Implementation of HER2 Testing in Endometrial Cancer, a Summary of Real-World Initial Experience in a Large Tertiary Cancer Center.

HER2-targeted therapies have transformed the management of advanced or recurrent serous endometrial cancer (EC), leading to an increased clinical demand for HER2 testing. Despite its adoption in select academic centers, the global extent of such tumor testing is unclear. In this study, we report on the initial two-year experience of HER2 testing at a major academic center with a reference gynecologic oncology service and biomarker reference laboratory. All patients who underwent HER2 testing based on physician discretion, reflex HER2 testing, and reference laboratory requests were included. From February 2021 to October 2023, HER2 testing was performed on 192 tumor tissue samples from 180 EC patients. Serous carcinoma constituted 52% of samples, reflecting diagnostic challenges and limited therapeutic options for advanced EC. HER2 positivity was found in 28% of all cases and 30% of p53-aberrant cases. An immunohistochemistry (IHC) score of 3+ was found in 15% of samples, while IHC 2+ was found in 45% (13% IHC 2+/ISH+ and 32% IHC 2+/ISH-). The newly identified 'HER2-low' category comprised 46% of the samples. Heterogeneity was noted in 42% of HER2-positive cases, with complex patterns in 3%. NGS and HER2 IHC-FISH showed a 24% discordance, attributed to intratumoral heterogeneity, tumor cellularity, a small number of amplified cells, and the HER2/CEP17 ratio near the cut-off. This study offers real-world insights into HER2 testing in EC, highlighting the challenges and underscoring the need for standardized guidelines in specimen handling, proficiency testing, and scoring criteria to enhance patient management and therapeutic decision-making.

Cystic neutrophilic granulomatous mastitis: sensitivity and specificity of 16s rRNA and Sanger sequencing for Corynebacterium spp.

AIMS: Cystic neutrophilic granulomatous mastitis (CNGM) is a subtype of granulomatous mastitis (GM) associated with Corynebacterium spp infection. We aimed to analyse the prevalence of Corynebacteria in CNGM and non-CNGM cases. METHODS: Breast specimens diagnosed as granulomatous inflammation between 2010 and 2020 were reviewed to identify a CNGM cohort and a non-CNGM cohort. Polymerase chain reaction-based identification of Corynebacteria by 16S ribosomal RNA (16S rRNA) primers, followed by confirmatory Sanger sequencing (SS), was performed on all cases. Clinical, radiological and microbiology data were retrieved from the electronic patient records. RESULTS: Twenty-eight CNGM cases and 19 non-CNGM cases were identified. Compared with the non-CNGM cohort, patients in the CNGM cohort were more likely to be multiparous (p=0.01), breast feeding (p=0.01) and presenting with a larger breast mass (p<0.01), spontaneous drainage (p=0.05) and skin irritation (p<0.01). No significant difference in the prevalence of Corynebacteria between the cohorts (7% vs 11%, p=0.68) by microbiological culture was identified. Compared with microbiology culture, the sensitivity and specificity of each Corynebacterial detection method were 50% and 81% for Gram stain, and 25% and 100% for 16S rRNA combined with SS. Regardless of the diagnosis, patients positive for Corynebacteria were more likely to have a persistent disease (p<0.01). CONCLUSION: CNGM presents as a large symptomatic breast mass in multiparous breastfeeding women. The importance of adequate sampling and repeated microbiology culture in conjunction with sequencing on all GM cases with persistent disease is paramount.

MLH1 Methylation Testing as an Integral Component of Universal Endometrial Cancer Screening-A Critical Appraisal.

