Immunohistochemical assessment of HER2 low breast cancer: interobserver reproducibility and correlation with digital image analysis.

PURPOSE: The recent findings from the DESTINY-Breast04 trial highlighted the clinical importance of distinguishing between HER2 immunohistochemistry (IHC) scores 0 and 1 + in metastatic breast cancer (BC). However, pathologist interpretation of HER2 IHC scoring is subjective, and standardized methodology is needed. We evaluated the consistency of HER2 IHC scoring among pathologists and the accuracy of digital image analysis (DIA) in interpreting HER2 IHC staining in cases of HER2-low BC. METHODS: Fifty whole-slide biopsies of BC with HER2 IHC staining were evaluated, comprising 25 cases originally reported as IHC score 0 and 25 as 1 +. These slides were digitally scanned. Six pathologists with breast expertise independently reviewed and scored the scanned images, and DIA was applied. Agreement among pathologists and concordance between pathologist scores and DIA results were statistically analyzed using Kendall coefficient of concordance (W) tests. RESULTS: Substantial agreement among at least five of the six pathologists was found for 18 of the score 0 cases (72%) and 15 of the score 1 + cases (60%), indicating excellent interobserver agreement (W = 0.828). DIA scores were highly concordant with pathologist scores in 96% of cases (47/49), indicating excellent concordance (W = 0.959). CONCLUSION: Although breast subspecialty pathologists were relatively consistent in evaluating BC with HER2 IHC scores of 0 and 1 +, DIA may be a reliable supplementary tool to enhance the standardization and quantification of HER2 IHC assessment, especially in challenging cases where results may be ambiguous (i.e., scores 0-1 +). These findings hold promise for improving the accuracy and consistency of HER2 testing.

Infiltrative pattern of invasion is independently associated with shorter survival and desmoplastic stroma markers FAP and THBS2 in mucinous ovarian carcinoma.

AIMS: Mucinous ovarian carcinoma (MOC) is a rare ovarian cancer histotype with generally good prognosis when diagnosed at an early stage. However, MOC with the infiltrative pattern of invasion has a worse prognosis, although to date studies have not been large enough to control for covariables. Data on reproducibility of classifying the invasion pattern are limited, as are molecular correlates for infiltrative invasion. We hypothesized that the invasion pattern would be associated with an aberrant tumour microenvironment. METHODS AND RESULTS: Four subspecialty pathologists assessed interobserver reproducibility of the pattern of invasion in 134 MOC. Immunohistochemistry on fibroblast activation protein (FAP) and THBS2 was performed on 98 cases. Association with survival was tested using Cox regression. The average interobserver agreement for the infiltrative pattern was moderate (kappa 0.60, agreement 86.3%). After reproducibility review, 24/134 MOC (18%) were determined to have the infiltrative pattern and this was associated with a higher risk of death, independent of FIGO stage, grade, and patient age in a time-dependent manner (hazard ratio [HR] = 10.2, 95% confidence interval [CI] 3.0-34.5). High stromal expression of FAP and THBS2 was more common in infiltrative MOC (FAP: 60%, THBS2: 58%, both P < 0.001) and associated with survival (multivariate HR for FAP: 1.5 [95% CI 1.1-2.1] and THBS2: 1.91 [95% CI 1.1-3.2]). CONCLUSIONS: The pattern of invasion should be included in reporting for MOC due to the strong prognostic implications. We highlight the histological features that should be considered to improve reproducibility. FAP and THBS2 are associated with infiltrative invasion in MOC.

TP53 as a Diagnostic Aid in the Distinction of Ovarian Mucinous Borderline Tumors From Mucinous Carcinoma.

Ovarian mucinous borderline tumors (MBTs) are clinically managed as benign neoplasms while the management of ovarian mucinous carcinomas (MC) is dependent on tumor stage. Despite the standardization of sampling of ovarian mucinous neoplasms, limited interobserver reproducibility between MBT and MC persists. Based on our recent finding that abnormal TP53 expression is associated with unfavorable outcome in MBT, we hypothesized that TP53 status might improve the reproducible distinction of MBT from MC. A virtual slide set of 85 consecutive ovarian mucinous neoplasms received at a single institution, with each case represented by 3 full sections, were reviewed by 3 pathologists in 2 iterations. The initial assessment was based solely on morphologic review, while the second iteration was performed with knowledge of TP53 status. The reproducibility of a trinary categorization (MBT, MBT with intraepithelial carcinoma [IEC], MC) significantly improved from a κ of 0.60 based on the initial morphologic assessment to a κ of 0.76 (t-test, P =0.0042) after consideration of TP53 immunohistochemistry (IHC) results. Six out of 85 patients died of disease, and in 2 of them, at least 1 pathologist assessed MBT with IEC and not MC even after integration of TP53 IHC. With the integration of TP53 IHC, substantial interobserver agreement for MBT and MC can be reached, particularly in cases with an uncertain degree of confluent growth. TP53 IHC can also be used to highlight and support the presence of IEC in MBT, however, discordances remained in 2 cases with adverse outcome.

