Aberrant P53 Research Articles

Translational Orthotopic Models of Glioblastoma Multiforme.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumorsdisplaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

P53 aberration and TFE3 gene amplification may be predictors of adverse prognosis in epithelioid angiomyolipoma of the kidney

BackgroundAlthough epithelioid angiomyolipoma of the kidney has been studied by several groups, the reported prevalence of malignant behavior remains uncertain and there are not yet definitive predictive biomarkers. We evaluated the behavior of renal epithelioid angiomyolipoma in a consecutive series in a single institution and investigated the prognostic value of aberrant p53 expression and TFE3 gene abnormality.MethodsWe retrospectively reviewed 14 epithelioid angiomyolipomas, most with pure or close to pure epithelioid components, comprising 12 consecutive cases who had attended our institution and two consultation cases. Fluorescence in situ hybridization with TFE3 break-apart probe was performed on 14 cases. The 14 cases were also labeled for p53 and TFE3 by immunohistochemistry. All cases were followed up.ResultsThree of the epithelioid angiomyolipomas were strongly positive for TFE3 and two had a mutant expression of p53. Although no TFE3 gene rearrangement was found, the two tumors with strong TFE3 expression showed TFE3 gene amplification. Follow-up details were available for seven of the 12 consecutive cases: two of them had developed metastases and died (29%), their mean overall survival was 41 months, and both had mutant p53 expression. The two consultation cases with TFE3 gene amplification developed recurrence/metastasis within 1 year after surgery.ConclusionsOur series study from a single institution presented the prevalence of malignant behavior in pure epithelioid angiomyolipomas, although the small number of cases with follow-up data greatly reduced the accuracy. p53 may be a prognostic marker for epithelioid angiomyolipoma. Cases with TFE3 gene amplification had poor prognoses.

Immunohistochemistry-based molecular classification of gastric cancer in India: How are we different?

462 Background: TCGA and ACRG have classified gastric cancer (GC) into molecular subgroups using genomics. We attempted to classify GC from our region using IHC (Immunohistochemistry) to understand its prognostic significance and compare with similar data from other parts of the world. Methods: This was a retrospective cohort of GC patients from years 2017-18. EBV (LMP), MSI (MLH1, MSH2, PMS2, MSH6),p53, E-cadherin and PIK3CA were assessed by IHC. Clinicopathological characteristics and survival were evaluated for association with molecular subtypes as defined by TCGA and ACRG classification system. Results: A total of 191patients were included with EBV positivity 35 (18.3 %), MSI 33(17.3 %), p-53 aberration 25(13.1%), E-cadherin negative (29.3%). On univariate analysis, p53 aberration was associated with higher age (>60 yrs), E- cadherin negative with diffuse histology & antro-pyloric region as predominant site. MSI with intestinal histology & antro-pyloric region as predominant site with MSI. The TCGA classification revealed EBV 35 (18%), MSI 28 (15 % ), GS 48 (25 %) and CIN 80 (42% ) subtypes respectively. CIN subtype was seen predominantly above 40 yrs of age (P=0.04), GS subtype was frequently associated with diffuse histology P<0.0001. The ACRG classification revealed MSI 33(17%), MSS/EMT 51 (26.7%), MSS/ p53 normal 91 (47.6%) and MSS/p53 aberrant 16 (8.4) subtypes. MSS/E-cad- subtype was associated with diffuse histology. PIK3CA positivity was higher in CIN (TCGA )& MSI (dmr) (ACRG) respectively. The median OS in MSI was 35 and 32 months higher compared to other subtypes (P<0.0001) in TCGA and ACRG classification respectively. Conclusions: The TCGA and ACRG classification identified molecular subtypes based on IHC from our region and significantly correlated with clinical factors and survival. PIK3CA as an additional marker may be further explored to be included in the classification system. Abbreviation: EBV - Epstein–Barr-virus, MSI – Microsatellite-Instability, GS- Genomic-instability, CIN- Chromosomal-instability, MSS- Microsatellite-stable, EMT-Epithelial-mesenchymal-transition.[Table: see text]

Paget cells of the esophagus: A clinicopathologic and immunohistochemical study of 10 cases.