MLH1/PMS2 loss due to MLH1 promoter hypermethylation (MLH1-PHM) is the most common cause of mismatch repair (MMR) deficiency in endometrial cancer (EC). This study aimed to determine the proportion of MLH1-deficient EC with PHM, assess the impact of the reflex MLH1-PHM testing strategy, and evaluate the associated costs within the publicly funded Canadian healthcare system. In a cohort of 2504 EC samples, 534 (21.4%) exhibited dual MLH1/PMS2 loss, prompting MLH1-PHM testing. Among 418 cases with available testing results, 404 (96.7%) were MLH1-hypermethylated, while 14 (3.3%) were non-methylated. The incidence of MLH1 non-methylated cases in our cohort was 14/2504 (0.56%) of all ECs, underscoring the prevalence of hypermethylation-driven MLH1/PMS2 loss in ECs universally screened for MMR deficiency. Reflex MLH1-PHM testing incurs substantial costs and resource utilization. Assay cost is CAD 231.90 per case, amounting to CAD 123,834.60 for 534 cases, with 30 tests needed per additional candidate for MLH1 germline analysis (CAD 6957.00 per candidate). This raises a provocative question: can we assume that the majority of the MLH1-deficient ECs are due to PHM and forgo further testing in healthcare systems with finite resources? It is imperative to assess resource utilization efficiency and explore optimized approaches that encompass clinical correlation, family history and judicious utilization of methylation testing to ensure it is provided only to those who stand to benefit from it.

Budget impact analysis of molecular subtype profiling in endometrial cancer.

OBJECTIVE: This study evaluated the costs associated with four approaches to classifying endometrial cancer (EC), including histomorphological, histomorphological with ancillary immunohistochemical assays, histomolecular and selective molecular classification. METHODS: Direct costs were determined per EC sample from the hospital's perspective. A budget impact analysis and sensitivity analysis were conducted to estimate the mean, minimum and maximum costs per sample and annual institutional costs in adjusted 2022 Canadian dollars. A provincial cost forecast was projected based on expected 2022 EC biopsies. RESULTS: In 2018, our institution performed 190 EC biopsies. The mean cost per biopsy was $158 ($156-$212) for histomorphological classification, $384 ($360-$514) for histomorphological classification with immunohistochemistry and $1297 ($1265-1833) for histomolecular classification. Total annual institutional cost for histomorphological classification was $29,980 and $72,950 with immunohistochemistry. For histomolecular classification, the first year cost was $246,521, accounting for initial educational learning curve, and $233,461 thereafter, assuming a consistent number of biopsies per year. Targeted implementation of histomolecular classification among high-grade, p53 abnormal and/or MMR-deficient ECs (56% of cases) cost $169,688 in the first year and $162,418 annually thereafter. With a projected 3400 EC biopsies in Ontario in 2022, histomorphological classification would annually cost $537,078 and $1,305,677 with immunohistochemistry. Histomolecular classification would cost $4,410,203 in the first year and $4,176,737 annually once established. Selective molecular classification would lead to a cost of $3,044,178 in the first year and $2,913,443 thereafter. CONCLUSIONS: The study highlights the need for informed decision-making when implementing molecular classification in clinical practice, given the substantial incremental healthcare costs associated with these approaches.

Utility of intraoperative pathology consultations of whipple resection specimens and their impact on final margin status.

The resection margin status is a significant surgical prognostic factor for the long-term outcomes of patients undergoing pancreaticoduodenectomy (Whipple procedure). As a result, surgeons frequently rely on intraoperative consults (IOCs) involving frozen sections to evaluate margin clearance during these resections. Nevertheless, the impact of this practice on final margin status and long-term outcomes remains a topic of debate. This study aimed to assess the impact of IOCs on the clearance rate of resection margins following Whipple procedure and distal pancreatectomy. A retrospective database review of all patients who underwent Whipple procedure or distal pancreatectomy at our institution between 2018 and 2020 was performed to evaluate the utility of IOCs by gastrointestinal surgeons and its correlation with final postoperative surgical margin status. A significant variation in the frequency of IOC requests for margins among surgeons was noted. However, the use of frozen section analysis for intraoperative margin assessment was not significantly associated with the clearance rate of final post-operative margins. More frequent use of IOC did not result in higher final margin clearance rate, an important prognostic factor following Whipple procedure.

Redefining and capturing pre-analytic deficiencies in an anatomical pathology laboratory: a quality improvement initiative.