Molecular Surrogate Subtypes of Ovarian and Peritoneal Low-grade Serous Carcinoma.

Low-grade serous carcinoma (LGSC) is an uncommon histotype of ovarian carcinoma, accounting for ~3% of cases. There is evidence that survival of peritoneal LGSC (pLGSC) is longer than that of ovarian LGSC (oLGSC). Key molecular alterations of LGSC have been established, including loss of CDKN2A and PR expression, MAPK pathway alterations, and loss of USP9X expression. We hypothesized that LGSC could be subclassified into clinically applicable molecular subtypes by a few surrogate tests similar to endometrioid carcinomas using a hierarchical decision tree based on the strength of the prognostic associations of the individual alterations. Our study included 71 LGSCs. Immunohistochemistry for CDKN2A, ER, PR, NF1, and USP9X and sequencing for KRAS , NRAS , and BRAF were performed. Our data showed the co-occurrence of key molecular alterations, and despite suggestive trends, hierarchical molecular subtyping did not provide significantly different stratification of patients according to survival in this cohort. We confirmed that patients diagnosed with pLGSC have a longer survival than high-stage oLGSC, with the intriguing observation that normal CDKN2A and PR status were associated with excellent survival in pLGSC. Therefore, CDKN2A and PR status might aid in the classification of indeterminate implants, where abnormal findings favor pLGSC over noninvasive implants. Molecular subtypes should be further evaluated in larger cohorts for their prognostic and potentially predictive value.

CCNE1 and survival of patients with tubo-ovarian high-grade serous carcinoma: An Ovarian Tumor Tissue Analysis consortium study.

BACKGROUND: Cyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC. METHODS: Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated. RESULTS: High-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08-1.47, p = .034, and HR, 1.18; 95% CI, 1.05-1.32, p = .015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR, 1.26; 95% CI, 1.09-1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1-SD increase; 95% CI, 0.94-1.06; p = .58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss. CONCLUSION: This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.

Increased FOXJ1 protein expression is associated with improved overall survival in high-grade serous ovarian carcinoma: an Ovarian Tumor Tissue Analysis Consortium Study.

BACKGROUND: Recently, we showed a >60% difference in 5-year survival for patients with tubo-ovarian high-grade serous carcinoma (HGSC) when stratified by a 101-gene mRNA expression prognostic signature. Given the varied patient outcomes, this study aimed to translate prognostic mRNA markers into protein expression assays by immunohistochemistry and validate their survival association in HGSC. METHODS: Two prognostic genes, FOXJ1 and GMNN, were selected based on high-quality antibodies, correlation with protein expression and variation in immunohistochemical scores in a preliminary cohort (n = 134 and n = 80, respectively). Six thousand four hundred and thirty-four (FOXJ1) and 5470 (GMNN) formalin-fixed, paraffin-embedded ovarian neoplasms (4634 and 4185 HGSC, respectively) represented on tissue microarrays from the Ovarian Tumor Tissue Analysis consortium underwent immunohistochemical staining and scoring, then univariate and multivariate survival analysis. RESULTS: Consistent with mRNA, FOXJ1 protein expression exhibited a linear, increasing association with improved overall survival in HGSC patients. Women with >50% expression had the most favourable outcomes (HR = 0.78, 95% CI 0.67-0.91, p < 0.0001). GMNN protein expression was not significantly associated with overall HSGC patient survival. However, HGSCs with >35% GMNN expression showed a trend for better outcomes, though this was not significant. CONCLUSION: We provide foundational evidence for the prognostic value of FOXJ1 in HGSC, validating the prior mRNA-based prognostic association by immunohistochemistry.

Survey of NF1 inactivation by surrogate immunohistochemistry in ovarian carcinomas.

