Immunofluorescence Staining for IgG Subclass: Cause for Discrepancy in the Detection of IgG1.

Introduction: Immunofluorescence (IF) staining for IgG subclasses plays an important role in the classification of kidney disease. However, widely used IgG subclass-specific antibodies are now commercially unavailable. Thus, we compared alternative antibodies for performing IgG subclass staining. Methods: A total of 21 cases were stained by 3 different methods: direct IF using fluorescein isothiocyanate (FITC)-conjugated polyclonal antibodies against IgG1-4 (commercially unavailable method), direct IF using FITC-conjugated monoclonal antibodies (clones HP-6091, 6014, 6050, and 6025), indirect IF using monoclonal antibodies (clones HP-6069, 6002, 6050, and 6025), and FITC-conjugated polyclonal secondary antibody. For cases with discrepancy in IgG1 staining, additional direct IF using FITC-conjugated monoclonal antibody (clone 4E3) was performed. Results: Of 21 cases, 11 (52%) had no staining for IgG1 by direct IF using the clone HP-6091 despite ≥1+ staining by the direct IF using polyclonal antibodies. Similarly, direct IF for IgG1 using the clone 4E3 had negative result in all 10 cases with available tissue. However, indirect IF for IgG1 using the clone HP-6069 had similar staining intensity (within 1 order of magnitude) as direct IF using the polyclonal antibodies (10 of 10). Results of IF for IgG2, IgG3, and IgG4 were similar in most cases. Conclusion: The choice of antibodies influences the result of IgG subclass staining, especially for anti-IgG1 antibodies, in which 2 monoclonal antibodies (HP6091 and 4E3) appear less sensitive. Although this may be due to unaccounted variables and requires confirmation, our results may partially explain the difference in IgG1 staining in the literature and underscore the need for careful validation.

Systemic induction of senescence in young mice after single heterochronic blood exchange.

ABSTACT: Ageing is the largest risk factor for many chronic diseases. Studies of heterochronic parabiosis, substantiated by blood exchange and old plasma dilution, show that old-age-related factors are systemically propagated and have pro-geronic effects in young mice. However, the underlying mechanisms how bloodborne factors promote ageing remain largely unknown. Here, using heterochronic blood exchange in male mice, we show that aged mouse blood induces cell and tissue senescence in young animals after one single exchange. This induction of senescence is abrogated if old animals are treated with senolytic drugs before blood exchange, therefore attenuating the pro-geronic influence of old blood on young mice. Hence, cellular senescence is neither simply a response to stress and damage that increases with age, nor a chronological cell-intrinsic phenomenon. Instead, senescence quickly and robustly spreads to young mice from old blood. Clearing senescence cells that accumulate with age rejuvenates old circulating blood and improves the health of multiple tissues.

PodoSighter: A Cloud-Based Tool for Label-Free Podocyte Detection in Kidney Whole-Slide Images.

BACKGROUND: Podocyte depletion precedes progressive glomerular damage in several kidney diseases. However, the current standard of visual detection and quantification of podocyte nuclei from brightfield microscopy images is laborious and imprecise. METHODS: We have developed PodoSighter, an online cloud-based tool, to automatically identify and quantify podocyte nuclei from giga-pixel brightfield whole-slide images (WSIs) using deep learning. Ground-truth to train the tool used immunohistochemically or immunofluorescence-labeled images from a multi-institutional cohort of 122 histologic sections from mouse, rat, and human kidneys. To demonstrate the generalizability of our tool in investigating podocyte loss in clinically relevant samples, we tested it in rodent models of glomerular diseases, including diabetic kidney disease, crescentic GN, and dose-dependent direct podocyte toxicity and depletion, and in human biopsies from steroid-resistant nephrotic syndrome and from human autopsy tissues. RESULTS: The optimal model yielded high sensitivity/specificity of 0.80/0.80, 0.81/0.86, and 0.80/0.91, in mouse, rat, and human images, respectively, from periodic acid-Schiff-stained WSIs. Furthermore, the podocyte nuclear morphometrics extracted using PodoSighter were informative in identifying diseased glomeruli. We have made PodoSighter freely available to the general public as turnkey plugins in a cloud-based web application for end users. CONCLUSIONS: Our study demonstrates an automated computational approach to detect and quantify podocyte nuclei in standard histologically stained WSIs, facilitating podocyte research, and enabling possible future clinical applications.