Pre-analytical deficiencies (PADs) are a major source of errors in anatomical pathology, accounting for about 70% of laboratory deficiencies. These can lead to incorrect diagnoses, delayed treatments, and increased healthcare costs. As part of a quality improvement initiative, we retrospectively identified and characterized 237 PADs documented over a 1-year period in a tertiary care academic center. The most common PADs were errors in specimen procurement (56%), handling of samples within the lab (16%), accessioning (10%), incomplete requisitions (9%), and transportation-related issues (7%). Strategies were then devised to mitigate these errors. Categorization of pre- and intra-laboratory PADs was refined into eight categories (collection, requisition, specimen container, transportation, receiving, accessioning, preparation, and communications) in the laboratory information system. Mandatory PAD documentation was implemented for accessioning staff. Post-implementation, prospective analysis identified that the most common PADs were related to surgical requisitions (75%). Among these, missing ordering physician's signature was the most common, accounting for 67.7% of requisition-related PADs and 50.8% of all PADs. Other common PADs included incomplete information of specimens, clinical information, patient information, physician information, source location, collection time, incorrect requisition forms, and illegible handwritten information. This study highlights the importance of identifying and addressing PADs in the anatomical pathology laboratory setting as well as the potential benefits of implementing standardized documentation and quality improvement processes to address these deficiencies.

Genomic profiling of dedifferentiated endometrial carcinomas arising in the background of high-grade carcinoma: a targeted next-generation sequencing study.

AIMS: Our understanding of dedifferentiated endometrial carcinoma (DEC), a rare and aggressive malignancy, mainly reflects undifferentiated carcinomas (UC) arising in the setting of low-grade endometrial cancer (DEC-LG). However, cases of UC arising in the setting of high-grade EC (DEC-HG) have been noted in the literature. Our knowledge of the genomics of DEC-HG is limited. To characterise the molecular landscape of DEC-HC, targeted genomic sequencing and immunohistochemical analysis was carried out on seven DEC-HG and four DEC-LG. METHODS AND RESULTS: DEC-HG and DEC-LG, including undifferentiated and differentiated components, both showed a similar frequency and spectrum of mutations. ARID1A mutations were identified in 6/7 (86%) DEC-HG and 4/4 (100%) DEC-LG, while SMARCA4 mutations were present in 4/7 (57%) DEC-HG and in 1/4 (25%) DEC-LG. Concurrent SMARCA4/BRG1 protein loss by immunohistochemistry was observed in 3/4 and 1/1 SMARCA4 mutated DEC-HG and DEC-LG, respectively. Neither genomic alterations nor protein loss in SMARCB1/INI1 were observed in any of our cases. TP53 mutations were detected in 4/7 (57%) DEC-HG and in 2/4 (50%) DEC-LG, while mutation-pattern p53 immunohistochemistry expression was observed in 2/7 (29%) DEC-HG and none of the DEC-LG. MLH1 mutations were observed in 1/7 (14%) DEC-HG and 1/4 (25%) DEC-LG. MSH2 and MSH6 mutations were each detected in 1/7 (14%) DEC-HG, but neither was associated with corresponding loss of protein expression. CONCLUSION: The findings support expanding the definition of DEC to include DEC-HG, a previously under-recognised phenomenon with genomic similarities to DEC-LG.

Mesonephric-like adenocarcinoma of the female genital tract: novel observations and detailed molecular characterisation of mixed tumours and mesonephric-like carcinosarcomas.

AIMS: To report novel observations in five mesonephric-like adenocarcinomas (MLAs) of the female genital tract. METHODS AND RESULTS: We report two endometrial MLAs in association with endometrioid carcinoma and atypical hyperplasia and three (one endometrial, two ovarian) cases with a sarcomatoid component (mesonephric-like carcinosarcoma). Pathogenic KRAS mutations, which are characteristic of MLA, were identified in all cases although interestingly, in one of the mixed carcinomas, this was confined to the endometrioid component. The concurrent MLA, endometrioid carcinoma and atypical hyperplasia components in one case harboured identical EGFR, PTEN and CCNE1 mutations, suggesting that the atypical hyperplasia gave rise to a Müllerian carcinoma with both endometrioid and mesonephric-like components. The carcinosarcomas all contained a component of MLA and a sarcomatous component with chondroid elements. In the ovarian carcinosarcomas, the coexisting epithelial and sarcomatous components shared some mutations including KRAS and CREBBP, suggesting that they are clonally related. Furthermore, in one case CREBBP and KRAS mutations detected in the MLA and sarcomatous components were also detected in an associated undifferentiated carcinoma component, suggesting that it was clonally related to the MLA and sarcomatous components. CONCLUSIONS: Our observations provide additional evidence that MLAs have a Müllerian origin and characterise mesonephric-like carcinosarcomas in which chondroid elements appear to be characteristic. In reporting these findings, we provide recommendations for distinction between a mesonephric-like carcinosarcoma and a MLA with a spindle cell component.