Extramammary Paget's disease (EMPD) of the esophagus is very rare and the clinicopathologic features are not well characterized. We analyzed 10 cases with reported presence of Paget or Pagetoid tumor cells in the esophageal specimens collected between 2005 and 2018 at our institution. The cohort included 7 males and 3 females with a median age of 67 years. Histologically, 7 cases were secondary Pagetoid spread of tumor cells directly from an underlying invasive adenocarcinoma (pADC) located at the distal esophagus, all CK7+with variable intracytoplasmic mucin. The clinical course of those secondary cases was dependent on the underlying malignancies. Only 3 cases were primary, including 2 Pagetoid squamous cell carcinoma in-situ (pSqCCis) and 1 Pagetoid adenocarcinoma in-situ (pADCis) with focalstromal invasion. The primary cases showed similar clinical and endoscopic presentations. Immunohistochemically, the singly dispersed Paget or Pagetoid tumor cells frequently showed loss of E-cadherin and gain of vimentin expression. Seven cases, including 5 pADC, 1 pSqCCis and the pADCis showed aberrant p53 expression. Four patients, all pADC, died of disease at a median follow-up of 10 months, while the others were alive. Paget or Pagetoid tumor cells in the esophagus can be primary or secondary to an invasive carcinoma. The clinical outcomes depend on the underlying malignancy and extensiveness of disease. Frequent p53 aberrancies and epithelium-mesenchymal transition likely play a role in the pathogenesis.

Аберрантная экспрессия р53 в карциноме желудка и ее связь с HER2 статусом

Introduction. p53 is a tumor suppressor, whose expression is actively studied in many tumors. However, scientists argue about aberrant p53 expression criteria and whether tumoral p53 expression correlates with various clinical and morphological parameters of gastric cancer and has a prognostic role. No data were published in Russia on the incidence of tumors with p53 overexpression. It remains unclear whether aberrant p53 expression is an independent prognostic sign in gastric cancer. The study aimed to evaluate the frequency of p53 expression in gastric adenocarcinomas in the Russian population, to give the definition for aberrant p53 expression, and to clarify the relationship between p53 expression, clinical and morphological tumor characteristics, HER2 status, and the impact of p53 expression on the prognosis. Materials and methods. We studied surgical pathology samples from 310 patients with verified gastric cancer. The age of the patients ranged from 22 to 85 years (mean 63 years). Each sample was stained immunohistochemically with antibodies to p53 (clone DO-7) and the HercepTest kit (Dako/Agilent Technologies). We compared the results with the main clinical and morphological characteristics of gastric cancer and patient survival data. Results.The frequency of aberrant p53 expression was 49.4%. Aberrant expression should include both cases with overexpression and cases with a complete absence of marker expression. In contrast to the normal p53 expression group, the group with aberrant p53 expression was characterized by more frequent proximal tumor location; fewer diffuse and infiltrative carcinoma forms; a significant predominance of tubular, papillary, and mixed histological types (p=0.000); significantly higher degrees of differentiation (p=0.011); a significantly lower number of cases with identified signet ring cells in tumors (p=0.000); a lower frequency of stage IV in patients; and a significant predominance of the intestinal and intermediate subtype according to P. Lauren classification (p=0.000). The overall five-year survival in patients with aberrant p53 expression was not significantly lower (p=0.392) than in patients with normal p53 expression (35.6%, median 36 months and 42.5%, median 51 months, respectively). According to the Cox proportional hazards regression model, the p53 expression level is not a significant prognostic sign (HR=1.281; CI: 0.818–2.008, p=0.280). In the group with aberrant p53 expression, a positive HER2 status was detected significantly more often (13.7%) than in the group with normal p53 expression (1.9%, p=0.000). Conclusion. The group with aberrant p53 expression can be considered as an immunohistochemical analog of the chromosomally unstable gastric cancer subtype (according to TCGA) and the MSS/TP53- subtype (according to ACRG). In the group with aberrant p53 expression, cases with a positive HER2 status were significantly more common. Keywords: р53, HER2/neu, ERBB2, gastric cancer, gastric adenocarcinoma, immunohistochemistry

The statuses of HER2 expression and mismatch repair in endometrial clear cell carcinoma.