A retrospective observational study of benign anthracotic lymphadenitis and its association with PET avid lymph nodes in patients undergoing cancer evaluation.

BACKGROUND: Accurate staging of newly diagnosed or recurrent malignancy is essential for effective treatment. An important first step in staging involves the use of PET/CT to identify areas of FDG avidity. PET/CT however has limitations, including false positive FDG uptake from benign causes. In this paper we characterize an uncommon yet clinically important cause of false positive PET/CTs, that of benign anthracotic lymphadenitis (BAL). We examine the clinical, radiographic and histologic characteristics of BAL in patients referred for endobronchial ultrasound (EBUS) guided biopsies and discuss its context in relation to existing literature. METHODS: We performed a retrospective observational case series of 20 patients who were referred for EBUS guided biopsies of PET positive mediastinal and hilar lymph nodes during the work-up or treatment of suspected malignancy. RESULTS: To be included, all patients received PET imaging as well as an EBUS guided biopsy of FDG avid lymph nodes which demonstrated anthracotic pigment as the only histologic abnormality. The key findings were that 90% of patients in this cohort were born outside of the US, 90% had bilateral FDG avid lymph nodes with an average standardized uptake value (SUV) of 7.9±2.2. Most patients, based on their history, had a likely exposure to biomass fuel or urban pollution. CONCLUSIONS: BAL may be an underrecognized cause for PET positive lymph nodes in patients undergoing work-up for malignancy. These findings support the importance of sampling mediastinal and hilar lymph nodes even when SUVs are highly suggestive of malignancy.

A multi-center retrospective cohort study defines the spectrum of kidney pathology in Coronavirus 2019 Disease (COVID-19).

Kidney failure is common in patients with Coronavirus Disease-19 (COVID-19), resulting in increased morbidity and mortality. In an international collaboration, 284 kidney biopsies were evaluated to improve understanding of kidney disease in COVID-19. Diagnoses were compared to five years of 63,575 native biopsies prior to the pandemic and 13,955 allograft biopsies to identify diseases that have increased in patients with COVID-19. Genotyping for APOL1 G1 and G2 alleles was performed in 107 African American and Hispanic patients. Immunohistochemistry for SARS-CoV-2 was utilized to assess direct viral infection in 273 cases along with clinical information at the time of biopsy. The leading indication for native biopsy was acute kidney injury (45.4%), followed by proteinuria with or without concurrent acute kidney injury (42.6%). There were more African American patients (44.6%) than patients of other ethnicities. The most common diagnosis in native biopsies was collapsing glomerulopathy (25.8%), which was associated with high-risk APOL1 genotypes in 91.7% of cases. Compared to the five-year biopsy database, the frequency of myoglobin cast nephropathy and proliferative glomerulonephritis with monoclonal IgG deposits was also increased in patients with COVID-19 (3.3% and 1.7%, respectively), while there was a reduced frequency of chronic conditions (including diabetes mellitus, IgA nephropathy, and arterionephrosclerosis) as the primary diagnosis. In transplants, the leading indication was acute kidney injury (86.4%), for which rejection was the predominant diagnosis (61.4%). Direct SARS-CoV-2 viral infection was not identified. Thus, our multi-center large case series identified kidney diseases that disproportionately affect patients with COVID-19 and demonstrated a high frequency of APOL1 high-risk genotypes within this group, with no evidence of direct viral infection within the kidney.

A Distributed System Improves Inter-Observer and AI Concordance in Annotating Interstitial Fibrosis and Tubular Atrophy.