OBJECTIVE: Inhibition of the MAPK pathway by MEK inhibitors (MEKi) is currently a therapeutic standard in several cancer types, including ovarian low-grade serous carcinoma (LGSC). A common MAPK pathway alteration in tubo-ovarian high-grade serous carcinoma (HGSC) is the genomic inactivation of neurofibromin 1 (NF1). The primary objectives of our study were to survey the prevalence of NF1 inactivation in the principal ovarian carcinoma histotype as well as to evaluate its associations with clinico-pathological parameters and key biomarkers including BRCA1/2 status in HGSC. METHODS: A recently commercialized NF1 antibody (clone NFC) was orthogonally validated on an automated immunohistochemistry (IHC) platform and IHC was performed on tissue microarrays containing 2140 ovarian carcinoma cases. Expression was interpreted as loss/inactivated (complete or subclonal) versus normal/retained. RESULTS: Loss of NF1 expression was detected in 250/1429 (17.4%) HGSC including 11% with subclonal loss. Survival of NF1-inactivated HGSC patients was intermediate between favorable BRCA1/2 mutated HGSC and unfavorable CCNE1 high-level amplified HGSC. NF1 inactivation was mutually exclusive with CCNE1 high-level amplifications, co-occurred with RB1 loss and occurred at similar frequencies in BRCA1/2 mutated versus wild-type HGSC. NF1 loss was found in 21/286 (7.3%) endometrioid carcinomas with a favorable prognostic association (p = 0.048), and in 4/64 (5.9%) LGSC, mutually exclusive with other driver events. CONCLUSIONS: NF1 inactivation occurs in a significant subset of BRCA1/2 wild-type HGSC and a subset of LGSC. While the functional effects of NF1 inactivation need to be further characterized, this signifies a potential therapeutic opportunity to explore targeting NF1 inactivation in these tumors.

Multicentre external validation of a commercial artificial intelligence software to analyse chest radiographs in health screening environments with low disease prevalence.

OBJECTIVES: To externally validate the performance of a commercial AI software program for interpreting CXRs in a large, consecutive, real-world cohort from primary healthcare centres. METHODS: A total of 3047 CXRs were collected from two primary healthcare centres, characterised by low disease prevalence, between January and December 2018. All CXRs were labelled as normal or abnormal according to CT findings. Four radiology residents read all CXRs twice with and without AI assistance. The performances of the AI and readers with and without AI assistance were measured in terms of area under the receiver operating characteristic curve (AUROC), sensitivity, and specificity. RESULTS: The prevalence of clinically significant lesions was 2.2% (68 of 3047). The AUROC, sensitivity, and specificity of the AI were 0.648 (95% confidence interval [CI] 0.630-0.665), 35.3% (CI, 24.7-47.8), and 94.2% (CI, 93.3-95.0), respectively. AI detected 12 of 41 pneumonia, 3 of 5 tuberculosis, and 9 of 22 tumours. AI-undetected lesions tended to be smaller than true-positive lesions. The readers' AUROCs ranged from 0.534-0.676 without AI and 0.571-0.688 with AI (all p values < 0.05). For all readers, the mean reading time was 2.96-10.27 s longer with AI assistance (all p values < 0.05). CONCLUSIONS: The performance of commercial AI in these high-volume, low-prevalence settings was poorer than expected, although it modestly boosted the performance of less-experienced readers. The technical prowess of AI demonstrated in experimental settings and approved by regulatory bodies may not directly translate to real-world practice, especially where the demand for AI assistance is highest. KEY POINTS: • This study shows the limited applicability of commercial AI software for detecting abnormalities in CXRs in a health screening population. • When using AI software in a specific clinical setting that differs from the training setting, it is necessary to adjust the threshold or perform additional training with such data that reflects this environment well. • Prospective test accuracy studies, randomised controlled trials, or cohort studies are needed to examine AI software to be implemented in real clinical practice.

Evaluation of fully automated commercial software for Agatston calcium scoring on non-ECG-gated low-dose chest CT with different slice thickness.

