Abnormal P53 Expression Research Articles

Aberrant p53 Expression in Gastric Biopsies and Resection Specimens Following Neoadjuvant Chemoradiation: A Diagnostic Pitfall.

Gastric mucosal biopsies and resections from patients treated with neoadjuvant radiation and/or chemotherapy are frequently encountered. These samples may show histologic features related to therapy including inflammation, ulceration, and epithelial atypia. In some cases, epithelial atypia may be marked, prompting the use of adjunct p53 immunohistochemistry. We examined p53 expression by immunohistochemistry in gastric mucosa following therapy. We evaluated the histology and p53 immunohistochemical expression in gastric mucosa from 5 7 resections and 3 mucosal biopsies, from 60 patients treated with radiation and/or chemotherapy for gastroesophageal carcinoma (n = 33) or pancreatic carcinoma (n = 27). We identified histomorphologic features of therapy-related epithelial changes in 50 of 60 cases (83%). Abnormal p53 expression was present at least focally in nearly half the cases (27 of 60 cases; 45%), all of which showed morphologic evidence of therapy-related epithelial changes . Neuroendocrine cell micronests were present in 37 of 60 cases (62%). Next-generation sequencing (NGS) of foci with therapy-related epithelial changes showing abnormal p53 expression and carcinoma from the same patient was attempted and yielded results in 1 patient. Interestingly, differing TP53 alterations in the patient's adenocarcinoma and in a histologically benign esophageal submucosal gland with therapy-related epithelial changes and abnormal p53 expression were identified. Our results demonstrate that abnormal p53 expression is relatively common in gastric mucosal samples following radiation and/or chemotherapy and suggest that p53 expression should be avoided when distinguishing therapy -related changes from dysplasia or carcinoma. Furthermore, our NGS results raise interesting biological questions, which may warrant further investigation.

Molecular-Based Immunohistochemical Algorithm for Uterine Leiomyosarcoma Diagnosis

The morphologic assessment of uterine leiomyosarcoma (LMS) may be challenging, and diagnostic immunohistochemical (IHC) analysis is currently lacking. We evaluated the genomic landscape of 167 uterine LMS by targeted next-generation sequencing (NGS) to identify common genomic alterations. IHC analyses corresponding to these genomic landmarks were applied to a test cohort of 16 uterine LMS, 6 smooth muscle tumors of uncertain malignant potential (STUMP), and 6 leiomyomas with NGS data and a validation cohort of 8 uterine LMS, 12 STUMP, 21 leiomyomas and leiomyoma variants, 7 low-grade endometrial stromal sarcomas, and 2 diagnostically challenging uterine smooth muscle tumors. IHC results were individually interpreted by 3 pathologists blinded to NGS data. Overall, 94% of LMS showed ≥1 genomic alteration involving TP53, RB1, ATRX, PTEN, CDKN2A, or MDM2, with 80% showing alterations in ≥2 of these genes. In the test cohort, an initial panel of p53, Rb, PTEN, and ATRX was applied, followed by a panel of DAXX, MTAP, and MDM2 in cases without abnormalities. Abnormal p53, Rb, PTEN, and ATRX IHC expression was seen in 75%, 88%, 44%, and 38% of LMS, respectively, in the test cohort. Two or more abnormal IHC results among these markers were seen in 81% of LMS. STUMPs demonstrated only 1 IHC abnormality involving these markers. No IHC abnormalities were seen in leiomyomas. In the validation cohort, abnormal p53, Rb, and PTEN IHC results were seen in LMS, whereas rare STUMP or leiomyomas with bizarre nuclei showed IHC abnormalities involving only 1 of the markers. Abnormalities in ≥2 markers were present in both diagnostically challenging smooth muscle tumors, confirming LMS. Concordance was excellent among pathologists in the interpretation of IHC (κ = 0.97) and between IHC and NGS results (κ = 0.941). Uterine LMS exhibit genomic landmark alterations for which IHC surrogates exist, and a diagnostic algorithm involving molecular-based IHC may aid in the evaluation of unusual uterine smooth muscle tumors.

Nuclear β-Catenin Expression in the Context of Abnormal p53 Expression Indicates a Nonserous Histotype in Endometrial Carcinoma.