Histologic examination of interstitial fibrosis and tubular atrophy (IFTA) is critical to determine the extent of irreversible kidney injury in renal disease. The current clinical standard involves pathologist's visual assessment of IFTA, which is prone to inter-observer variability. To address this diagnostic variability, we designed two case studies (CSs), including seven pathologists, using HistomicsTK- a distributed system developed by Kitware Inc. (Clifton Park, NY). Twenty-five whole slide images (WSIs) were classified into a training set of 21 and a validation set of four. The training set was composed of seven unique subsets, each provided to an individual pathologist along with four common WSIs from the validation set. In CS 1, all pathologists individually annotated IFTA in their respective slides. These annotations were then used to train a deep learning algorithm to computationally segment IFTA. In CS 2, manual and computational annotations from CS 1 were first reviewed by the annotators to improve concordance of IFTA annotation. Both the manual and computational annotation processes were then repeated as in CS1. The inter-observer concordance in the validation set was measured by Krippendorff's alpha (KA). The KA for the seven pathologists in CS1 was 0.62 with CI [0.57, 0.67], and after reviewing each other's annotations in CS2, 0.66 with CI [0.60, 0.72]. The respective CS1 and CS2 KA were 0.58 with CI [0.52, 0.64] and 0.63 with CI [0.56, 0.69] when including the deep learner as an eighth annotator. These results suggest that our designed annotation framework refines agreement of spatial annotation of IFTA and demonstrates a human-AI approach to significantly improve the development of computational models.

Automated Computational Detection of Interstitial Fibrosis, Tubular Atrophy, and Glomerulosclerosis.

BACKGROUND: Interstitial fibrosis, tubular atrophy (IFTA), and glomerulosclerosis are indicators of irrecoverable kidney injury. Modern machine learning (ML) tools have enabled robust, automated identification of image structures that can be comparable with analysis by human experts. ML algorithms were developed and tested for the ability to replicate the detection and quantification of IFTA and glomerulosclerosis that renal pathologists perform. METHODS: A renal pathologist annotated renal biopsy specimens from 116 whole-slide images (WSIs) for IFTA and glomerulosclerosis. A total of 79 WSIs were used for training different configurations of a convolutional neural network (CNN), and 17 and 20 WSIs were used as internal and external testing cases, respectively. The best model was compared against the input of four renal pathologists on 20 new testing slides. Further, for 87 testing biopsy specimens, IFTA and glomerulosclerosis measurements made by pathologists and the CNN were correlated to patient outcome using classic statistical tools. RESULTS: The best average performance across all image classes came from a DeepLab version 2 network trained at 40× magnification. IFTA and glomerulosclerosis percentages derived from this CNN achieved high levels of agreement with four renal pathologists. The pathologist- and CNN-based analyses of IFTA and glomerulosclerosis showed statistically significant and equivalent correlation with all patient-outcome variables. CONCLUSIONS: ML algorithms can be trained to replicate the IFTA and glomerulosclerosis assessment performed by renal pathologists. This suggests computational methods may be able to provide a standardized approach to evaluate the extent of chronic kidney injury in situations in which renal-pathologist time is restricted or unavailable.

Cancer of Unknown Primary in the Molecular Era.

Cancer of unknown primary (CUP) is a rare malignancy that presents with metastatic disease and no identifiable site of origin. Most patients have unfavorable features and attempts to treat based on tissue-of-origin identification have not yielded a survival advantage compared with empiric chemotherapy. Next-generation sequencing has revealed genomic alterations that can be targeted in selected cases, suggesting that CUP represents a unique malignancy in which the genomic aberrations may be integral to the diagnosis. Recent trials focusing on tailored combination therapy matched to the genomic alterations in each cancer are providing new avenues of clinical investigation. Here, we discuss recent findings on molecular aberrations in CUP and how the genomic and immune landscape can be leveraged to optimize therapy.

Anti-LDL Receptor-Related Protein 2 Nephropathy with Synchronous Primary Kidney Extranodal Marginal Zone Lymphoma.