High-grade endometrial carcinomas (HGEC) are difficult to classify. With the current use of HER2-based therapy in serous carcinoma, a diagnosis of clear cell carcinoma (CCC) has the potential to exclude patients from receiving therapy. Therefore, we examined HER2 expression in our CCC patients. The preparations of 8 patients with CCC who underwent hysterectomy, bilateral salpingo-oophorectomy, and lymph node dissection were re-evaluated. Patients did not have any prior treatment. Histopathologic parameters that were evaluated include cytoplasmic clearing, nuclear atypia, mitotic activity, hobnail architecture, hyalinized cores, hyaline globules, stratification of epithelial lining papillae, or glandular structures, and highly atypical cell layers. Immunohistochemically, HER2, ER, PR, HNF1β, Napsin A, MLH1, MSH2, MSH6 and PMS2 were applied. HER2 staining pattern, ASCO/CAP protocol used for endometrial carcinom was used. HER2 was positive in 3 of our 8 CCC patients (37.5%). While all of our HER2+cases were Napsin A and HNF1β positive, MMR proteins were intact and ER and PR were negative. Two patients had wild type p53 and 1 patient had aberrant p53 staining. Considering that there is not always a consensus between SC and CCC, even among gynecopathologists, tumor heterogeneity and different tumor components may exist, and while patients may be diagnosed with CCC and benefit from HER2 therapy, there is also a possibility that they may not benefit from the treatment. The fact that 37.5% of our CCC cases were HER2+is a finding with strong implications for the therapeutic approach. As a result of our study, in patients with CCC, if MMR is intact and ER-PR is negative, regardless of the p53 staining pattern, HER2 testing may be an objective screening method for patients who are likely to benefit from HER-targeted therapy. Consequently, patients with a diagnosis of CCC can be candidates for future clinical trials of HER2-targeted therapy.

Expression of p53 as Potential Biomarker in Oral Submucous Fibrosis: An Immunohistochemical Study.

Oral submucous fibrosis (OSMF) is potentially malignant disorder known to transform into oral cancer. The malignant transformation is often associated with changes at the genetic level that in turn is reflected by the altered expression of proteins related to cell cycle, proliferation, and apoptosis. Expression of p53 tumor suppressor gene is one of the common findings in human cancers including the oral cancer. Therefore, the early detection of potentially malignant OSMF has been crucial in the inhibition of oral cancer. To determine the main pathological logical factors and expression of aberrant p53 in OSMF, oral squamous cell carcinomas (OSCC) and in normal patients, to study correlation between p53 expression with clinical staging and histological grading of OSMF. An immunohistochemical (IHC) study was performed for p53 expression on 35 cases of OSMF, 10 cases of OSCC with history of habits and 10 normal patients without any habits. The expression of p53 showed a significant difference between normal oral mucosa, OSMF and OSCC samples. The study demonstrated a high incidence of p53 over expression in OSMF and OSCC. The results indicate that p53 over expression may play a role in pathogenesis of OSMF and in the development of Oral squamous cell carcinoma. With early detection of the high-risk patients with OSMF, we can expect to develop more intensive treatment modalities, leading to the reduction in cancer transformation rate from OSMF.

MicroRNA 200a as a histologically independent marker for meningioma recurrence: Results of a four microRNA panel analysis in meningiomas.

Meningiomas are mostly benign neoplasms of the central nervous system. Nevertheless there are recurrences in about 20% after surgical resection. Previous studies could reveal several predictors of meningioma recurrence. Tumor progression often is associated with a specific pattern of chromosome losses. Our study investigated the potential function of selected microRNAs as markers of tumor progression. By real-time polymerase chain reaction the expressions of microRNA 21-3p, 34a-3p, 200a-3p, and 409-3p were analyzed in solid tumor and in blood samples of 51 meningioma patients as well as in blood samples of 20 healthy individuals. Additionally, aberrations of parts of chromosomes 1, 14, 18, and 22 were analyzed by FISH. Tumor and blood samples were statistically analyzed, using Spearman's rank correlation coefficient as well as Mann-Whitney U- and Kruskal-Wallis-Test. MicroRNA 200a showed significantly lower expressions in recurrent meningiomas than in newly diagnosed ones. MicroRNA 409 in meningiomas was correlated significantly with tumor volume and showed a significant negative correlation with patient age. Significance was found between the expression patterns of microRNAs 34a and 200a with the respective aberrations of chromosome 1p and the microRNA 409 with aberration of chromosome 14. In the male cohort the expression of microRNA 200a in blood was significantly upregulated in patients compared to healthy volunteers. By our research the function of microRNA 200a was proved to detect meningioma patients by liquid biopsy. We detected microRNA 200a as a new biomarker to indicate meningioma recurrences. Future transferability to blood could be important for patient follow-up.

