Is p53 immunohistochemistry alone useful for delineating adjuvant endometrial treatment in low-middle-income countries?

OBJECTIVE: Endometrial cancer (EC) treatment changed substantially with the introduction of molecular classification. Low-middle income (LMIC) countries will face barriers to including molecular classification to guide treatment. This study aims to analyse the value of p53 immunohistochemistry to delineate adjuvant treatment in FIGO stages I and II. METHODS: Patients with EC treated between 2010 and 2016 were retrospectively evaluated. Patients included in this analysis must have reviewed FIGO stage I/II high-grade histologies (endometrioid grade 3, serous, clear cell, carcinosarcoma, mixed and undifferentiated). Samples were subjected to p53 immunohistochemistry. Recurrence-free and overall survival were analysed using the Kaplan-Meier method and log-rank test. Cox proportional hazards regression was performed for multivariable analysis. RESULTS: From 2010 to 2016, 265 patients met the inclusion criteria. Patients with aberrant p53 (71.4 %) were associated with older age (59.7 % vs 77.8 % with more than 60 years), relapse (12.5 vs 29.6 %) and death (22.2 vs 46.7 %). The pattern of relapse was not different, with most being at extrapelvic sites (55.5 % vs 62.3 % for p53 wild type and aberrant, respectively). The median overall survival was not reached versus 92.2 months for p53 wild type and aberrant, respectively (p = 0.003). In multivariate analysis, chemotherapy decreased death (p = 0.014) in p53 aberrant tumours, a benefit not seen in the wild-type cohort (p = 0.22). CONCLUSION: This retrospective analysis corroborates the finding of worse outcomes for p53 aberrant tumours in stage I/II EC and the benefit of more aggressive adjuvant treatment (systemic therapy and radiotherapy). Although not ideal as a sole molecular marker, p53 immunohistochemistry could complement the classical anatomopathological features and be part of the decision-making process with patients in LMIC.

[Precancers of the oral mucosa: clinic, diagnostics]. / Predraki slizistoi obolochki rta: klinika i diagnostika.

OBJECTIVE: The aim of the study. Improving the efficiency of diagnosis and detailing the features of the clinic of «potentially malignant¼ diseases of the oral mucosa. MATERIALS AND METHODS: Clinical and laboratory examination of 124 patients of the department of oral mucosa diseases aged 35 to 80 years, among whom there were 75 women and 49 men, with diseases such as erythroplakia - 12 patients, verrucous leukoplakia - 52 patients, erosive form of leukoplakia - 35 patients, cheilitis Manganotti - 25 patients. Histological and immunohistochemical methods of investigation were used as diagnostics. To assess the proliferative activity of epithelial cells, the determination of the Ki-67 index was used. The synthesis of keratin 15 (K15) in epithelial layers was determined as a diagnostic criterion for the severity of neoplasia. The expression of human papillomavirus type 16 (HPV 16) antigens and p16INK4a protein in epithelial cells was studied, as well as the expression of p53 protein. RESULTS: A high prevalence of p53 mutations was observed in patients with erythroplakia. In leukoplakia, the expression of the Ki-67 protein was detected in the cell nuclei in both the basal and parabasal layers of the multilayer squamous epithelium, in 77% of cases, the expression of the p16INK4a protein in the epithelial nuclei with varying degrees of dysplastic changes was noted, and a positive reaction to HPV16 was also observed in the cell nuclei and cytoplasm of epithelial cells in the basal, parabasal and spiny epithelial layers. The appearance of K15 in the cytoplasm of cells above the basal layer with abrasive precancerous cheilitis was found in 48% of cases. CONCLUSION: To diagnose early manifestations of neoplastic processes in «potentially malignant¼ diseases of the oral mucosa, it is necessary to use both classical histological and immunohistochemical methods of investigation with various markers.

Enhancing upper tract urothelial carcinoma diagnosis: Utility of cytokeratin 17 and CK20/CD44/p53 immunohistochemical panel.

Upper tract urothelial carcinoma (UTUC) presents diagnostic challenges due to small biopsy specimen size, poor orientation, and technical obstacles that can yield equivocal diagnoses. This uncertainty often mandates repeated biopsies to evaluate the necessity of nephroureterectomy. Prior studies have suggested cytokeratin 17 (CK17) immunostain as an adjunctive tool for diagnosing bladder urothelial neoplasia in both urine cytology and tissue biopsy specimens. We evaluated the utility of CK17 in differentiating UTUC from benign urothelium and its ability to stratify low-grade from high-grade neoplasia. Our study involved a cohort of previously diagnosed cytology (n = 29) and tissue specimens from biopsies and resections (n = 85). We evaluated CK17 staining percentage in cytology and tissue samples and localization patterns in biopsy/resection samples. Our findings showed a statistically significant distinction (p < 0.05) between UTUC and benign tissue specimens based on full thickness localization pattern (odds ratio 8.8 [95% CI 1.53-67.4]). The percentage of CK17 staining failed to significantly differentiate neoplastic from non-neoplastic cases in cytology or tissue samples. Additionally, based on prior research showing the efficacy of CK20/CD44/p53 triple panel in bladder urothelial neoplasia, we utilized tissue microarrays to evaluate if these markers could distinguish UTUC from benign urothelium. We found that CK20/CD44/p53, individually or in combination, could not distinguish urothelial neoplasia from non-neoplasia. Full thickness CK17 urothelial localization by immunohistochemistry was highly reproducible with excellent interobserver agreement and may play a supplementary role in distinguishing upper tract urothelial neoplasia from benign urothelium.