Introduction: Anti-LDL receptor-related protein 2 (anti-LRP2) nephropathy is a rare but progressive form of autoimmune-mediated tubulointerstitial nephritis and glomerular disease, characterized by a classic pattern of immune complex deposition in the kidney. A theoretic link between autoimmune disease and lymphoproliferative diseases exists, and therefore, in some cases autoimmune-mediated inflammation and lymphoproliferative neoplasm can co-exist in the same site. Case Presentation: An elderly man presented with 6 months of weight loss and fatigue. Subsequent workup showed an elevated serum creatinine and subnephrotic range proteinuria. Kidney biopsy was performed which revealed anti-LRP2 nephropathy with concurrent primary kidney extranodal marginal zone lymphoma. He was subsequently treated with rituximab but remains dialysis-dependent (12 months after his initial diagnosis, at time of publication of this report). Conclusion: We discuss the bidirectional relationship between autoimmune disease and lymphoma in the kidney, along with a brief review of the literature pertaining to these rare lesions. Our case report highlights the diagnostic difficulties faced by pathologists when encountering this entity. We also attempt to spread awareness about the co-existence of tubulointerstitial inflammation and lymphoproliferative disorder, which may be under-recognized.

Progression of proliferative glomerulonephritis with monoclonal IgG deposits in pediatric patients.

BACKGROUND: Proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) is a glomerular disease defined by non-organized glomerular deposits of heavy and light chain-restricted immunoglobulin and is rarely reported in children. METHODS: We characterized a series of nine pediatric patients from two academic centers with biopsy-proven PGNMID and additionally describe two patients with monotypic IgG in the setting of IgM deposition. RESULTS: Each patient presented with hematuria and/or proteinuria; however, only five had elevated serum creatinine. Prodromal or concurrent infection was identified in six patients, low C3 in five, and alternate complement pathway gene variants in two. No monoclonal serum proteins were identified in five tested patients. Seven patients had monotypic deposits composed of IgG3-λ, two showed IgG3-κ, and one each IgG1 and IgG3 with lambda dominance in the setting of IgM deposition. The glomerular pattern was predominantly mesangial proliferative or membranoproliferative glomerulonephritis (MPGN). Treatment and outcomes were variable; four patients have recent PGNMID diagnoses and therefore minimal follow up, one had relatively stable kidney function for over a decade, and six experienced kidney failure, with four receiving transplants. Recurrent deposits of the same isotype were identified in five of six transplanted kidneys, corresponding to three of four transplanted patients. One of these patients developed PGNMID recurrences in three separate kidney allografts over a 20-year disease course. CONCLUSIONS: Our study emphasizes the need for upfront IgG subclass investigation in pediatric mesangial or MPGN with IgG deposition and monotypic or biased light-chain staining. Furthermore, this pediatric experience suggests expanded pathogenic considerations in PGNMID. Graphical abstract.

Clinicopathologic Characteristics of JC Virus Nephropathy in Kidney Transplant Recipients.

BACKGROUND: The vast majority of polyomavirus nephropathy (PVN) is due to BK virus, but rare cases result from JC virus reactivation. To date, only a handful of biopsy-proven JC-PVN cases have been reported. Here, we describe the clinical and pathologic findings in 7 patients with biopsy-proven JC-PVN. METHODS: Search of the pathology archives at 2 institutions found 7 cases of JC-PVN. Clinical data were extracted from the electronic medical records, and the biopsies were reviewed. RESULTS: Four cases were diagnosed at 6 y posttransplant or later. The remaining 3 cases presented within approximately 2 y posttransplant, of which 2 showed subclinical JC-PVN on surveillance biopsy. Two early presenting patients were treated for acute rejection just before acquiring JC-PVN. Late presenting patients had higher chronicity, which correlated to worse outcome. All but 1 biopsy showed nonspecific inflammation within areas of interstitial fibrosis without significant inflammation in unscarred cortex. The earliest presenting patient was the exception and showed active inflammation with tubulitis. Viral cytopathic changes were detected in all cases with moderate or high-histologic viral load (pvl), showing preference for the distal tubules and medulla. The 2 cases with low pvl did not demonstrate cytopathic changes but were SV40 positive. CONCLUSIONS: JC-PVN can be insidious in presentation, which may cause delayed or missed diagnosis. Unlike BK-PVN, which typically occurs early in the posttransplant period, JC-PVN can occur both early and late following transplant. Overreliance on negative plasma and urine BK viral loads to exclude PVN can be a pitfall.