Molecular correlates of invasion pattern in HPV-associated endocervical adenocarcinoma: emergence of two distinct risk-stratified tiers.

BACKGROUND: The pattern-based (Silva) classification of invasive human papilloma virus (HPV)-associated endocervical adenocarcinomas (HPVA) is an established and reproducible method to predict outcomes for this otherwise stage-dependent group of tumours. Previous studies utilising targeted sequencing have shown a correlation between mutational profiles and an invasive pattern. However, such correlation has not been explored using comprehensive molecular testing. DESIGN: Clinicopathologic data including invasive pattern (Silva groups A, B, and C) was collected for a cohort of invasive HPVA, which previously underwent massive parallel sequencing using a panel covering 447 genes. Pathogenic alterations, molecular signatures, tumour mutational burden (TMB), and copy number alterations (CNA) were correlated with pattern of invasion. RESULTS: Forty five HPVA (11 pattern A, 17 pattern B, and 17 pattern C tumours) were included. Patients with pattern A presented at stage I with no involved lymph nodes or evidence of recurrence (in those with >2 months of follow-up). Patterns B and C patients also mostly presented at stage I with negative lymph nodes, but had a greater frequency of recurrence; 3/17 pattern B and 1/17 pattern C HPVAs harboured lymphovascular space invasion (LVI). An APOBEC mutational signature was detected only in Silva pattern C tumours (5/17), and pathogenic PIK3CA changes were detected only in destructively invasive HPVA (patterns B and C). When cases were grouped as low-risk (pattern A and pattern B without LVI) and high-risk (pattern B with LVI and pattern C), high-risk tumours were enriched in mutations in PIK3CA, ATRX, and ERBB2. There was a statistically significant difference in TMB between low-risk and high-risk pattern tumours (P = 0.006), as well as between Pattern C tumours with and without an APOBEC signature (P = 0.002). CNA burden increased from pattern A to C. CONCLUSION: Our findings further indicate that key molecular events in HPVA correlate with the morphologic invasive properties of the tumour and their aggressiveness. Pattern B tumours with LVI clustered with pattern C tumours, whereas pattern B tumours without LVI approached pattern A genotypically. Our study provides a biologic foundation for consolidating the Silva system into low-risk (pattern A + B without LVI) and high-risk (pattern B with LVI and pattern C) categories.

The Impact of the Pathologist in Multidisciplinary Cancer Conferences on Patient Care : Evidence From the Literature.

OBJECTIVES: Multidisciplinary cancer conferences (MCCs) are important tools in the treatment of patients with complex health issues, helping clinicians achieve optimal outcomes in oncological practice. To explore the role of pathologists at MCCs, we conducted a review of prior research on this topic. METHODS: We conducted a scoping review by searching MEDLINE, EMBASE, and the Cochrane Library for English-language qualitative, quantitative, or multiple/mixed methods studies on the role and impact of pathologists on MCCs. We used Microsoft Excel to extract data. RESULTS: Of 76 research results, we included only 3 studies that involved review of cancer cases by pathologists for MCCs. All 3 studies showed that expert pathology review improved the accuracy of diagnosis and refined disease staging, leading to changes in the management of melanoma, breast cancer, and gynecologic cancer. No studies explored the barriers to pathologists participating in MCCs or the strategies or interventions employed to promote or support pathologist involvement. CONCLUSIONS: We identified a paucity of studies on the role of pathologists in MCCs. Given the positive impact of MCCs involving pathologists on the accuracy of diagnosis and optimization of treatment, future research is warranted to further establish the role and impact of pathologists in MCCs and how to promote or support pathologists' involvement.

Cytohistologic immunohistochemical correlation of epithelial tubo-ovarian neoplasms: Can cell blocks substitute for tissue?