The interobserver reproducibility is poor for histotyping within the p53-abnormal molecular category of endometrial carcinomas (ECs); therefore, biomarkers that improve histologic classification are useful. β-catenin has been proposed to have prognostic significance in specific clinicopathologic and molecular contexts. The diagnostic utility for β-catenin expression patterns in determining the histotype of p53-abnormal ECs has not been well studied. We identified ECs molecularly classified as "p53-abnormal." The p53-abnormal classification was assigned when (1) no POLE exonuclease domain hotspot mutations identified, (2) mismatch-repair protein expression was retained, and (3) abnormal p53 expression (null or overexpression) was present. Morphology was re-reviewed and β-catenin immunohistochemistry was scored as abnormal (nuclear) or normal (membranous, non-nuclear). Eighty ECs were identified in the "p53-abnormal" category; 27 (33.75%) were uterine serous carcinomas, and 53 were of nonserous histotype: 28 uterine carcinosarcomas (35%), 16 endometrioid carcinomas (20%), 2 clear cell carcinomas (2.5%), and 7 high-grade EC with ambiguous morphology (8.75%). All 27 uterine serous carcinomas demonstrated membranous β-catenin staining. Of the 53 nonserous ECs, 11 (21%) showed abnormal β-catenin expression: 6 endometrioid carcinomas, 4 uterine carcinosarcoma, and 1 high-grade EC with ambiguous morphology. The specificity of abnormal β-catenin expression for nonserous EC is high (100%) but the sensitivity is low (21%) with positive and negative predictive values of 100% and 60%, respectively. Our data shows that abnormal β-catenin expression in the context of p53-abnormal EC is highly specific, but not sensitive, for nonserous ECs and may be of value as part of a panel in classifying high-grade EC, particularly to exclude uterine serous carcinoma when nuclear staining is present.

P53 immunohistochemistry in endometrial cancer: clinical and molecular correlates in the PORTEC-3 trial

AbstractStandard molecular classification of endometrial cancers (EC) is now endorsed by the WHO and identifies p53-abnormal (p53abn) EC as the subgroup with the poorest prognosis and the most likely to benefit from adjuvant chemo(radio)therapy. P53abn EC are POLE wildtype, mismatch repair proficient and show abnormal immunohistochemical (IHC) staining for p53. Correct interpretation of routinely performed p53 IHC has therefore become of paramount importance. We aimed to comprehensively investigate abnormal p53 IHC patterns and their relation to clinicopathological and molecular features. Tumor material of 411 molecularly classified high-risk EC from consenting patients from the PORTEC-3 clinical trial were collected. p53 IHC was successful in 408 EC and was considered abnormal when the tumor showed a mutant expression pattern (including subclonal): overexpression, null or cytoplasmic. The presence of pathogenic mutations was determined by next generation sequencing (NGS). Abnormal p53 expression was observed in 131/408 (32%) tumors. The most common abnormal p53 IHC pattern was overexpression (n = 89, 68%), followed by null (n = 12, 9%) and cytoplasmic (n = 3, 2%). Subclonal abnormal p53 staining was observed in 27 cases (21%), which was frequently but not exclusively, associated with POLE mutations and/or MMRd (n = 22/27; p < 0.001). Agreement between p53 IHC and TP53 NGS was observed in 90.7%, resulting in a sensitivity and specificity of 83.6% and 94.3%, respectively. Excluding POLEmut and MMRd EC, as per the WHO-endorsed algorithm, increased the accuracy to 94.5% with sensitivity and specificity of 95.0% and 94.1%, respectively. Our data shows that awareness of the abnormal p53 IHC patterns are prerequisites for correct EC molecular classification. Subclonal abnormal p53 expression is a strong indicator for POLEmut and/or MMRd EC. No significant differences in clinical outcomes were observed among the abnormal p53 IHC patterns. Our data support use of the WHO-endorsed algorithm and combining the different abnormal p53 IHC patterns into one diagnostic entity (p53abn EC).

Association of immunohistochemical profiles with histotypes in endometrial carcinomas

ObjectiveAlthough a large number of endometrial cancer patients are cured with surgery alone, there are significant numbers of patients with more aggressive variants of endometrial carcinoma for whom the prognosis remains poor. We investigated the effects of prevalence, histotypes, and immunohistochemical profiles on prognostic value in a hospital-based population. Materials and methodsA retrospective study of surgically resected primary endometrial carcinoma was included. Immunohistochemical stains were performed on formalin-fixed paraffin-embedded tissue microarray sections for β-Catenin, estrogen receptor (ER), progesterone receptor (PR), HER-2, MLH1, MSH2, MSH6, PMS2, and p53. ResultsLoss of mismatch repair expression was detected in 25.4% of samples (29/114, mean age 57 years) of the tumors. The following loss of expression was observed in patients: MLH1/PMS2 in 16.6% of patients, MSH6 in 7.0% of patients, MLH1 in 0.9% of patients, and MSH6/PMS2/MLH1 in 0.9% of patients. Immunohistochemistry of p53 was analyzed for 111 patients. A total of 13 patients (11.7%, mean age 64 years) had p53-abnormal expression (absent, cytoplasmic or diffuse strong positive patterns), and more than half (9/13, 69.2%) had endometrioid histotype. Abnormalities in p53 were significantly associated with histotype (p = 0.001), advanced tumor stage (p = 0.038), death of disease (p = 0.002), PR percentage (p = 0.002), and HER-2 expression (p = 0.018). Immunohistochemical nuclear localization of β-Catenin was detected in 7.1% of the cohort. The combination of p53 and nuclear β-Catenin expressions was not significantly predictive of disease-free or overall survival. ConclusionThe results of this study are useful for management of endometrial cancer in patients with DNA mismatch repair, abnormal p53 expression, or nuclear localization of β-Catenin.