Membranous nephropathy in patients with HIV: a report of 11 cases.

BACKGROUND: Membranous nephropathy (MN) has been recognized to occur in patients with human immunodeficiency virus (HIV) infection since the beginning of the HIV epidemic. The prevalence of phospholipase A2 receptor (PLA2R)-associated MN in this group has not been well studied. METHODS: We conducted a retrospective review of electronic pathology databases at three institutions to identify patients with MN and known HIV at the time of renal biopsy. Patients with comorbidities and coinfections known to be independently associated with MN were excluded. RESULTS: We identified 11 HIV-positive patients with biopsy-confirmed MN meeting inclusion and exclusion criteria. Patient ages ranged from 39 to 66 years old, and 10 of 11 patients (91%) were male. The majority of patients presented with nephrotic-range proteinuria, were on anti-retroviral therapy at the time of biopsy and had low or undetectable HIV viral loads. Biopsies from 5 of 10 (50%) patients demonstrated capillary wall staining for PLA2R. Measurement of serum anti-PLA2R antibodies was performed in three patients, one of whom had positive anti-PLA2R antibody titers. Follow-up data was available on 10 of 11 patients (median length of follow-up: 44 months; range: 4-145 months). All patients were maintained on anti-retroviral therapy (ARV) and 5 patients (52%) received concomitant immunosuppressive regimens. Three patients developed end-stage renal disease (ESRD) during the follow-up period. CONCLUSIONS: MN in the setting of HIV is often identified in the setting of an undetectable viral loads, and similar to other chronic viral infection-associated MNs, ~ 50% of cases demonstrate tissue reactivity with PLA2R antigen, which may be seen without corresponding anti-PLA2R serum antibodies.

#FNAFriday: How cytopathologists learn, teach, and share knowledge on Twitter.

BACKGROUND: Twitter is an expanding social media network among cytopathologists to share knowledge. Tweets are made up of text which may also include images or video. All tweets labeled under a hashtag can be tracked. The #FNAFriday hashtag was created in 2015 by one of the authors (X.J.) to build a community of individuals, to educate and share interesting cases, and highlight a variety of diagnoses with FNA specimens. METHODS: We retrospectively extracted all tweets labeled with #FNAFriday from April 2015 to mid-February 2019 (47 mo) using the Twitter search engine. The data point included: author, number of figures, type of cytology-stain, use of immunocytochemistry, histochemistry or molecular techniques, and the subspeciality. The educational content was categorized as: live-tweeting, training activities, and publication references. The number if comments, retweets and likes was also recorded. RESULTS: A total of 349 original tweets using #FNAFriday were tracked with an average of 7.43 tweets/month. We describe the "top three" countries with most tweets, active users and subspecialties. The most frequent stain was Papanicolau and part of the content of the tweets was using cellblock (14.04%), histologic correlation (10.03%), immunocytochemistry (8.60%), molecular tests (2.01%), gross pictures (4.58%), and radiologic pictures (3.4%). CONCLUSION: The presence of cytopathologists on Twitter who want to share their cases has increased. The weekly FNAFriday label with other cytology hashtags is a specific keyword for those interested in the field.

Cytologic processing of ureteral microbiopsies is associated with higher sensitivity for detection of urothelial carcinoma compared with conventional biopsy processing.