BACKGROUND: Cytologic specimens often represent the initial diagnostic material for tubo-ovarian neoplasms resulting from therapeutic paracentesis for patients presenting with high-volume ascites. However, subtyping and immunohistochemical (IHC) characterization, which have implications in preoperative management and downstream ancillary testing, are not routinely performed in many institutions. This study aims to perform cytohistologic correlation of commonly used IHC stains to establish their reliability in peritoneal fluids/washing specimens. METHODS: A retrospective search of the laboratory information systems was performed to identify peritoneal fluid/washing specimens involved by borderline or malignant epithelial tubo-ovarian neoplasms and concurrent/subsequent surgical resection specimens. Cell blocks and tissue were stained for PAX8, WT-1, p53, p16, Napsin-A, estrogen receptor, and progesterone receptor, and staining between cytological and surgical specimens was compared. RESULTS: A total of 56 case pairs were included, with the following final diagnoses on histological examination: 37 high-grade serous carcinomas, eight clear cell carcinomas, one endometrioid adenocarcinoma, two low-grade serous carcinomas, and eight serous borderline tumors. There was perfect cytohistologic correlation for PAX8 (Lin's concordance correlation coefficient [LINCCC] = 1.00) and WT-1 (LINCCC = 1.00), substantial/good correlation for p53 (LINCCC = 0.96), p16 (LINCCC = 0.93), napsin-A (LINCCC = 0.91) and ER (LINCCC = 0.77), and moderate correlation for PR (LINCCC = 0.54). CONCLUSIONS: Immunohistochemical correlation between peritoneal fluid and surgical resection specimens for tubo-ovarian neoplasms is high. Common subtypes of tubo-ovarian carcinomas can be reliably distinguished on fluids using IHC.

Development of a Clinically Applicable NanoString-Based Gene Expression Classifier for Muscle-Invasive Bladder Cancer Molecular Stratification.

Transcriptional profiling of muscle-invasive bladder cancer (MIBC) using RNA sequencing (RNA-seq) technology has demonstrated the existence of intrinsic basal and luminal molecular subtypes that vary in their prognosis and response to therapy. However, routine use of RNA-seq in a clinical setting is restricted by cost and technical difficulties. Herein, we provide a single-sample NanoString-based seven-gene (KRT5, KRT6C, SERPINB13, UPK1A, UPK2, UPK3A and KRT20) MIBC molecular classifier that assigns a luminal and basal molecular subtype. The classifier was developed in a series of 138 chemotherapy naïve MIBCs split into training (70%) and testing (30%) datasets. Further, we validated the previously published CK5/6 and GATA3 immunohistochemical classifier which showed high concordance of 96.9% with the NanoString-based gene expression classifier. Immunohistochemistry-based molecular subtypes significantly correlated with recurrence-free survival (RFS) and disease-specific survival (DSS) in univariable (p = 0.006 and p = 0.011, respectively) and multivariate cox regression analysis for DSS (p = 0.032). Used sequentially, the immunohistochemical- and NanoString-based classifiers provide faster turnaround time, lower cost per sample and simpler data analysis for ease of clinical implementation in routine diagnostics.

Combining CAPRA-S With Tumor IDC/C Features Improves the Prognostication of Biochemical Recurrence in Prostate Cancer Patients.

BACKGROUND: Intraductal carcinoma and cribriform (IDC/C) tumor features are well-established prognosticators of biochemical recurrence (BCR), metastasis, and prostate cancer (PCa)-specific mortality. However, approximately 70% of PCa patients undergoing a radical prostatectomy are IDC/C negative, yet up-to 20% of these patients progress and experience BCR. Thus, tumor histopathologic characteristics such as IDC/C alone are limited in their ability to predict disease progression. Conversely, several nomograms such as Cancer of the Prostate Risk Assessment-Surgery (CAPRA-S) have been developed to aid in the prognostication of BCR, but not yet widely applied in clinical settings. MATERIALS AND METHODS: In this study, we assessed the combined prognostic utility of IDC/C, and CAPRA-S for BCR in 3 PCa patient cohorts. RESULTS: CAPRA-S+IDC/C improved the predictive accuracy of BCR in all 3 cohorts (P < .001). Specifically, among IDC/C negative cases, CAPRA-S improved the prognostication of BCR in low-risk (Cohort 1; P < .001, Cohort 2; P < .001, Cohort 3; P = .003), intermediate (Cohort 1; P < .001, Cohort 2; P = .006, Cohort 3; P = .03) and high-risk (Cohort 1-3; P < .001) patients. Conversely, IDC/C improved the prognostication of BCR among CAPRA-S low-risk (Cohorts 1; P < .001 and Cohort 3; P = .003) patients. CONCLUSION: Our results suggest the investigation of histopathological IDC/C features in CAPRA-S low-risk patients and conversely, nomogram CAPRA-S among IDC/C negative patients improves the identification of patients likely to experience BCR, which would otherwise be missed through current assessment regimens. These patients can be offered more intensive monitoring and adjuvant therapies upfront to circumvent the development of recurrent cancer or overtreatment at the time of surgery.