OBJECTIVES: To evaluate commercial deep learning-based software for fully automated coronary artery calcium (CAC) scoring on non-electrocardiogram (ECG)-gated low-dose CT (LDCT) with different slice thicknesses compared with manual ECG-gated calcium-scoring CT (CSCT). METHODS: This retrospective study included 567 patients who underwent both LDCT and CSCT. All LDCT images were reconstructed with a 2.5-mm slice thickness (LDCT2.5-mm), and 453 LDCT scans were reconstructed with a 1.0-mm slice thickness (LDCT1.0-mm). Automated CAC scoring was performed on CSCT (CSCTauto), LDCT1.0-mm, and LDCT2.5-mm images. The reliability of CSCTauto, LDCT1.0-mm, and LDCT2.5-mm was compared with manual CSCT scoring (CSCTmanual) using intraclass correlation coefficients (ICCs) and Bland-Altman analysis. Agreement, in CAC severity category, was analyzed using weighted kappa statistics. Diagnostic performance at various Agatston score cutoffs was also calculated. RESULTS: CSCTauto, LDCT1.0-mm, and LDCT2.5-mm demonstrated excellent agreement with CSCTmanual (ICC [95% confidence interval, CI]: 1.000 [1.000, 1.000], 0.937 [0.917, 0.952], and 0.955 [0.946, 0.963], respectively). The mean difference with 95% limits of agreement was lower with LDCT1.0-mm than with LDCT2.5-mm (19.94 [95% CI, -244.0, 283.9] vs. 45.26 [-248.2, 338.7]). Regarding CAC severity, LDCT1.0-mm achieved almost perfect agreement, and LDCT2.5-mm achieved substantial agreement (kappa [95% CI]: 0.809 [0.776, 0.838], 0.776 [0.740, 0.809], respectively). Diagnostic performance for detecting Agatston score ≥ 400 was also higher with LDCT1.0-mm than with LDCT2.5-mm (F1 score, 0.929 vs. 0.855). CONCLUSIONS: Fully automated CAC-scoring software with both CSCT and LDCT yielded excellent reliability and agreement with CSCTmanual. LDCT1.0-mm yielded more accurate Agatston scoring than LDCT2.5-mm using fully automated commercial software. KEY POINTS: • Total Agatston scores and all vessels of CSCTauto, LDCT1.0-mm, and LDCT2.5-mm demonstrated excellent agreement with CSCTmanual (all ICC > 0.85). • The diagnostic performance for detecting all Agatston score cutoffs was better with LDCT1.0-mm than with LDCT2.5-mm. • This automated software yielded a lower degree of underestimation compared with methods described in previous studies, and the degree of underestimation was lower with LDCT1.0-mm than with LDCT2.5-mm.

Validated biomarker assays confirm that ARID1A loss is confounded with MMR deficiency, CD8+ TIL infiltration, and provides no independent prognostic value in endometriosis-associated ovarian carcinomas.

ARID1A (BAF250a) is a component of the SWI/SNF chromatin modifying complex, plays an important tumour suppressor role, and is considered prognostic in several malignancies. However, in ovarian carcinomas there are contradictory reports on its relationship to outcome, immune response, and correlation with clinicopathological features. We assembled a series of 1623 endometriosis-associated ovarian carcinomas, including 1078 endometrioid (ENOC) and 545 clear cell (CCOC) ovarian carcinomas, through combining resources of the Ovarian Tumor Tissue Analysis (OTTA) Consortium, the Canadian Ovarian Unified Experimental Resource (COEUR), local, and collaborative networks. Validated immunohistochemical surrogate assays for ARID1A mutations were applied to all samples. We investigated associations between ARID1A loss/mutation, clinical features, outcome, CD8+ tumour-infiltrating lymphocytes (CD8+ TILs), and DNA mismatch repair deficiency (MMRd). ARID1A loss was observed in 42% of CCOCs and 25% of ENOCs. We found no associations between ARID1A loss and outcomes, stage, age, or CD8+ TIL status in CCOC. Similarly, we found no association with outcome or stage in endometrioid cases. In ENOC, ARID1A loss was more prevalent in younger patients (p = 0.012) and was associated with MMRd (p < 0.001) and the presence of CD8+ TILs (p = 0.008). Consistent with MMRd being causative of ARID1A mutations, in a subset of ENOCs we also observed an association with ARID1A loss-of-function mutation as a result of small indels (p = 0.035, versus single nucleotide variants). In ENOC, the association with ARID1A loss, CD8+ TILs, and age appears confounded by MMRd status. Although this observation does not explicitly rule out a role for ARID1A influence on CD8+ TIL infiltration in ENOC, given current knowledge regarding MMRd, it seems more likely that effects are dominated by the hypermutation phenotype. This large dataset with consistently applied biomarker assessment now provides a benchmark for the prevalence of ARID1A loss-of-function mutations in endometriosis-associated ovarian cancers and brings clarity to the prognostic significance. © 2021 The Pathological Society of Great Britain and Ireland.

Endometrial neuroendocrine carcinoma and undifferentiated carcinoma are distinct entities with overlap in neuroendocrine marker expression.