Prognostic and Predictive Value of Aberrant P53 Status in Human Cancers: Systematic Review and Meta-Analysis

Background: Many studies have attempted to correlate p53 abnormality including p53 gene mutation and p53 protein over expression with prognosis or therapeutic response in adjuvant chemotherapy but have yielded controversial results. To investigate whether p53 aberrations have different impacts on survival and outcomes of adjuvant chemotherapy among cancer patients, we conducted a meta-analysis. Methods: We performed comprehensive search before September 18, 2022. Hazard Ratio (HR) with 95% Confidence Interval (CI) was effective measure, and Stata 16.0 was applied for data analysis. Results: A total of 14 articles were enrolled in this meta-analysis. p53 protein over expression detected by immunohistochemistry was a risk factor of 5-year Overall Survival (OS) among cancer patients after Radical Surgery (RS). Furthermore, p53 protein over expressed patients displayed inferior response to adjuvant chemotherapy with unfavorable 5-year Disease Free Survival (DFS) (HR = 1.61, 95% CI: 1.12 ~ 2.32, P = 0.011). p53 gene mutation was a negative indicator of OS in adjuvant chemotherapy (HR = 1.41, 95% CI: 1.19 ~ 1.69, P< 0.001). Furthermore, we performed subgroup analysis according to year of publication, the number of patients, detection method of abnormal p53 and tumor types, consistent result was observed. Conclusion: p53 protein over expression appears to serve as a predictive and prognostic biomarker in adjuvant chemotherapy setting. p53 gene mutation is a potential predictor and could identify high-risk patients who obtain limited overall benefit from adjuvant chemotherapy and support clinical decision-making.

Upregulated MYC expression and p53 mutations may contribute to the oncogenesis of canine Meibomian gland carcinomas.

Sebaceous carcinomas of the human ocular adnexa commonly exhibit pagetoid spread, mutations in tumor-suppressor genes, and protooncogene copy number gain. Sebaceous carcinomas are rarely reported in other species, and while the Meibomian gland (MG) represents the most common ocular adnexal structure of the canine eyelid to develop neoplasia, most are clinically and histologically benign. The objective of this study was to compare molecular features of canine MG carcinomas and adenomas. Two retrospectively identified MG carcinomas were subject to immunohistochemistry and qPCR. When compared with normal glands, MYC was upregulated in benign and malignant MG neoplasms. Aberrant p53 expression was restricted to the nuclei of intraepithelial neoplastic cells in MG carcinomas. Adipophilin expression was diminished in MG neoplasms compared with the normal MG. Our findings, if confirmed in a larger cohort of cases, could suggest that MG oncogenesis in a dog may exhibit similar molecular features as their human counterparts.

Correlative Assessment of p53 Immunostaining Patterns and TP53 Mutation Status by Next-Generation Sequencing in High-Grade Endometrial Carcinomas.