Metabolomic Analysis Points to Bioactive Lipid Species and Acireductone Dioxygenase 1 (ADI1) as Potential Therapeutic Targets in Poor Prognosis Endometrial Cancer.

Simple SummaryUterine serous carcinoma is considered a rare and aggressive variant of endometrial cancer that accounts for 10% of all endometrial cancers diagnosed but is responsible for 40% of endometrial cancer-related deaths. Unfortunately, current treatments for serous endometrial carcinoma are ineffective. Therefore, there is a need to find new therapeutic targets. The aim of this study was to analyse the metabolic profile of serous cancer in order to identify new molecules and thereby define potential therapeutic targets. We observed that most of the differential metabolites are lipid species (suggesting the important role of the lipid metabolism). In addition, we found an increase in 2-Oxo-4-methylthiobutanoic acid (synthesised by the ADI1 enzyme) in serous carcinomas. Using public database analysis and immunohistochemistry, we established a correlation between elevated ADI1 levels and serous carcinoma. Furthermore, the ectopic modification of ADI1 expression in vitro revealed the ability of ADI1 to induce pathological cell migration and invasion capabilities.Metabolomic profiling analysis has the potential to highlight new molecules and cellular pathways that may serve as potential therapeutic targets for disease treatment. In this study, we used an LC-MS/MS platform to define, for the first time, the specific metabolomic signature of uterine serous carcinoma (SC), a relatively rare and aggressive variant of endometrial cancer (EC) responsible for 40% of all endometrial cancer-related deaths. A metabolomic analysis of 31 ECs (20 endometrial endometrioid carcinomas (EECs) and 11 SCs) was performed. Following multivariate statistical analysis, we identified 232 statistically different metabolites among the SC and EEC patient samples. Notably, most of the metabolites identified (89.2%) were lipid species and showed lower levels in SCs when compared to EECs. In addition to lipids, we also documented metabolites belonging to amino acids and purine nucleotides (such as 2-Oxo-4-methylthiobutanoic acid, synthesised by acireductone dioxygenase 1 (ADI1) enzyme), which showed higher levels in SCs. To further investigate the role of ADI1 in SC, we analysed the expression protein levels of ADI1 in 96 ECs (67 EECs and 29 SCs), proving that the levels of ADI1 were higher in SCs compared to EECs. We also found that ADI1 mRNA levels were higher in p53 abnormal ECs compared to p53 wild type tumours. Furthermore, elevated ADI1 mRNA levels showed a statistically significant negative correlation with overall survival and progression-free survival among EEC patients. Finally, we tested the ability of ADI1 to induce migration and invasion capabilities in EC cell lines. Altogether, these results suggest that ADI1 could be a potential therapeutic target in poor-prognosis SCs and other Ecs with abnormal p53 expression.

Pancreatic ductal adenocarcinoma with a predominant large duct pattern has better recurrence-free survival than conventional pancreatic ductal adenocarcinoma: a comprehensive histopathological, immunohistochemical, and mutational study

Large duct pattern of pancreatic ductal adenocarcinomas (PDACs) comprises occasional large cancer glands (>0.5mm in size), along with conventional smaller cancer glands. They histologically mimic intraductal papillary mucinous neoplasms. However, the clinicopathologic significance of PDACs with predominant large duct pattern (PLDP) has not been systematically evaluated. A total of 41 cases of PDACs with PLDP, which were defined as irregularly-shaped cancer glands >0.5mm in size occupied >50% of tumor volume, were enrolled and their clinicopathological, immunohistochemical, and targeted exome-wise mutational characteristics were compared with 298 conventional PDACs. PDACs with PLDP had cancers with larger tumor sizes (P=0.025), which were more frequently well to moderately differentiation (P<0.001), with less lymphovascular invasion (P=0.013) and had a higher T category (P=0.023) than conventional PDACs. Immunohistochemically, PDACs with PLDP showed similar abnormal p53 (61%) and SMAD4 (59%) expression patterns as conventional PDACs. In addition, PDACs with PLDP showed diffuse MUC1 (88%), MUC5AC (100%), MUC6 (66%), and focal MUC2 (20%) expressions. More frequent ROS1 mutations were observed in PDACs with PLDP. PDAC patients with PLDP had a better overall and recurrence-free survival (OS and RFS; median, 42 and 34 months) than that of patients with conventional PDACs (34 and 16 months) as per univariate (P = 0.037 and P = 0.001) and multivariate (P = 0.031 and P = 0.034) analyses. PDACs with PLDP showed mutational patterns similar to those of conventional PDACs. They had unique histologic features and longer OS and RFS compared to those of conventional PDACs. Therefore, PDACs with PLDP could be considered a histologic subtype of PDACs.