INTRODUCTION: Because of the high rates of false-negative or nondiagnostic ureteral Piranha microbiopsies associated with low cellularity, we assessed the effect of processing these using cytology. MATERIALS AND METHODS: We included 2 groups of 44 consecutive microbiopsies processed from formalin as a standard surgical biopsy and 22 processed by cytology. All samples were from the ureter or renal pelvis or calyx. The cytology samples were collected in alcohol-based media and were prepared with a Cellient cell block only (n = 9) or with a Cellient cell block for the visible particles, together with ThinPrep, to capture the remaining desquamated cells (n = 13). RESULTS: Malignancy was diagnosed in 5 of 44 conventionally processed microbiopsies (11%) compared with 14 of 22 cytologically processed microbiopsies (64%; P < 0.001), including 1 case with invasion. Nineteen site-matched biopsies from 2 patients had undergone both cytologic and surgical processing, with 8 of 8 cytologically processed biopsies diagnosed as malignant. None of the 11 surgically processed biopsies from the same patients matched for site were diagnosed as malignant. Of the 11, 2 (18%) were suspicious for high-grade urothelial carcinoma and 6 (55%) were considered atypical. Increased sensitivity from cytologic processing appears related to increased cell recovery; large numbers of well-preserved urothelial cells were identified in the ThinPrep (range, 1000-25,000 cells/slide), and a nonsignificant trend was found toward increased urothelium (defined as >200 cells/profile) in the Cellient cell blocks (14 of 22 [64%]) compared with the histologic biopsies (17 of 44 [39%]; P = 0.070). CONCLUSIONS: Cytologic processing of ureteral microbiopsies showed superior sensitivity for detecting high-grade urothelial carcinoma, apparently owing to the increased cellular recovery.

Computational Segmentation and Classification of Diabetic Glomerulosclerosis.

BACKGROUND: Pathologists use visual classification of glomerular lesions to assess samples from patients with diabetic nephropathy (DN). The results may vary among pathologists. Digital algorithms may reduce this variability and provide more consistent image structure interpretation. METHODS: We developed a digital pipeline to classify renal biopsies from patients with DN. We combined traditional image analysis with modern machine learning to efficiently capture important structures, minimize manual effort and supervision, and enforce biologic prior information onto our model. To computationally quantify glomerular structure despite its complexity, we simplified it to three components consisting of nuclei, capillary lumina and Bowman spaces; and Periodic Acid-Schiff positive structures. We detected glomerular boundaries and nuclei from whole slide images using convolutional neural networks, and the remaining glomerular structures using an unsupervised technique developed expressly for this purpose. We defined a set of digital features which quantify the structural progression of DN, and a recurrent network architecture which processes these features into a classification. RESULTS: Our digital classification agreed with a senior pathologist whose classifications were used as ground truth with moderate Cohen's kappa κ = 0.55 and 95% confidence interval [0.50, 0.60]. Two other renal pathologists agreed with the digital classification with κ1 = 0.68, 95% interval [0.50, 0.86] and κ2 = 0.48, 95% interval [0.32, 0.64]. Our results suggest computational approaches are comparable to human visual classification methods, and can offer improved precision in clinical decision workflows. We detected glomerular boundaries from whole slide images with 0.93±0.04 balanced accuracy, glomerular nuclei with 0.94 sensitivity and 0.93 specificity, and glomerular structural components with 0.95 sensitivity and 0.99 specificity. CONCLUSIONS: Computationally derived, histologic image features hold significant diagnostic information that may augment clinical diagnostics.

De novo leukocyte chemotactic factor 2 amyloidosis in a pediatric renal allograft, 15 years post-transplant.

Leukocyte chemotactic factor 2 amyloidosis (ALECT2) is a recently described form of systemic amyloidosis, which most commonly affects the kidney and liver. The LECT2 protein is produced during inflammatory processes, but its precise function in renal diseases in unclear. ALECT2, however, is known to be a relatively common form of renal amyloidosis, after amyloid light chain and serum amyloid A types and is most often seen in patients of Hispanic ethnicity. ALECT2 can occur de novo or as recurrent disease in kidney transplants. We present the first case, to our knowledge, of de novo ALECT2 in a pediatric kidney transplant patient, 15 years post-transplant.

Estrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor-2 Testing in Breast Cancer: Assessing the Value of Repeated Centralized Testing in Excision Specimens.