TP53 mutations are frequently identified in the copy number-high molecular subgroup of endometrial carcinomas (ECs). P53 immunohistochemistry (IHC) is a widely used surrogate marker reflecting the mutational status of TP53 , and recent reports have shown ~95% concordance between the two methods in ECs. While these results are promising, studies evaluating the correlation between different p53 IHC staining patterns and comprehensive next-generation sequencing results are still limited. We compared the p53 IHC staining patterns, scored as wild-type, diffuse nuclear overexpression, null/complete absence, and cytoplasmic, to next-generation sequencing results reported by FoundationOneCDx in 43 high-grade ECs: 20 serous ECs, 9 mixed ECs with a serous component, 4 carcinosarcomas with a serous component, and 10 grade 3 endometrioid ECs. The concordance of p53 IHC and TP53 mutation status was 100% (43/43) overall, including 100% (33/33) concordance in tumors with a serous component and 100% (10/10) in endometrioid ECs. Among the 35 tumors with aberrant p53 expression the most commonly observed pattern was diffuse nuclear overexpression seen in 69% (24/35), followed by cytoplasmic staining in 17% (6/35), and complete absence of staining (null) in 14% (5/35) of tumors. Of the 6 tumors with cytoplasmic staining, 4 corresponded to missense mutations within the DNA binding domain (V157F in 2 tumors, and S127P and R280S, in 2 tumor each), while 2 corresponded to nonsense mutations in the tetramerization domain (p.E339*). Our results further support that p53 IHC can serve as an accurate predictor of TP53 alterations in ECs to aid the molecular-based tumor classification and the distinction between tumor histotypes, both of which play an important role in the assessment of clinical prognosis and therapeutic decision making. In addition, our data suggest, that the type and position of TP53 mutation may not directly correlate with the observed p53 IHC pattern in all tumors, and that there may be alternative mechanisms for cytoplasmic localization (other than mutations involving the nuclear localization domain), possibly due to conformational changes or posttranslational modifications of the aberrant p53 protein.

Management of patients with early-stage ovarian clear cell carcinoma: risk stratification and fertility conservation

ObjectiveWe sought to describe clinicopathologic and treatment factors associated with oncologic outcomes in patients with early-stage ovarian clear cell carcinoma undergoing complete staging and in a sub-set of these patients...

Aberrant p53 expression is associated with neoplastic progression in Barrett oesophagus diagnosed as indefinite for dysplasia.

The aim of this study was to investigate the role of immunohistochemical (IHC) expression of p53 and other potential clinical parameters as prognostic markers for predicting neoplastic progression in Barrett oesophagus (BE) patients diagnosed as indefinite for dysplasia (IND). The study included patients with established BE of any extent who had a diagnosis of IND accompanied by concurrent p53 immunohistochemistry (IHC) stain at the index endoscopic procedure and at least one follow-up examination between 2000 and 2021. Correlation between disease progression from IND to higher-grade dysplasia [low-grade dysplasia (LGD), high-grade dysplasia (HGD) and oesophageal adenocarcinoma (EAC)] and clinicopathological parameters were analysed. A total of 149 patients (99 males; mean age 63.3 ± 10.0 years, range=35-89) were included in the final analysis. Median follow-up was 37.1 months [interquartile range (IQR)=20.5-59.1 months]. Progression rates from IND to LGD and HGD were 12.1% (18 of 149) and 2.7% (four of 149), respectively. On multivariate analysis, the number of IND diagnoses was significantly associated with progression to both LGD and HGD (P= 0.016 and P< 0.001, respectively). Cox regression analysis showed that aberrant p53 expression was significantly associated with progression to LGD [hazard ratio (HR)=4.87, 95% confidence interval (CI)=1.91-12.45, P= 0.001] and HGD (HR=21.81, 95% CI=1.88-253.70, P= 0.014). Kaplan-Meier survival analysis also demonstrated that aberrant p53 expression was significantly associated with progression to LGD (P< 0.001) and HGD (P= 0.001). Our results suggest that frequency of IND diagnoses and status of p53 expression can help to stratify risk of neoplastic progression in BE patients with IND.

Clinicopathological Characteristics of Advanced Epstein-Barr Virus-associated Gastric Cancer Highlighting Aberrant p53 Expression.