Upregulation of IFNÉ£-mediated chemokines dominate the immune transcriptome of muscle-invasive urothelial carcinoma.

Tumor inflammation is prognostically significant in high-grade muscle-invasive bladder cancer (MIBC). However, the underlying mechanisms remain elusive. To identify inflammation-associated immune gene expression patterns, we performed transcriptomic profiling of 40 MIBC archival tumors using the NanoString nCounter Human v.1.1 PanCancer Panel. Findings were validated using the TCGA MIBC dataset. Unsupervised and supervised clustering identified a distinctive immune-related gene expression profile for inflammation, characterized by significant upregulation of 149 genes, particularly chemokines, a subset of which also had potential prognostic utility. Some of the most enriched biological processes were lymphocyte activation and proliferation, leukocyte adhesion and migration, antigen processing and presentation and cellular response to IFN-γ. Upregulation of numerous IFN-γ-inducible chemokines, class II MHC molecules and immune checkpoint genes was detected as part of the complex immune response to MIBC. Further, B-cell markers linked to tertiary lymphoid structures were upregulated, which in turn is predictive of tumor response to immunotherapy and favorable outcome. Our findings of both an overall activated immune profile and immunosuppressive microenvironment provide novel insights into the complex immune milieu of MIBC with inflammation and supports its clinical significance for predicting prognosis and immunotherapeutic responsiveness, which warrants further investigation. This may open novel opportunities to identify mechanisms for developing new immunotherapeutic strategies.

Three-antibody classifier for muscle invasive urothelial carcinoma and its correlation with p53 expression.

AIMS: To assess the utility of a three-antibody immunohistochemistry panel to classify muscle invasive bladder cancers (MIBCs) in correlation with morphological features and p53 status. METHODS: A retrospective review of 243 chemotherapy naïve MIBC cystectomy specimens was performed to assess morphological features. A tissue microarray was sequentially stained with CK5/6, GATA-3 and p16. Subgroups were assigned as basal-like (CK5/6+, GATA3-) and luminal (CK5/6-, GATA3+), with the latter subdivided into genomically unstable (GU, p16+) and urothelial like (Uro, p16-) subgroups. p53 staining was assessed as abnormal/wild type. Cases from the The Cancer Genome Atlas (TCGA) portal were assessed as external validation. RESULTS: We identified 78.8% luminal, 21.2% basal cases within our cohort and 63.4% luminal, 36.6% basal in the TCGA dataset. Divergent differentiation (p<0.001) was significantly associated with basal-subtype cases in both cohorts. Within the luminal subgroup (n=186), 81 cases were classified as GU and 105 as Uro. Abnormal p53 staining was noted in 48.0% of basal, 80.2% GU and 38.1% Uro cases. Further, basal-subtype tumours significantly correlated with disease-specific death compared with Uro cases in multivariate survival analysis. CONCLUSIONS: This retrospective study demonstrates the potential utility of a three-antibody immunohistochemistry panel to differentiate luminal and basal MIBC.

Development of a multiplex immuno-oncology biomarker and digital pathology workflow for assessment of urothelial carcinoma.

