variant, followed by NOS. Follow-up data was available for 103 cases,72.8% had complete remission, 10.6% had partial remission, and in 16.5 % there was no remission. Relapses were observed in 6.7% cases, two patients (1.9%) succumbed, and 4.8% cases progressed to chronic kidney disease. Conclusion This study showed that perihilar variant was less prevalent, with tip and cellular variants being more prevalent in Indian subcontinent compared to Western literature. Collapsing variant was also less common.

Proliferative Glomerulonephritis with Monoclonal Immunoglobulins in a Child with Recurrence in the Allograft.

Proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID) is among the spectrum of monoclonal protein-associated renal diseases, with only about 15 case reports in children. We report a 7-year-old boy with biopsy-proven crescentic PGNMID who progressed to end-stage renal disease within a few months of presentation. He then received a renal transplant with his grandmother as a donor. Proteinuria was detected at 27 months post-transplant and an allograft biopsy revealed a recurrent disease.

Banff Classification from 1991 to 2019. A Significant Contribution to Our Understanding and Reporting of Allograft Renal Biopsies.

The Banff schema of classification of renal allograft biopsies, first proposed at the meeting in Banff, Canada in 1991 has evolved through subsequent meetings held once in two years and is the internationally accepted scheme of classification which is consensual, current, validated and in clinical use. This review traces the evolution of the classification and our understanding of renal transplant pathology, with emphasis on alloimmune reactions. The proceedings of the meetings and the important studies which have shaped the classification are covered.

Myoglobinuria-induced Acute Kidney Injury Secondary to CovishieldTM Vaccination.

Vaccination is the best strategy for the development of herd immunity and for the control of the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. As the number of immunizations across the globe reaches a record number, random cases of diverse adverse effects of the vaccines are being documented. We report a case of renal biopsy-proven myoglobin-induced acute tubular injury requiring dialytic support post-CovishieldTM vaccination. Awareness of this rare complication is necessary so that it can be recognized early, and renal injury avoided.

Whole slide imaging vs eyeballing: The future in quantification of tubular atrophy in routine clinical practice.

Introduction: Histologic assessment of interstitial fibrosis and tubular atrophy is an accepted method of assessing chronic damage to the kidney and correlates with renal function in native and allograft renal biopsies. The challenge, however, is to quantify the interstitial fibrosis and tubular atrophy with accuracy and to minimize the inter-observer variability. Though "eyeballing" on light microscopy is the most commonly practised method used for the quantification of tubular atrophy, it may not be very accurate. To complement this method, Whole Slide Imaging (WSI) techniques that have more accurate results and have a higher reproducibility can be used. There is not much data on the correlation of the results obtained by the 'eyeballing' technique with those by digital WSI. Methods: Tubular atrophy in 151 consecutive adequate native kidney biopsies were graded 0 to III by 'conventional' eyeballing by a single experienced renal pathologist. These results were compared with the grades obtained on the same cases by WSI and digital marking of the atrophy. Results: The concordance of the two groups in the entire cohort was only 66.2% with over grading in 30.4% and under grading in 3.3%. Whilst accuracy of grading was over 74% in all grades, the sensitivity in grades I and II were low at 52% and 47.3% respectively as was the positive predictive value at 32.5 and 44% respectively. Conclusion: Assessment of tubular atrophy on digital images will be the way forward for accurate quantification.

Renal Lymphoma Diagnosed on Kidney Biopsy Presenting as Acute Kidney Injury.

Introduction: Renal manifestations associated with hematolymphoid malignancies are known. Primary or secondary involvement of the kidney by lymphomatous infiltration has various clinical presentations. Acute kidney injury is not an uncommon finding in relation to lymphomatous interstitial infiltration proven on kidney biopsy. However, diagnosing it solely on renal biopsy remains a challenge and needs expertise and aid of immunohistochemistry as the prognosis is dismal. Methods: This is a retrospective study of kidney biopsy-proven cases of renal lymphoma presenting with acute kidney injury. Results: The study included 12 patients with ages ranging from 4 to 50 years who presented with serum creatinine ranging 2.1-9.6 mg%. Renal biopsy findings showed interstitial lymphomatous infiltrate. Two cases were diagnosed as primary lymphoma and the other 10 as secondary lymphomas. Among the 12 cases, nine were B-cell non-Hodgkin lymphoma, of which diffuse large B-cell lymphoma was diagnosed in six (50%), low-grade B-cell type in two (16.6%), chronic lymphocytic leukemia in one (8.3%), and three were T-cell-type. Two were acute T-cell lymphoblastic lymphoma and one other was a high-grade T-cell lymphoma. Four patients succumbed. The other four patients are alive; one is on chemotherapy, while two of them are on hemodialysis. Conclusion: Acute kidney injury as a presenting feature with lymphomatous infiltration of renal parenchyma is not uncommon. The patchy involvement makes it challenging on kidney biopsy with definitive diagnosis being made with the help of immunohistochemistry. Appropriate multidisciplinary involvement improves patient outcome.

Fetal sepsis: a cause of stillbirth.

Infection is considered a leading cause of fetal death, responsible for approximately 20% of cases. Such estimates are derived from the frequency of acute histological chorioamnionitis and funisitis in cases of fetal death rather than direct detection of microorganisms in the fetal compartment. We report a case of clinically unexplained fetal death at 38 weeks of gestation in an uncomplicated pregnancy resulting in delivery of an appropriate-for-gestational-age fetus. The mother did not have any clinical signs of infection. Overwhelming bacterial invasion in multiple fetal organs, including the heart, liver, spleen, and kidneys, was observed despite the lack of evidence of maternal clinical infection. The bacteria were visualized by using standard histologic techniques (e.g. H&E/ tissue Gram stain) highlighting the value of autopsy in determining the cause of death.