Epstein-Barr virus (EBV)-associated gastric cancer is a distinct subtype of stomach adenocarcinoma. Although previous studies have investigated its clinicopathological characteristics, there is a lack of research focusing on advanced EBV-associated gastric cancer. In this study, we performed a comprehensive review of advanced EBV-associated gastric cancer cases. We retrospectively collected 18 consecutive cases of surgically resected advanced EBV-associated gastric cancer. Clinicopathological parameters were investigated using histological review, immunohistochemistry, and a review of the electronic medical records of the hospital. The predominant histological pattern of advanced EBV-associated gastric cancer, according to the Laurén classification, was intestinal-type adenocarcinoma with varying degrees of differentiation. However, focal areas showing conventional gastric carcinoma with a lymphoid stromal pattern were found in all cases except one. In addition to the previously described histological patterns of EBV-associated gastric cancer, one case displayed chronic granulomatous inflammation-like histology with barely identifiable malignant epithelial cells. Another case had a pure signet-ring cell carcinoma component showing nuclear positivity for the EBV-encoded RNA in situ hybridization assay. Remarkably, five out of 18 cases (27.8%) showed aberrant p53 expression on immunostaining, which is known to occur rarely in EBV-associated gastric cancer. All cases with aberrant p53 expression had intestinal-type adenocarcinoma-like components. Advanced EBV-associated gastric cancer had distinct histology and a higher rate of aberrant p53 immunostaining pattern than conventional EBV-associated gastric cancer. Therefore, their biological behavior should be investigated separately.

Molecular Characteristics of Metastatic Lesions Have Superior Prognostic Impact on Endometrial Cancer

Background/Aim: In endometrial cancer (EC), lymph node (LN) metastasis significantly impacts prognosis. Thus far, no studies have reported the molecular genetics of each metastatic lesion. This study aimed to investigate the molecular characteristics of primary and metastatic LNs and their association with clinical outcomes. Patients and Methods: The clinicopathological and molecular characteristics of 33 patients with EC with regional LN metastasis (FIGO stage IIIC) were investigated; we evaluated the mutational status of p53 and DNA mismatch repair (MMR) proteins in the primary lesion, all the positive LNs (102 lesions), mutational variation between primary and paired metastatic lesions, inter-lesion heterogeneity, and their association with clinical outcomes. Results: Immunohistochemically, 12 patients (36.4%) displayed aberrant p53 expression in metastatic lesions, and a concordant rate of 93.4% was observed between primary and metastatic lesions. Inter-lesion heterogeneity was observed in 20 cases (60.6%). In Kaplan–Meier analysis, patients with aberrant p53 expression in metastatic LNs exhibited worse progression-free survival (PFS) than those with wild-type p53 expression (p=0.008). Wild-type p53 expression in primary lesion with inter-lesion heterogeneity had a significantly worse PFS (p=0.049) than those without heterogeneity. In the Cox univariate analysis, p53 expression in metastatic LNs was significantly associated with recurrence (p=0.013). Genetic diversity between primary and metastatic lesions and among metastases was validated by evaluating the p53 and MMR proteins using immunohistochemistry analysis. Conclusion: The molecular characteristics of metastatic lesions in addition to those of primary lesions could provide beneficial prognostic information in patients with EC with regional LN metastasis.

Feasibility and clinical applicability of genomic profiling based on cervical smear samples in patients with endometrial cancer.

PurposeCervical smear samples are easily obtainable and may effectively reflect the tumor microenvironment in gynecological cancers. Therefore, we investigated the feasibility of genomic profiling based on tumor DNA analysis from cervical smear samples from endometrial cancer patients.Materials and methodsPreoperative cervical smear samples were obtained via vaginal sampling in 50 patients, including 39 with endometrial cancer and 11 with benign uterine disease. Matched blood samples were obtained simultaneously. Genomic DNA (gDNA) from cervical smear and/or cell-free DNA from whole blood were extracted and sequenced using the Pan100 panel covering 100 endometrial cancer-related genes.ResultsCervical swab-based gDNA analysis detected cancer with 67% sensitivity and 100% specificity, showing a superior performance compared to that of the matched blood or Pap smear tests. Cervical swab-based gDNA effectively identified patients with loss of MSH2 or MSH6 and aberrant p53 expression based on immunohistochemistry. Genomic landscape analysis of cervical swab-based gDNA identified PTEN, PIK3CA, TP53, and ARID1A as the most frequently altered genes. Furthermore, 26 endometrial cancer patients could be classified according to the Proactive Molecular Risk Classifier for Endometrial Cancer.ConclusionCervical swab-based gDNA test showed an improved detection potential and allowed the classification of patients, which has both predictive and prognostic implications.