P53 and Rb immunohistochemistry staining reveal subtypes of gastric neuroendocrine carcinoma with distinct prognosis.

e16210 Background: Due to the rarity, research on gastric neuroendocrine carcinoma (NEC) is limited. The treatment has been extrapolated from small-cell lung cancer (SCLC) data. However, a previous study found gastric NECs had a genetic feature characterized by a lower frequency of RB1 mutations than SCLC. Several studies also showed RB1mutation or loss of Rb expression could predict the efficacy of SCLC treatment in NEC. Our study investigated a subtyping method by p53 and Rb immunohistochemistry (IHC) staining in gastric NEC, which might help identify patients responding to SCLC treatment. Methods: Patients with gastric NEC who underwent radical surgery in Peking Union Medical College Hospital (PUMCH) from 2013 to 2021 were identified. The expression of Rb, p53, PD-L1 and CD8 T cell infiltration were accessed by IHC stain. The mutations of RB1 and TP53, tumor mutation burden (TMB), and microsatellite instability (MSI) were detected by whole-exom sequencing (WES). Fisher’s exact test and Mann–Whitney test were used to detect differences between groups. Spearman's test was used to detect correlations between variables. Kaplan-Meier and Cox hazards methods were applied to the survival analysis. Samples of gastric adenocarcinoma, gastric neuroendocrine tumor (NET), and SCLC were also included. Results: A total of 41 patients with gastric NEC were included. WES was carried out in 28 patients. TP53 mutation was detected in 14 patients. RB1 mutation was detected in 1 patient. A classification method based on gene mutation was not able to be further investigated. In IHC of 41 patients, 46.2% showed Rb expression absence. 68.4% had abnormal p53 expression. 39 patients were classified based on IHC results. Patients with both abnormal expression of p53 and Rb were defined as SCLC-like type, accounting for 38.5%.The remaining patients were defined as non-SCLC-like type. SCLC-like gastric NEC had an increased number of lymph nodes metastasis (P = 0.001) and higher TNM stage (P &lt; 0.001). The SCLC-like group's overall survival (OS) was significantly lower than the non-SCLC-like group (P = 0.023). Classification based on p53 and Rb expression was an independent predictor of OS (P = 0.021), HR = 4.690, 95%CI (1.263, 17.421). The classification results of gastric NEC were significantly different from those in gastric NET(P = 0.001), gastric adenocarcinoma(P = 0.002), but not in SCLC(P = 0.464). In 39 classified patients, the positive rate of PD-L1 expression was 23.1%; CD8 T cells high infiltration (&gt;25 % of the stromal area) was 46.7%; the median TMB was 4.9 Muts/MB, and MSI was not detected. There was no significant difference in the expression of immunotherapy-related markers between the two types. Conclusions: Our study revealed subtypes of gastric NEC based on p53 and Rb expression with different prognoses. Further research on the treatment efficacy predictive role of this subtyping method is warranted.

Latent membrane proteins from EBV differentially target cellular pathways to accelerate MYC-induced lymphomagenesis

AbstractMYC translocations in association with Epstein-Barr virus (EBV) infection are often observed in B-cell lymphomas. A subset of Burkitt lymphoma (BL) expresses EBV latent membrane proteins 1 and 2A (LMP1 and LMP2A) in addition to the typical restricted EBV latent gene expression. EBV-associated diffuse large B-cell lymphoma (DLBCL) typically exhibits latency type II or III and expresses LMP1. Here, we investigate the role of LMP1 in MYC-driven lymphomagenesis in our murine model. λ-MYC mice develop tumors having a “starry sky” appearance and have abnormal p53 expression that is also observed in human BL. LMP2A/λ-MYC double-transgenic mice develop tumors significantly faster than mice only expressing MYC. Similar to LMP2A/λ-MYC mice, LMP1/λ-MYC mice also have accelerated MYC-driven lymphomagenesis. As observed in LMP2A/λ-MYC mice, p27kip1 was degraded in LMP1/λ-MYC pretumor and tumor B cells. Coexpression of LMP1 and LMP2A resulted in the enhancement of B cell proliferation. In contrast to LMP2A, the inhibition of Syk or cyclin-dependant kinase (CDK)4/6 activity did not effectively inhibit LMP1-mediated MYC lymphomagenesis. Also, in contrast to LMP2A, LMP1 did not lessen abnormal p53 expression in λ-MYC tumors. To investigate the significance of LMP1 expression in human BL development, we reanalyzed RNA sequencing (RNA-Seq) data of primary human BL from previous studies. Interestingly, p53 mutations were less observed in LMP1-expressing BL, although they were not significantly changed by EBV infection, indicating LMP1 may lessen p53 mutations in human primary BL. This suggests that LMP1 effects in EBV-associated human BL vary from what we observe in our murine model. Finally, our studies suggest a novel pathogenic role of LMP1 in lymphomagenesis.

Pure erythroid leukemia is characterized by biallelic TP53 inactivation and abnormal p53 expression patterns in de novo and secondary cases.

Not available.