BACKGROUND: Immune checkpoint inhibitors (ICI) therapies have demonstrated significant benefit in the treatment of many tumors including high grade urothelial cancer (HGUC) of the bladder. However, variability in patients' clinical responses highlights the need for biomarkers to aid patient stratification. ICI relies on an intact host immune response. In this context, we hypothesize that key players in the antitumor immune response such as markers of activated cytotoxic T lymphocytes (CD8, granzyme-B) and immune suppression (FOXP3) may help to identify patients who will derive the greatest therapeutic benefit from ICI. A major obstacle for deployment of such a strategy is the limited quantities of tumor-derived biopsy material. Therefore, in this technical study, we develop a multiplex biomarker with digital workflow. We explored the (1) concordance of conventional single stain results using digital image analysis, and (2) agreement between digital scoring versus manual analysis. METHODS: (1) For concordance study of single and multiplex stains, triplicate core tissue microarrays of 207 muscle invasive, HGUC of bladder had sequential 4-micron sections cut and stained with CD8, FOXP3 and granzyme-B. An inhouse developed tri-chromogen multiplex immunohistochemistry (m-IHC) assay consisting of CD8 (green), granzyme B (brown), and FOXP3 (red) was used to stain the next sequential tissue section. (2) Agreement between manual and digital analysis was performed on 19 whole slide sections of HGUC cystectomy specimens. All slides were scanned using Aperio ScanScope AT Digital Scanner at 40X. Quantitative digital image analysis was performed using QuPath version 0.2.3 open-source software. Scores from triplicate cores were averaged for each HGUC specimen for each marker. Intraclass correlation coefficients were used to compare percent positive cells between the single- and multi-plex assays. Lin's concordance correlation coefficients were used for manual versus digital analysis. RESULTS AND CONCLUSIONS: m-IHC offers significant advantages in characterizing the host immune microenvironment particularly in limited biopsy tissue material. Utilizing a digital image workflow resulted in significant concordance between m-IHC and individual single stains (p < 0.001 for all assessments). Moderate to good agreements were achieved between manual and digital scoring. Our technical work demonstrated potential uses of multiplex marker in assessing the host immune status and could be used in conjunction with PD-L1 as a predictor of response to ICI therapy.

Immune gene expression profiles in high-grade urothelial carcinoma of the bladder: a NanoString study.

AIMS: The advent of immune checkpoint inhibitor therapy has proven beneficial in a subset of high-grade urothelial carcinomas (HGUC) of the bladder. Although treatment selection is currently largely determined by programmed death-ligand 1 (PD-L1) status, multiple factors in the immune system may modulate the host immune response to HGUC and immunotherapy. In this pilot study, we used a transcriptomic approach to identify the immune milieu associated with PD-L1 expression to enhance our understanding of the HGUC immune evasion network. METHODS: The immune transcriptome of 40 HGUC cystectomy cases was profiled using the NanoString nCounter Human V.1.1 PanCancer Panel. All cases were assessed for associated PD-L1 status (SP263) using whole tissue sections. PD-L1 status was determined as high or low using 25% tumour and/or immune cell staining. RESULTS: The most significantly differentially expressed gene was PD-L1 messenger RNA (CD274), which strongly correlated with protein expression (r=0.720, p<0.001). The sensitivity, specificity, positive and negative predictive values of CD274 for PD-L1 expression were 85%, 96%, 92% and 93%, respectively. The PD-L1 associated gene signature also included complement components C1QA and CD46 and NOD2 (innate immune system), proinflammatory cytokines CXCL14, CXCL16, CCL3, CCL3L1 and OSM along with the immune response mediator SMAD3, among others. Pathway analysis determined enrichment of these genes in interleukin-10 production, lymphocyte chemotaxis and aberrant IFNγ, NF-κB and ERK signalling networks. CONCLUSIONS: We report key genes and pathways in the immune transcriptome and their association with PD-L1 status, which may be involved in immune evasion of HGUC and warrants further investigation.

CTNNB1 Mutations and Aberrant ß-Catenin Expression in Ovarian Endometrioid Carcinoma: Correlation With Patient Outcome.