AIMS: Dedifferentiated endometrial carcinomas (DDECs)/undifferentiated endometrial carcinomas (UDECs) frequently harbour genomic activation of switch/sucrose non-fermentable (SWI/SNF)-complex proteins, and can show histological overlap with neuroendocrine carcinoma (NEC). The aim of this study was to compare the extent of the expression of neuroendocrine markers, SWI/SNF proteins and mismatch repair (MMR) proteins in DDEC/UDEC and NEC. METHODS AND RESULTS: The extent of expression of synaptophysin, chromogranin, CD56, ARID1A, ARID1B, SMARCA4, SMARCB1 and MMR proteins was evaluated by immunohistochemistry on 44 SWI/SNF-deficient DDECs/UDECs and 15 NECs. Thirty-three of 44 (75%) DDECs/UDECs showed expression of at least one neuroendocrine marker, with 18 of 44 (41%) expressing two or more neuroendocrine markers, whereas all 15 NECs showed expression of at least one neuroendocrine marker, with 14 of 15 (93%) expressing two or more neuroendocrine markers. Neuroendocrine marker expression in DDECs/UDECs was typically focal when present, with average extents of 17%, 4% and 8% for synaptophysin, chromogranin and CD56 in the positive cases, respectively, in contrast to 73%, 40% and 62% in the positive NEC cases, respectively. All 15 NECs showed intact expression of SWI/SNF-complex proteins, except for one that showed isolated loss of ARID1A. Thirty-eight of 44 DDECs/UDECs were MMR-abnormal (34 with loss of MLH1 and PMS2, and four with loss of PMS2 alone), whereas all NECs retained MMR protein expression. CONCLUSIONS: Our study demonstrates frequent but typically focal neuroendocrine marker expression in SWI/SNF-deficient DDECs/UDECs, whereas NECs typically express two or more neuroendocrine markers, with diffuse expression of at least one marker. ARID1B, SMARCA4 and SMARCB1 immunohistochemistry can be used to aid in the differentiation between DDEC/UDEC and NEC.

Differentiating pulmonary metastasis from benign lung nodules in thyroid cancer patients using dual-energy CT parameters.

OBJECTIVES: To explore the importance of quantitative characteristics of dual-energy CT (DECT) between pulmonary metastasis and benign lung nodules in thyroid cancer. METHODS: In this retrospective study, we identified 63 patients from our institution's database with pathologically proven thyroid cancer who underwent DECT to assess pulmonary metastasis. Among these patients, 22 had 55 pulmonary metastases, and 41 had 97 benign nodules. If nodules showed increased iodine uptake on I-131 single-photon emission computed tomography-computed tomography or increased size in follow-up CT, they were considered metastatic. We compared the clinical findings and DECT parameters of both groups and performed a receiver operating characteristic analysis to evaluate the optimal cutoff values of the DECT parameters. RESULTS: Patients with metastases were significantly older than patients with benign nodules (p = 0.048). The DECT parameters of the metastatic nodules were significantly higher than those of the benign nodules (iodine concentration [IC], 5.61 ± 2.02 mg/mL vs. 1.61 ± 0.98 mg/mL; normalized IC [NIC], 0.60 ± 0.20 vs. 0.16 ± 0.11; NIC using pulmonary artery [NICPA], 0.60 ± 0.44 vs. 0.15 ± 0.11; slope of the spectral attenuation curves [λHU], 5.18 ± 2.54 vs. 2.12 ± 1.39; and Z-effective value [Zeff], 10.0 ± 0.94 vs. 8.79 ± 0.75; all p < 0.001). In the subgroup analysis according to nodule size, all DECT parameters of the metastatic nodules in all subgroups were significantly higher than those of the benign nodules (all p < 0.05). The cutoff values for IC, NIC, λHU, NICPA, and Zeff for diagnosing metastases were 3.10, 0.29, 3.57, 0.28, and 9.34, respectively (all p < 0.001). CONCLUSIONS: DECT parameters can help to differentiate metastatic and benign lung nodules in thyroid cancer. KEY POINTS: • DECT parameters can help to differentiate metastatic and benign lung nodules in patients with thyroid cancer. • DECT parameters showed a significant difference between benign lung nodules and lung metastases, even for nodules with diameters ≥ 3 mm and < 5 mm. • Among the DECT parameters, the highest diagnostic accuracy for differentiating pulmonary metastases from benign lung nodules was achieved with the NIC and IC, followed by the NICPA and λHU, and their cutoff values were 0.29, 3.10, 0.28, and 3.57, respectively.

Prognostic and Theranostic Biomarkers in Ovarian Clear Cell Carcinoma.