Developmental toxicity of glyphosate on embryo-larval zebrafish (Danio rerio)

Glyphosate (GLY) induces developmental toxicity in fish, but research on the toxicity mechanism is limited. In this study, zebrafish embryos were exposed for 120 hpf to 0.7, 7, and 35 mg L-1 GLY. The results show that GLY treatment induced developmental toxicity in the fish, including premature hatching, reduced heartbeats, pericardial and yolk sac oedema, swim bladder deficiency, and shortened body length, which was possibly due to a significantly decreased triiodothyronine (T3)/thyroxine (T4) ratio and the abnormal expression patterns of hypothalamic-pituitary-thyroid (HPT) (crh, tshβ, tr α, tr β, and t tr ) and growth hormone/insulin-like growth factor (GH/IGF) axis-related genes (gh, ghrα, ghrβ, igf1, igf1rα, and igf1rβ) in larvae exposed to GLY. In addition, GLY exposure altered the levels of SOD and CAT, increased ROS, promoted malondialdehyde (MDA) content, and significantly altered the levels of endoplasmic reticulum (ER) stress signalling pathway factors (perk, eif2α, gadd34, atf4, ire1α, xbp1, atf6, hspa5, and chop), suggesting that GLY treatment induced oxidative injury and ER stress in the larvae. Further research showed that treatment with a higher concentration of GLY upregulated the levels of iNOS, IL-1β, and TNF-α while inhibiting the expression of IL-10 and TGF-β, suggesting that GLY causes an inflammatory reaction in the larvae. In addition, we also found that apoptosis was induced in the larvae, which was determined by acridine orange staining and abnormal expression of p53, caspase-3, -8, and -9. Taken together, our results demonstrate that GLY exposure altered the T3/T4 ratio, disturbed the expression patterns of HPT and GH/IGF axis-related genes, and induced oxidative and ER stress, inflammatory reactions, and apoptosis in the zebrafish larvae. This investigation contributes to improved understanding of the developmental toxicity mechanism of GLY in fish.

DNA Methylation Profiles of Ovarian Clear Cell Carcinoma.

Ovarian clear cell carcinoma (OCCC) is a rare ovarian cancer histotype that tends to be resistant to standard platinum-based chemotherapeutics. We sought to better understand the role of DNA methylation in clinical and biological subclassification of OCCC. We interrogated genome-wide methylation using DNA from fresh frozen tumors from 271 cases, applied nonsmooth nonnegative matrix factorization (nsNMF) clustering, and evaluated clinical associations and biological pathways. Two approximately equally sized clusters that associated with several clinical features were identified. Compared with Cluster 2 (N = 137), Cluster 1 cases (N = 134) presented at a more advanced stage, were less likely to be of Asian ancestry, and tended to have poorer outcomes including macroscopic residual disease following primary debulking surgery (P < 0.10). Subset analyses of targeted tumor sequencing and IHC data revealed that Cluster 1 tumors showed TP53 mutation and abnormal p53 expression, and Cluster 2 tumors showed aneuploidy and ARID1A/PIK3CA mutation (P < 0.05). Cluster-defining CpGs included 1,388 CpGs residing within 200 bp of the transcription start sites of 977 genes; 38% of these genes (N = 369 genes) were differentially expressed across cluster in transcriptomic subset analysis (P < 10-4). Differentially expressed genes were enriched for six immune-related pathways, including IFNα and IFNγ responses (P < 10-6). DNA methylation clusters in OCCC correlate with disease features and gene expression patterns among immune pathways. This work serves as a foundation for integrative analyses that better understand the complex biology of OCCC in an effort to improve potential for development of targeted therapeutics.

Quantitative single-cell analysis of immunofluorescence protein multiplex images illustrates biomarker spatial heterogeneity within breast cancer subtypes

BackgroundThe extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed. As an initial evaluation of the extent of breast cancer cellular heterogeneity, we conducted quantitative and spatial imaging of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor-2 (HER2), Ki67, TP53, CDKN1A (P21/WAF1), CDKN2A (P16INK4A), CD8 and CD20 of a tissue microarray (TMA) representing subtypes defined by St. Gallen surrogate classification.MethodsQuantitative, single cell-based imaging was conducted using an Immunofluorescence protein multiplexing platform (MxIF) to study protein co-expression signatures and their spatial localization patterns. The range of MxIF intensity values of each protein marker was compared to the respective IHC score for the TMA core. Extent of heterogeneity in spatial neighborhoods was analyzed using co-occurrence matrix and Diversity Index measures.ResultsOn the 101 cores from 59 cases studied, diverse expression levels and distributions were observed in MxIF measures of ER and PR among the hormonal receptor-positive tumor cores. As expected, Luminal A-like cancers exhibit higher proportions of cell groups that co-express ER and PR, while Luminal B-like (HER2-negative) cancers were composed of ER+, PR- groups. Proliferating cells defined by Ki67 positivity were mainly found in groups with PR-negative cells. Triple-Negative Breast Cancer (TNBC) exhibited the highest proliferative fraction and incidence of abnormal P53 and P16 expression. Among the tumors exhibiting P53 overexpression by immunohistochemistry, a group of TNBC was found with much higher MxIF-measured P53 signal intensity compared to HER2+, Luminal B-like and other TNBC cases. Densities of CD8 and CD20 cells were highest in HER2+ cancers. Spatial analysis demonstrated variability in heterogeneity in cellular neighborhoods in the cancer and the tumor microenvironment. ConclusionsProtein marker multiplexing and quantitative image analysis demonstrated marked heterogeneity in protein co-expression signatures and cellular arrangement within each breast cancer subtype. These refined descriptors of biomarker expressions and spatial patterns could be valuable in the development of more informative tools to guide diagnosis and treatment.