Molecular subtyping of gastroesophageal dysplasia heterogeneity according to TCGA/ACRG classes

Gastric adenocarcinoma has recently been classified into several subtypes on the basis of molecular profiling, which has been successfully reproduced by immunohistochemistry (IHC) and in situ hybridization (ISH). A series of 73 gastroesophageal dysplastic lesions (37 gastric dysplasia and 36 Barrett dysplasia; 44 low-grade dysplasia and 29 high-grade dysplasia) was investigated for mismatch repair proteins, E-cadherin, p53, and EBER status, to reproduce The Cancer Genome Atlas (TCGA) and Asian Cancer Research Group (ACRG) molecular clustering. Overall, the dysplastic lesions were classified as follows: according to TCGA classification, EBV, 0/73 (0%), MSI, 6/73 (8.2%), GS, 4/73 (5.5%), CIN, 63/73 (86.3%); according to ACRG molecular subtyping, MSI, 6/73 (8.2%), MSS/EMT, 4/73 (5.5%), MSS/TP53−, 33/73 (45.2%), MSS/TP53+, 30/73 (41.1%). A positive association was found between MSS/TP53− and Barrett dysplasia (p = 0.0004), between MSS/TP53+ and LG dysplasia (p = 0.001) and between MSS/TP53+ and gastric dysplasia (p = 0.0018). Gastroesophageal dysplastic lesions proved to be heterogenous in terms of TCGA/ACRG classes, but with a different distribution from that of cancers, with no EBV-positive cases, an increasing presence of mismatch repair deficiency from low grade to high grade lesions, and a prevalence of p53 aberrations in Barrett dysplasia. The present study further demonstrated that gastroesophageal dysplastic lesions may be characterized by alterations in predictive/prognostic biomarkers, and this should be considered in routine diagnostic.

ORF1p Is a Potential Novel Diagnostic Marker for Differentiated Vulvar Intraepithelial Neoplasia.

Accurate diagnosis of differentiated vulvar intraepithelial neoplasia (dVIN) can be challenging as histomorphologic features may be subtle and overlap with nondysplastic lesions. In practice, aberrant p53 expression supports the diagnosis, but a substantial percentage retains wild-type p53. Recently, the retrotransposon long interspersed nuclear element 1 has been detected in distinct cancer types. We have now investigated the expression of the long interspersed nuclear element 1 encoded protein ORF1p in dysplastic and nondysplastic vulvar samples to assess its diagnostic value. Specimens of dVIN (n=29), high-grade squamous intraepithelial lesions (n=26), inflammatory vulvar lesions (n=20), lichen sclerosus (n=22), and normal vulvar epithelia (n=29) were included. ORF1p and p53 expression was determined using immunohistochemistry. The majority of dVIN [27/29 (93%)] and high-grade squamous intraepithelial lesions [20/26 (77%)] showed distinct (i.e. moderate or strong) ORF1p expression in the basal and suprabasal or all epithelial layers, respectively. Of note, ORF1p was present in all 4 cases of dVIN with wild-type p53 staining pattern. In contrast, ORF1p was negative or weakly expressed in most inflammatory lesions [14/20 (70%)] and lichen sclerosus [18/22 (82%), P <0.001]. Normal control epithelium exhibited negative staining in all cases. In conclusion, ORF1p might be a useful diagnostic marker for dVIN, especially in cases with retained wild-type p53.

Molecular classification of endometrial carcinomas: a singleinstitution review (084)