CTNNB1 mutations and aberrant ß-catenin expression have adverse prognosis in endometrial endometrioid carcinoma, and recent evidence suggests a prognostic role of ß-catenin in ovarian endometrioid carcinoma. Thus, we aimed to determine the prognostic value of the CTNNB1 mutational status, and its correlation with ß-catenin expression, in a well-annotated cohort of 51 ovarian endometrioid carcinomas. We performed immunohistochemistry for ß-catenin and developed an 11-gene next-generation sequencing panel that included whole exome sequencing of CTNNB1 and TP53. Results were correlated with clinicopathologic variables including disease-free and disease-specific survival. Tumor recurrence was documented in 14 patients (27%), and cancer-related death in 8 patients (16%). CTNNB1 mutations were found in 22 cases (43%), and nuclear ß-catenin in 26 cases (51%). CTNNB1 mutation highly correlated with nuclear ß-catenin (P<0.05). Mutated CTNNB1 status was statistically associated with better disease-free survival (P=0.04, log-rank test) and approached significance for better disease-specific survival (P=0.07). It also correlated with earlier International Federation of Gynecology and Obstetrics stage (P<0.05). Nuclear ß-catenin, TP53 mutations, age, ProMisE group, surface involvement, tumor grade and stage also correlated with disease-free survival. There was no association between membranous ß-catenin expression and disease-free or disease-specific survival. CTNNB1 mutations and nuclear ß-catenin expression are associated with better progression-free survival in patients with OEC. This relationship may be in part due to a trend of CTNNB1-mutated tumors to present at early stage. ß-catenin immunohistochemistry may serve as a prognostic biomarker and a surrogate for CTNN1B mutations in the evaluation of patients with ovarian endometrioid neoplasia, particularly those in reproductive-age or found incidentally without upfront staging surgery.

Can immune markers help identify fast relapse in patients with muscle invasive bladder cancer?

The aim of this pilot study was to assess the role of immune markers in fast relapse (<2 years) of high-grade muscle invasive urothelial carcinomas of the bladder (HGUC) treated by cystectomy. A series of 40 such cases was investigated for immune protein (CD3, CD4, CD8, CD20, CD68, CD163, FOXP3 and PD-1) status by immunohistochemistry. Decreased expression of all immune cell markers was observed in tumors of patients who relapsed quickly. In Kaplan-Meier (log-rank test) analysis, low CD3, CD4 and CD8 expression was associated with fast relapse (P = 0.005, 0.028, 0.036 respectively). Additional evaluation of the immune transcriptome by NanoString Human PanCancer Immune Panel v.1.1 has identified 5 differentially expressed genes significantly associated with fast relapse. Among these, KLRB1 and HLA-DQA1 were also significant on Kaplan-Meier analysis (log-rank test P = 0.007 and 0.006, respectively). These findings strengthen the potential clinical utility and, hence, the need for further evaluation of immune markers in HGUC prognostication.

The prognostic role of horizontal and circumferential tumor extent in cervical cancer: Implications for the 2019 FIGO staging system.

OBJECTIVE: The FIGO 2019 update on cervical cancer staging removed horizontal tumor extent (HZTE) as a staging variable. Evidence is needed to substantiate this change. The prognostic significance of HZTE and a related variable, circumferential tumor extent (%CTE), is similarly unknown. We aimed to investigate the association of HZTE and %CTE with survival outcomes in cervical cancer patients. METHODS: We identified patients treated with primary surgery for stage I cervical cancer in a single institution during a 9-year period. HZTE and, when available, %CTE were obtained from pathology records. Cases were staged using 2019 FIGO staging. Correlations between HZTE, %CTE and FIGO stage with recurrence-free (RFS) and disease-specific survival (DSS) were determined using univariable and multivariable analyses. RESULTS: 285 patients were included with a median follow-up of 48 (range 7-123) months. HZTE was statistically associated with RFS and DSS on univariate and multivariate analysis. None of the 168 stage IA patients in our series had tumor recurrence or death during follow-up, including 42 with HZTE ≥7 mm. None of the patients with a tumor horizontal extent <7 mm experienced recurrence or death. %CTE correlated only with RFS on univariate analysis. 2019 FIGO stage did not independently correlate with RFS or DSS in our sample. CONCLUSIONS: HZTE is an independent predictor of survival in cervical carcinoma. In stage IA tumors, however, HZTE does not offer superior prognostic value, supporting the 2019 FIGO recommendations to remove this variable from staging in these cases. HZTE may be useful in larger tumors in which staging depends on maximum tumor size. %CTE is not an independent prognostic variable in cervical cancer, and we advise against its use.