Clinical factors and biomarker profiles associated with patient outcome in endometrioid ovarian carcinoma - Emphasis on tumor grade

ObjectiveThe role of clinicopathological factors and molecular markers in prognostic classification of endometrioid ovarian carcinoma (EnOC) is not established. Tumor grade is used in risk assessment, but the role of current 3-tier grading system has been challenged. MethodsClinicopathological factors and 12 immunohistochemical biomarkers (PR, ER, β-catenin, vimentin, ARID1A, HNF1-β, p53, p16, MIB-1, E-cadherin, c-erb-B2 and L1CAM) were analyzed as regards patient outcome in 215 contemporarily classified EnOCs. ResultsOf clinical parameters, grade and stage appeared as strong independent prognostic factors both for disease-free and disease-specific overall survival. Grades 1–3 distinguished clearly from each other in the survival analysis, whereas stages I-II and stages III-IV clustered with each other. PR, ER, nuclear β-catenin and vimentin positivity were associated with favorable overall outcome and clinical parameters, whereas abnormal expression of p53, overexpression of p16 and L1CAM positivity were associated with aggressive disease characteristics and poor survival. The frequency of good-prognosis markers PR and β-catenin gradually decreased and poor-prognosis markers p53, p16 and L1CAM gradually increased from grade 1–3. However, vimentin and ER were expressed at similar frequencies across different grades and presented with independent prognostic significance. ConclusionsWe found histological grade and disease stage, but not residual tumor, to be independent clinical prognostic factors in EnOC. A set of good-prognosis markers (PR, ER, β-catenin and vimentin) and poor-prognosis markers (p53, p16 and L1CAM) were identified. Our findings support continuation of the use of the 3-tier grading system for EnOC and provide clinically feasible IHC biomarkers for prognostic profiling.

Does Lymph Node Assessment Change the Prognostic Significance of Extensive LVSI and p53 Mutation in Endometrial Cancer?

While high-intermediate risk (HIR) early stage endometrial cancer patients achieve excellent outcomes with adjuvant vaginal brachytherapy (VBT), the recent PORTEC2 update suggested factors such as extensive lymphovascular space invasion (LVSI) and abnormal p53 expression predict for poorer outcomes and that these patients should be treated with external beam radiation therapy (EBRT), rather than VBT. Our study aims to determine if patients with these risk factors who undergo a lymph node assessment prior to receiving adjuvant therapy show similarly high recurrence rates.We identified 97 patients with FIGO 2009 stage IA grade 3, stage IB grade 1-2, and stage IIIC (with positive LN only but no other stage II or III factors) endometrial cancer who underwent surgical resection including lymph node assessment from 2009 to 2018 at our institution. We reviewed the LVSI status and extent, and the p53 status in these patients. Local failure (LF), regional failure (RF) distant metastasis (DM), and overall survival (OS) were compared using Kaplan-Meier.Median follow up was 52 months. The median OS was not reached. The 3-year cumulative incidence of failure were: 3.3% LF, 7.1% RF and 2.2% DF. Patients with any LVSI had poorer OS (P < 0.01, increased rates of LF (P < 0.02) and increased rates of RF (P < 0.04). Extent of LVSI also correlated with OS, LF, RF and DM, where patients with extensive LVSI has worse outcomes when compared with patients with limited LVSI (P = 0.04, P = 0.04, P = 0.04, P = 0.01, respectively). Abnormal p53 correlated with increased rates of LF (P < 0.02). Patients who had both abnormal p53 and any LVSI had worse OS (P > 0.0010), increased LF (P = 0.047) and RF (P < 0.01), compared to patients who had neither risk factor. 47% of the entire patient cohort and 50% of patients with extensive LVSI or abnormal p53 received radiation therapy. No patients who completed EBRT experienced in-field recurrences.Patients with HIR early stage endometrial cancer treated at our institution have had excellent outcomes. However, a small group of patients with unfavorable risk factors such as abnormal p53 expression and/or LVSI have worse outcomes, even with lymph node assessment, suggesting that these factors can aid in the selection of patients who may derive benefit from intensification of treatment. EBRT may be beneficial in this high risk population, as no patients who completed EBRT in our study had in field recurrences.C. Hui: None. M. Mendoza: None. R. Von Eyben: None. P. Hammer: None. B. Howitt: None. E.A. Kidd: Advisory Board; Varian Brachytherapy Advisory Board.