<b>Objectives:</b> To describe endometrial carcinoma (EC) molecular classification utilizing clinical next-generation sequencing (NGS) and immunohistochemistry (IHC) and to assess the clinical, pathologic, and survival outcomes based on molecular data. <b>Methods:</b> We identified all primary and recurrent EC samples of 341-505 cancer-related genes undergoing NGS (MSK-Integrated Mutation Profiling of Actionable Cancer Targets; MSK-IMPACT) from test inception in 2014 through 2020. Samples were classified into the Cancer Genome Atlas (TCGA) molecular subtypes utilizing MSK-IMPACT results for <i>POLE</i> hotspot mutations, <i>TP53</i> mutations, and MSIsensor score; and mismatch repair (MMR) and p53 IHC results collected from pathology reports. Tumors not classified per TCGA based on available NGS and IHC data were excluded from the analysis. Clinical, pathologic, and survival data were obtained for primary ECs undergoing initial staging surgery or treatment at our institution after staging surgery. Only NGS performed prior to documented recurrence was used for survival analysis, to avoid ascertainment bias. Appropriate statistical tests were applied. <b>Results:</b> Clinical MSK-IMPACT sequencing was performed on 2174 ECs. Except for 40 unclassifiable cases, 2134 tumors corresponded to 2066 unique patients, including 1712 (80%) primary and 409 (19%) recurrent tumors. Molecular classification was distributed as 5% <i>POLE</i> (<i>n</i>=113), 22% microsatellite instability-high (MSI-H; <i>n</i>=479), 38% copy-number high (CN-H) (<i>n</i>=818), and 34% CN-low (<i>n</i>=724). MMR-deficiency by IHC was found in 16% of <i>POLE</i>, and IHC-based aberrant p53 expression in 12% of <i>POLE</i> and 2% of MSI-H ECs. Due to the hierarchy of our hybrid classification algorithm, this did not affect molecular designation. Otherwise, all available MMR and p53 IHC results were concordant with NGS results to determine classification. CN-H was 91% non-endometrioid with 67% FIGO G3 within the endometrioid type, while <i>POLE</i>, MSI, and CN-L were 10-32% and 17-21%, respectively (p<0.05). Criteria for survival analysis using primary tumor NGS results were met by 1118 patients. The median follow-up was 22.5 months (0.5-214 months). Three-year progression-free survival was 97% for <i>POLE</i> (SE 2.0), 90% CN-L (SE 2.1), 80% MSI-H (SE 3.1), and 46% CN-H (SE 3.5), (p<0.01). Three-year overall survival was 100% for <i>POLE</i>, 94% MSI-H (SE 2.1), 93% CN-L (SE 2.2), and 71% CN-H (SE 3.6), (p<0.01). CN-H classification remained prognostic for recurrence outcomes when adjusting for age, stage, and histology (HR: 6.2, 95% CI: 1.5-26.6; p=0.04). <b>Conclusions:</b> Our approach to molecular classification involves a hybrid strategy utilizing combined NGS and IHC analysis as a surrogate for whole-exome sequencing employed by TCGA. Our current database is mostly CN-H, which may reflect the referral-based nature of our institution. When applying appropriate criteria for survival analysis, CN-H portends the worst outcomes. Future studies should consider the histological heterogeneity of CN-H when considering treatment options.Fig. 1

Molecular characterization of gastric adenocarcinoma diagnosed in patients previously treated for Hodgkin lymphoma or testicular cancer.

IntroductionThe risk of developing gastric cancer is increased in patients treated with radiotherapy for Hodgkin lymphoma (HL) or testicular cancer (TC). This study aims to assess if gastric adenocarcinoma after treatment for HL/TC (t-GC) is molecularly different from gastric adenocarcinoma in the general population.MethodsPatients were diagnosed with t-GC ≥5 years after treatment for HL/TC. Four molecular subtypes were identified using immunohistochemical and molecular analyses: Epstein-Barr virus (EBV), mismatch repair (MMR) deficiency or microsatellite instability (MSI), aberrant p53 staining as surrogate for chromosomal instability (sCIN), and a surrogate for genomic stability (sGS) without these aberrations. Results were compared with gastric cancer in the general population (p-GC) described in literature.ResultsMolecular subtyping of 90 t-GCs resulted in 3% EBV, 8% MSI, 36% sCIN and 53% sGS. 3/6 of MSI t-GCs had MLH1 promoter methylation and 2/6 were explained by double somatic mutations in MMR genes. T-GCs were more frequently sGS than p-GCs (53% vs. 38%, p = 0.04). T-GC was more frequently sGS in HL/TC patients diagnosed before 1990, than after 1990 (63% vs. 38%, p = 0.03). T-GCs located in the antrum, an area that receives high irradiation doses, were more frequently sGS (61% vs. 28% in p-GCs, p = 0.02).ConclusionOur results demonstrate that t-GCs are more frequently of the sGS subtype than p-GCs. An association of t-GC of the sGS subtype with prior anticancer treatment is suggested by the high frequency in HL/TC patients who were treated before 1990, a time period in which HL/TC treatments were more extensive.