At some tertiary breast care centers, where many patients are referred from other institutions, it is routine to repeat testing for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2/neu) in excision specimens if these tests were performed on the preceding biopsy at the referring facility. The goal of this study is to assess the value of this practice. We documented results from ER, PR, and HER2 testing in 541 consecutive invasive breast cancers excised over a 2.5-year period and analyzed the subset (n=153) for which testing was performed on the excision specimen solely due to the fact that testing on the preceding biopsy was performed at an outside institution. The rates and directions of biopsy-to-excision change were as follows: ER [1.3% (2/153), 100% from (+) to (-)]; PR [4% (6/153), 83% from (+) to (-)]; HER2/neu assessed by immunohistochemistry [21% (29/137)]; HER2/neu assessed by fluorescence in situ hybridization [3.3% (2/61); 50% from amplified to nonamplified and 50% vice versa]. There were no ER(-) and PR(-) biopsy cases that became ER and/or PR(+) in the excision. By coordinate analysis for the hormone receptors [ie, ER and/or PR(+) being indicative of "hormone receptor" (HR) positivity], there were no cases that changed from HR(+) in the biopsy to HR(-) in the excision (or vice versa), which suggests that repeat testing for ER and PR in this setting is of limited value. In an analysis that incorporated both immunohistochemistry and in situ fluorescence hybridization results, there were 2 cases with a clinically significant biopsy-to-excision change in HER2/neu status in which that change was detected primarily because the excision was retested. These findings provide baseline data for formulating policies on whether repeat testing should routinely be performed in the described scenario.

Does a p53 "Wild-type" Immunophenotype Exclude a Diagnosis of Endometrial Serous Carcinoma?

An aberrant p53 immunophenotype may be identified in several histotypes of endometrial carcinoma, and is accordingly recognized to lack diagnostic specificity in and of itself. However, based on the high frequency with which p53 aberrations have historically been identified in endometrial serous carcinoma, a mutation-type immunophenotype is considered to be highly sensitive for the histotype. Using an illustrative case study and a review of the literature, we explore a relatively routine diagnostic question: whether the negative predictive value of a wild-type p53 immunophenotype for serous carcinoma is absolute, that is, whether a p53-wild type immunophenotype is absolutely incompatible with a diagnosis of serous carcinoma. The case is an advanced stage endometrial carcinoma that was reproducibly classified by pathologists from 3 institutions as serous carcinoma based on its morphologic features. By immunohistochemistry, the tumor was p53-wild type (DO-7 clone), diffusely positive for p16 (block positivity), and showed retained expression of PTEN, MSH2, MSH6, MLH1, and PMS2. Next generation sequencing showed that there indeed was an underlying mutation in TP53 (D393fs*78, R213*). The tumor was microsatellite stable, had a low mutational burden (4 mutations per MB), and displayed no mutations in the exonuclease domain of DNA polymerase epsilon (POLE) gene. Other genomic alterations included RB1 mutation (R46fs*19), amplifications in MYST3 and CRKL, and ARID1A deletion (splice site 5125-94_5138del108). A review of the recent literature identified 5 studies in which a total of 259 cases of serous carcinoma were whole-exome sequenced. The average TP53 mutational rate in endometrial serous carcinoma was only 75% (range, 60 to 88). A total of 12 (33%) of 36 immunohistochemical studies reported a p53-aberrant rate of <80% in endometrial serous carcinoma. We discuss in detail several potential explanations that may underlie the scenario of serous carcinoma-like morphology combined with p53-wild-type immunophenotype, including analytic limitations, a nonserous histotype displaying morphologic mimicry of serous carcinoma, and true biological phenomena (including the possibility of a TP53-independent pathway of endometrial serous carcinogenesis). Ultimately, our central thematic question is provisionally answered in the negative. At present, the available data would not support a categorical conclusion that a p53 alteration is a necessary and obligate component in the genesis and/or diagnosis of endometrial serous carcinoma. On the basis of their collective experience, the authors proffer some recommendations on the use of p53 immunohistochemistry in the histotyping of endometrial carcinomas.