In this study, we aimed to test whether prognostic biomarkers can achieve a clinically relevant stratification of patients with stage I ovarian clear cell carcinoma (OCCC) and to survey the expression of 10 selected actionable targets (theranostic biomarkers) in stage II to IV cases. From the population-based Alberta Ovarian Tumor Type study, 160 samples of OCCC were evaluated by immunohistochemistry and/or silver-enhanced in situ hybridization for the status of 5 prognostic (p53, p16, IGF2BP3, CCNE1, FOLR1) and 10 theranostic biomarkers (ALK, BRAF V600E, ERBB2, ER, MET, MMR, PR, ROS1, NTRK1-3, VEGFR2). Kaplan-Meier survival analyses were performed. Cases with abnormal p53 or combined p16/IFG2BP3 abnormal expression identified a small subset of patients (6/54 cases) with stage I OCCC with an aggressive course (5-yr ovarian cancer-specific survival of 33.3%, compared with 91.5% in the other stage I cases). Among theranostic targets, ERBB2 amplification was present in 11/158 (7%) of OCCC, while MET was ubiquitously expressed in OCCC similar to a variety of normal control tissues. ER/PR showed a low prevalence of expression. No abnormal expression was detected for any of the other targets. We propose a combination of 3 biomarkers (p53, p16, IGF2BP3) to predict prognosis and the potential need for adjuvant therapy for patients with stage I OCCC. This finding requires replication in larger cohorts. In addition, OCCC could be tested for ERBB2 amplification for inclusion in gynecological basket trials targeting this alteration.

Targeting the actin/tropomyosin cytoskeleton in epithelial ovarian cancer reveals multiple mechanisms of synergy with anti-microtubule agents.

BACKGROUND: Anti-microtubule agents are widely used to treat ovarian cancers, but the efficacy is often compromised by drug resistance. We investigated co-targeting the actin/tropomyosin cytoskeleton and microtubules to increase treatment efficacy in ovarian cancers and potentially overcome resistance. METHODS: The presence of tropomyosin-3.1 (Tpm3.1) was examined in clinical specimens from ovarian cancer patients using immunohistochemistry. Combinatorial effects of an anti-Tpm3.1 compound, ATM-3507, with vinorelbine and paclitaxel were evaluated in ovarian cancer cells via MTS and apoptosis assays. The mechanisms of action were established using live- and fixed-cell imaging and protein analysis. RESULTS: Tpm3.1 is overexpressed in 97% of tumour tissues (558 of 577) representing all histotypes of epithelial ovarian cancer. ATM-3507 displayed synergy with both anti-microtubule agents to reduce cell viability. Only vinorelbine synergised with ATM-3507 in causing apoptosis. ATM-3507 significantly prolonged vinorelbine-induced mitotic arrest with elevated activity of the spindle assembly checkpoint and mitotic cell death; however, ATM-3507 showed minor impact on paclitaxel-induced mitotic defects. Both combinations substantially increased post-mitotic G1 arrest with cyclin D1 and E1 downregulation and an increase of p21Cip and p27Kip. CONCLUSION: Combined targeting of Tpm3.1/actin and microtubules is a promising treatment strategy for ovarian cancer that should be further tested in clinical settings.

Refined cut-off for TP53 immunohistochemistry improves prediction of TP53 mutation status in ovarian mucinous tumors: implications for outcome analyses.

TP53 mutations are implicated in the progression of mucinous borderline tumors (MBOT) to mucinous ovarian carcinomas (MOC). Optimized immunohistochemistry (IHC) for TP53 has been established as a proxy for the TP53 mutation status in other ovarian tumor types. We aimed to confirm the ability of TP53 IHC to predict TP53 mutation status in ovarian mucinous tumors and to evaluate the association of TP53 mutation status with survival among patients with MBOT and MOC. Tumor tissue from an initial cohort of 113 women with MBOT/MOC was stained with optimized IHC for TP53 using tissue microarrays (75.2%) or full sections (24.8%) and interpreted using established criteria as normal or abnormal (overexpression, complete absence, or cytoplasmic). Cases were considered concordant if abnormal IHC staining predicted deleterious TP53 mutations. Discordant tissue microarray cases were re-evaluated on full sections and interpretational criteria were refined. The initial cohort was expanded to a total of 165 MBOT and 424 MOC for the examination of the association of survival with TP53 mutation status, assessed either by TP53 IHC and/or sequencing. Initially, 82/113 (72.6%) cases were concordant using the established criteria. Refined criteria for overexpression to account for intratumoral heterogeneity and terminal differentiation improved concordance to 93.8% (106/113). In the expanded cohort, 19.4% (32/165) of MBOT showed evidence for TP53 mutation and this was associated with a higher risk of recurrence, disease-specific death, and all-cause mortality (overall survival: HR = 4.6, 95% CI 1.5-14.3, p = 0.0087). Within MOC, 61.1% (259/424) harbored a TP53 mutation, but this was not associated with survival (overall survival, p = 0.77). TP53 IHC is an accurate proxy for TP53 mutation status with refined interpretation criteria accounting for intratumoral heterogeneity and terminal differentiation in ovarian mucinous tumors. TP53 mutation status is an important biomarker to identify MBOT with a higher risk of mortality.