Abstracts and Case Studies for the College of American Pathologists 2021 (CAP21) Annual Meeting

Abstracts and Case Studies for the College of American Pathologists 2021 (CAP21) Annual Meeting

Abstract 2045: Aneuploidy, immunohistochemical overexpression of MSH-2, P53, P21^WAF1/CIP1, P16^INK4A, and correlation to epithelial ovarian carcinoma patient prognosis

Abstract Aneuploidy, immunohistochemical overexpression of MSH-2, P53, P21WAF1/CIP1, P16INK4A, and correlation to epithelial ovarian carcinoma patient prognosis Overexpression of MSH-2, P53, P21WAF1/CIP1, P16INK4A and DNA status in epithelial ovarian carcinoma (EOC) was determined in paraffin-embedded tissue (PET) to predict prognosis. Immunoreactivity, DNA and % S content were evaluated for their potential role in survival and relapse-free survival (RFS) in 112 patients diagnosed with EOC at Saskatoon Cancer Center, Canada. Immunostaining studies were with the following mouse monoclonal antibodies: MSH-2, FE11; P53, D07; P21WAF1/CIP1, EA 10; except for P16INK4A, rabbit, G175-405, 13251A. DNA and % S content (median, &amp;lt; % 5 = low-risk or &amp;gt; % 5 = high-risk) were assessed by flow-cytometric analysis1. Survival curves were determined by Kaplan-Meier method, mean survival time, and log rank test. P-values &amp;lt; 0.05 were considered significant; X2-test examined association between variables. Patients with aneuploid DNA had 59 months mean survival time, compared to cases with diploid DNA which had 89 months, (p = 0.03). RFS in those with diploid DNA lasted longer (84 months) while aneuploid cases had shorter time to progression (47 months, p = 0.01). % S content in the high-risk group, 47 cases, had worse survival (57 months, p = 0.0008) and RFS (52 months, p = 0.004). Aneuploidy was not related to overall survival within the subsets of early and advanced stage patients. Grade 2 patients with abnormal DNA and P53 expression were adversely associated with survival and RFS. Diploid DNA was observed in all mucinous tumors except in one case with aneuploid DNA. P53 immunoreactivity was related to shorter survival and RFS in 44 cases. P21WAF1/CIP1 overexpression was associated with P53 overexpression in 11 cases (p = 0.04) and shorter median survival (29 months) compared to negative P21WAF1/CIP1 staining (median survival time 62 months, p = 0.03). 11 grade 2 patients with P53 positivity had worse survival (59 months) and RFS (47 months) than the P53 negative tumors (p = 0.05, and p = 0.01 respectively). P16INK4A overexpression (46 cases) had no adverse effect on survival and RFS. No significant prognostic difference was found between MSH-2 positive and negative tumors. We conclude that % S content was a more beneficial prognostic marker than ploidy status. In grade 2 EOC, the effects of DNA content and P53 overexpression were significant variables related to outcome. For pathologists, these variables in grade 2 tumors can be adjunctive aid for reaching an agreement to diagnose the grade of EOC patients.Footnotes1 A. Geissel and J. L. Griffin, “Preparation of nuclei for flow cytometry,” in AFIP Advances in Laboratory Methods in histology and Pathology, ed. Mikel UV (Washington DC: American Registry of Pathology, 1944), 111-121. Citation Format: Kavitha Advikolanu Rao, Antony M. Magliocco, Andrew W. Maksymiuk. Aneuploidy, immunohistochemical overexpression of MSH-2, P53, P21^WAF1/CIP1, P16^INK4A, and correlation to epithelial ovarian carcinoma patient prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2045.

PD-L1, PARP1, and MMRs as potential therapeutic biomarkers for neuroendocrine cervical cancer.

ObjectiveNeuroendocrine cervical cancer (NECC) is a rare cervical cancer with high aggressivity that causes poor prognosis even in the early stage. Given other neuroendocrine carcinomas and other types of cervical cancer have been proved to have expression of programmed cell death protein 1 ligand 1(PD‐L1) and poly ADP‐ribose polymerase‐1(PARP1), we would measure and analyze these proteins in this invasive cancer. The purpose of this study is to investigate the application value of PD‐1/PD‐L1 and PARP1 inhibitors in NECC.MethodsThe NECC cases in our center with formalin‐fixed paraffin‐embedded tissue blocks were collected, and immunohistochemical (IHC) staining of PD‐L1, PARP1, Mismatch repair proteins (MMRs), and P53 was performed. Chi‐square test was used to analyze associations between various protein expressions. We analyzed the efficacy of immunotherapy in a recent patient with secondary recurrence after two courses of chemotherapy.ResultsAfter rigorous screening, 20 cases were finally included. Three cases did not undergo surgical treatment because of their advanced stage. Twelve (60%) developed distant metastases or relapsed within five years, and most of them within two years. The positive rate of PD‐L1 and PARP1 were 70% and 75% respectively. Among all the cases, microsatellite instability (MSI) was seen in six cases (30%) and abnormal p53 expression was in 15 patients (75%). PD‐L1 was associated with PARP1 expression in the MSI subgroup. The patient treated with chemotherapy + VEGF inhibitor (VEGFi) + programmed cell death protein 1(PD‐1) inhibitor had an excellent improvement in clinical symptoms, tumor markers, and mass size.ConclusionThe IHC results of PD‐L1, PARP1, and MMRs suggested that NECC was the target of immunotargeted therapy. Our case confirmed that immune checkpoint therapy was effective in patients with PD‐L1 positive and MMRs loss. Considering the clinical practicability, more cases should be collected, and effective biomarkers still need to be further searched.