Loss of ARID1B and SMARCB1 expression are specific for the diagnosis of dedifferentiated/undifferentiated carcinoma in tumours of the upper gynaecological tract and cervix.

AIMS: Genomic inactivation of ARID1B in ARID1A-inactivated tumour and genomic inactivation of SMARCB1 represent two recurrent mechanisms, core SWItch/sucrose non-fermentable (SWI/SNF) complex inactivation, that are associated with de-differentiation in endometrial carcinoma. Approximately one-third of dedifferentiated/undifferentiated endometrial carcinomas (DDEC/UEC) show loss of ARID1B expression with a minor subset showing loss of SMARCB1 expression, but little is known regarding the specificity of ARID1B or SMARCB1 loss in gynaecological tract tumours in general. The aim of this study was to examine the frequency of ARID1B and SMARCB1 loss by immunohistochemistry in a series of gynaecological tract epithelial/mesenchymal neoplasms. METHODS AND RESULTS: We evaluated 1849 tumours that included 748 endometrial carcinomas, 101 uterine carcinosarcomas/adenosarcomas, 64 uterine sarcomas, 221 cervical carcinomas and 715 ovarian carcinomas/borderline tumours by tissue microarrays (TMA). We observed ARID1B loss in 35 of 86 (41%) and SMARCB1 loss in three of 86 (3%) DDEC/UEC, but not in any other uterine tumour types examined. ARID1B-deficient DDEC/UEC also showed concurrent loss of ARID1A expression. All SMARCB1-deficient tumours showed loss of MLH1 and PMS2, while 29 of 35 ARID1B-deficient tumours showed loss of MLH1 and PMS2 or loss of MSH6. All ovarian carcinomas/borderline tumours and cervical carcinomas showed intact expression of ARID1B and SMARCB1. CONCLUSION: Our findings indicate that the loss of expression of ARID1B or SMARCB1 by immunohistochemistry is highly specific for undifferentiated carcinoma among tumours of the upper gynaecological tract and cervix, and therefore can be used to identify these highly aggressive malignant tumours.

Prognostic significance of T cells, PD-L1 immune checkpoint and tumour associated macrophages in clear cell carcinoma of the ovary.

OBJECTIVE: To define the pre-treatment tumour immune landscape of clear cell carcinoma of the ovary (CCOC). METHODS: We investigated the infiltration profiles of selected immune cell populations and immune checkpoint proteins that have been previously shown to have prognostic relevance in high grade serous carcinoma of the ovary to determine their association with clinical outcomes in CCOC patients. Using multiplex immunohistochemistry, we evaluated the density of CD3+, FoxP3+, CD8+ T cells, CD20+ B cells and expression of PD-1, PD-L1 and IDO1 immune checkpoints in a cohort of 162 CCOC tumour specimens on a tissue microarray. RESULTS: Increased infiltration of CD3+ CD8- (helper T) cells, CD8+ (cytotoxic T) cells, and CD68+ macrophages significantly associated with shorter disease-free survival, recurrence-free survival and overall survival. Importantly, higher expression of PD-L1 and IDO-1 immune checkpoints was associated with better clinical outcomes. CONCLUSION: Findings from our study are foundational towards the development of immune classifiers and biomarkers of response to immune checkpoint blockade therapy in CCOC.

Nitrates administered by spray versus tablet: comparison of coronary vasodilation on CT angiography.

OBJECTIVES: To compare the coronary vasodilation effects of spray with those induced by tablet administration in coronary CT angiography (CCTA). METHODS: A total of 2024 patients who underwent CCTA were identified for this retrospective study, including 828 patients with spray (spray group) and 1169 with tablets (tablet group). Of these, 93 patients underwent CCTA at least twice using both spray and tablets. The number of measurable segments and diameters of all 18 segments was measured. The number of measurable segments was compared between groups. RESULTS: No statistically significant differences were evident between these two groups in terms of clinical characteristics. All coronary segments except the ramus intermedius (RI) and left posterior descending artery (L-PDA) were significantly larger in the spray group than in the tablet group (all p < 0.001). In peripheral and branch vessels, as well as in central and main coronary arteries, the diameters were significantly larger in the spray group than in the tablet group (all p < 0.001). Although not always statistically significant, all coronary segments tended to be more measurable on CCTA with spray than with tablet. In the subgroup that underwent CCTA twice using both spray and tablets, all coronary segments except the RI, obtuse marginal artery 2 (OM2), and L-PDA were significantly larger in the spray group than in the tablet group (all p < 0.05). CONCLUSION: Lingual isosorbide dinitrate (ISDN) spray was more efficacious than sublingual nitroglycerin (NTG) tablets in coronary vasodilation for CCTA. Therefore, lingual ISDN spray should be preferred over sublingual NTG tablets for CCTA. KEY POINTS: • Lingual ISDN spray was more efficacious than sublingual NTG tablet for coronary vasodilation in coronary CT angiography, even in elderly patients. • The diameters of all coronary segments except RI and L-PDA were significantly larger, and there were significantly more coronary segments greater than 1.5 mm, except RI and L-PDA, in the spray group than in the tablet group in the whole study group. • Even in peripheral and branch vessels, the diameters of coronary arteries were significantly larger in the spray group than in the tablet group, and they were also larger in elderly patients.