The TCGA Molecular Classification of Endometrial Cancer and Its Possible Impact on Adjuvant Treatment Decisions.

Simple SummaryThe Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) is a recently developed tool to identify four distinct molecular subgroups of endometrial cancer. Patients identified as Polymerase Epsilon exonuclease domain mutated (POLE EDM) or p53-mutated have significantly altered prognosis compared to patients allocated to the mismatch repair deficient (MMRd) or p53 wt groups. The aim of this review is to give a broad overview over the initial development and refinement of the classifier as well as possible effects on the recommended adjuvant treatment. We have summarized the clinical data of 8 studies including 3650 endometrial cancer patients and analyzed the distribution of tumor stage and adjuvant treatment received in respect to the molecular subgroups. Based on the findings of the summarized studies treatment de-escalation might be feasible for POLE EDM patients while p53 abn patients should receive adjuvant (chemo-)radiotherapy.Adjuvant treatment decisions for endometrial cancer (EC) are based on stage, the histological grade of differentiation, histological subtype, and few histopathological markers. The Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) identified four risk groups of EC patients using a combination of immunohistochemistry and mutation analysis: Polymerase Epsilon exonuclease domain mutated (POLE EDM), mismatch repair deficient (MMRd), p53 wild-type/copy-number-low (p53 wt), and p53-mutated/copy-number-high (p53 abn). Patients allocated to the POLE or abnormal p53 expression subtype are faced with a significantly altered outcome possibly requiring a modified adjuvant treatment decision. Within this review, we summarize the development of ProMisE, characterize the four molecular subtypes, and finally discuss its value in terms of a patient-tailored therapy in order to prevent significant under or overtreatment.

Down-regulation of the Expression of Senescence Proteins P16 and P21 by Activating Connexin 43 in the Smooth Muscle of Spiral Modiolar Artery of Guinea Pigs

To analyze the correlation between connexin 43 (Cx43) and the expression of P16 and P21, aging-related proteins, and to investigate the possible role of Cx43 in the development of cell senescence with an aging model prepared by D-galactose (D-gal) intervention in the vascular smooth muscle cells (VSMCs) of guinea pig spiral modiolar artery (SMA). The VSMCs of guinea pig SMA were cultured with the adhesion method, and the markers of VSMCs were detected with immunofluorescence technique. The experiment has a control group, a D-gal group, and a group that received D-gal and gap junction agonist AAP10 intervention, hereafter referred to as the AAP10 group. Cell Counting Kit-8 (CCK-8) was used to check VSMC activity and to determine the concentration and duration of D-gal intervention. The mRNA expression of Cx43 in each group was checked with qRT-PCR. The expression of Cx43, P16 and P21 proteins in each group was examined with the Western blot. The expression and distribution of P16 and P21 proteins were examined with immunofluorescence assay. Immunofluorescence results showed that the positive expression rate of cell actin (α-SM-actin) was over 90%. CCK-8 results showed that the optimal concentration of D-gal intervention was 30 mg/mL and the intervention duration was 48 h. qRT-PCR test showed that the mRNA expression of Cx43 in VSMCs in the D-gal group was significantly lower than that in the control group ( P<0.01), while it is higher in the AAP10 group than that of the D-gal group ( P<0.01); Western blot assay showed that the Cx43 expression level in VSMCs in the D-gal group was significantly lower than that in the control group ( P<0.01) and the expression of P16 and P21 was significantly higher than that in the control group ( P<0.01), the expression of Cx43 protein in AAP10 group was significantly up-regulated compared with that in the D-gal group ( P<0.01), while the expression of P16 and P21 was down-regulated significantly ( P<0.01); The results of immunofluorescence showed that P16 and P21 were mainly expressed in the cell nucleus. Semi-quantitative analysis of fluorescence intensity showed that the level of P16 and P21 protein in the D-gal group was significantly higher than that in the control group, and the fluorescence intensity of AAP10 group was significantly lower than that in the D-gal group ( P<0.01). Up-regulation of Cx43 expression can reverse the D-gal-induced abnormal expression of P16 and P21, two aging-related proteins, in SMA. It is suggested that Cx43 on SMA may be involved in D-gal-induced cell senescence, which provides a theoretical basis and possible intervention target for the delay of cell senescence.