The Many Uses of p53 Immunohistochemistry in Gynecological Pathology: Proceedings of the ISGyP Companion Society Session at the 2020 USCAP Annual9 Meeting.

Ancillary immunohistochemistry (IHC) has become a reliable adjunct for subclassification of gynecological neoplasms. An important recent development was optimization and validation of p53 IHC, where 3 abnormal IHC patterns (nuclear overexpression, complete absence, cytoplasmic) were shown to predict underlying TP53 mutations with high accuracy in ovarian carcinomas. p53 IHC now helps in distinguishing high-grade serous from low-grade serous carcinomas. Thereafter, the new interpretation of p53 IHC was quickly adapted for other purposes and similar accuracies were shown in endometrial carcinomas, vulvar squamous cell carcinomas, and ovarian mucinous tumors. However, it required further refinement of the p53 IHC interpretation criteria for each tumor site. A proportion of endometrial endometrioid carcinomas shows an ultramutated or hypermutated genotype due to underlying POLE mutations or mismatch repair deficiency sometimes causing subclonal TP53 mutations, and their distribution can be visualized by p53 IHC. Squamous cell carcinomas and ovarian mucinous tumors show a phenomenon called terminal differentiation where basal cells demonstrate an abnormal pattern of p53 IHC but apical cells do not despite an underlying TP53 mutation. High-grade progression of adult granulosa cell tumors due to a subclonal TP53 mutation has been recently described. Another use of p53 IHC is triaging gynecological sarcomas for molecular testing based on the assumption that TP53-mutated gynecological sarcomas do not harbor cancer driving translocations. Therefore, familiarity with interpretation of p53 IHC is becoming increasingly important for the practicing gynecological pathologist. Furthermore, local optimization of the p53 IHC assay using validated protocols including appropriate low expressing control tissues (eg, tonsil) is vital in order to achieve high diagnostic accuracy, especially for abnormal staining patterns such as complete absence or cytoplasmic, and interlaboratory concordance. p53 IHC is a reliable diagnostic adjunct for histotyping and molecular subtyping of ovarian and endometrial carcinomas, and it paves the way for large-scale studies to validate the prognostic value of p53 IHC in several gynecological tumor types. The technical advances, validated interpretation criteria, and its growing versatility in identifying high-risk neoplasms paired with its widespread availability in pathology departments make p53 IHC perhaps the single most useful IHC stain in gynecological pathology.

Efficacy of auditory evoked potential follow-up in viral meningitis of infants.

PURPOSE: To examine the rate of occurrence of hearing impairments among the infants who had recovered from viral meningitis under 1 year of age through auditory evoked potential (AEP) test and to investigate the efficacy of the follow-up AEP test in viral meningitis infants. METHODS: Two hundred twenty infants (440 ears) were examined through AEP test once, and 47 (94 ears) of them went back for a second examination and were diagnosed with viral meningitis. The first AEP tests were compared with the second results in 47 infants. I latency, V latency, I-III interpeak latency (IPL), and III-V IPL were checked. RESULTS: In the first AEP test conducted on 440 ears, the average values of I and V latency and I-III IPL were delayed as compared with normal values. The second AEP results were conducted on 47 infants 92.36 days after the first exam. I latency and V latency of second exam were improved significantly (p < 0.05), but I-III and III-V IPL showed no significant changes. Two hearing impaired patients (4 ears) were confirmed through chart reviews. CONCLUSION: The AEP test is a helpful study for early detection of hearing problem. However, in this study, AEP test was too sensitive in acute period, and later, the incidence rate of hearing impairment was relatively low. Therefore, age of onset, severity of neurologic symptom, and clinical examination must be considered before the AEP test.