P53 Overexpression May Represent an Early Marker of Clinicopathologic Progression in Vasculogenic Mesenchymal Lesions of Germ Cell Tumor Origin.

Vasculogenic mesenchymal lesions (VMLs) of germ cell tumor origin are thought to originate in postpubertal-type yolk sac tumor components and include a spectrum of lesions from teratoma with vasculogenic stroma (TVS), to low and high-grade vasculogenic mesenchymal tumors (VMTs). VMLs exhibit rudimentary to well-developed neoplastic vessels within primitive mesenchyme, being considered a neoplastic reiteration of embryonic vasculogenesis in the splanchnic mesoderm of the yolk sac. They occur in patients with primary mediastinal germ cell tumors after chemotherapy, and a subset progresses to "somatic-type" sarcomas [including angiosarcoma (AS)], with high-grade VMTs likely portending a higher risk. Recently, we encountered a low-grade VMT that progressed to metastatic AS during follow-up. In this case, both the low-grade VMT and the subsequent AS demonstrated p53 overexpression, suggesting that p53 alterations may precede histopathologic transformation. To test this hypothesis, we evaluated neoplasms representing the entire spectrum of VMLs using p53 immunohistochemistry (IHC; clone DO-7, Dako). Overexpression was defined as nuclear positivity in > 80% of neoplastic cells. Because the distinction between high-grade VMT and AS can be subjective in some cases, they were grouped together in a single category. Thirty-nine VMLs were assessed: 16 high-grade VMT/AS, 19 low-grade VMT, and 4 TVS. Patient age ranged from 19 to 46 years (mean, 30 years; male = 97%). Four high-grade VMT/AS and one low-grade VMT showed p53 overexpression (5/39 VMLs, 13%; 4/16 high-grade VMT/AS, 25%). These tumors included 1 unequivocal AS and 1 high-grade VMT/AS with progression to rhabdomyosarcoma. The only low-grade VMT with p53 overexpression demonstrated progression to AS. Another high-grade VMT that progressed to sarcoma demonstrated p53 overexpression in the sarcoma component, but it was excluded because the VMT was not represented in the material available at the time of the study. Lesions with intratumoral grade heterogeneity (classified based the highest grade), demonstrated more pronounced p53 overexpression in the high-grade components. P53 overexpression is associated with disease progression in a subset of VMTs and may precede morphologic transformation to sarcoma. Routine evaluation of VMTs with p53 IHC seems justified, with overexpressors likely requiring an close clinical surveillance.

Presence of Merkel cell polyomavirus DNA and large-T antigen in keratinocyte carcinomas and its correlation with immunohistochemical markers p16, p53 and ki67.

BACKGROUND: Merkel cell polyomavirus (MCPyV), a human polyomavirus that is unequivocally linked to merkel cell carcinoma (MCC), has been found in association with keratinocytes carcinomas (KC), especially basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). Nevertheless, there is scarce information about the possible involvement of MCPyV in the development of KC. OBJECTIVES: To assess the presence of MCPyV DNA and Large-T Antigen (LT-Ag) via Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC) in cases of KC, and to correlate its presence with immunohistochemical markers p16, p53, and ki67, tumor type and subtype, sun-exposed location, and epidemiological data. METHODS: The prevalence of MCPyV DNA, LT-Ag, and immunohistochemical markers p16, p53, and ki67 was assessed by PCR and Immunohistochemistry (IHC) in 127 cases of KC, these results were correlated with tumor type and subtype, sun-exposed location, and epidemiological data. RESULTS: The MCPyV DNA was detected in 42.57% (43 of 101) cases by PCR, the LT-Ag was detected in 16.4% (20 of 122) of cases, p16 in 81.5% (97 of 119), p53 in 66.4% (83 of 125), ki67 in 89% (73 of 82). No correlation between MCPyV LT-Ag and DNA confronted with tumor type, subtype, location site, and immunohistochemical markers was found. A single correlation between the MCPyV LT-Ag and cSCC tumors and peri-tumoral lymphocyte cells was noted. STUDY LIMITATIONS: Further steps need to be taken to better evaluate the MCPyV influence and its possible role in KC carcinogenesis, as the evaluation of the virus genome state, the gene sequence that encodes LT-Ag in the KC tumor cells, and in situ hybridization for viral DNA or RNA in these cells. CONCLUSIONS: Despite the frequent detection of MCPyV in KC, the data available so far does not support the hypothesis of a causal relationship between them.

Characteristics and Prognosis of Sporadic Neoplasias Detected in Patients with Ulcerative Colitis.

INTRODUCTION: Patients with ulcerative colitis (UC) develop not only UC-associated neoplasias but also sporadic neoplasias (SNs). However, few studies have described the characteristics of SNs in patients with UC. Therefore, this study aimed to evaluate the clinical features and prognosis of SNs in patients with UC. METHODS: A total of 141 SNs in 59 patients with UC, detected by surveillance colonoscopy at Hiroshima University Hospital between January 1999 and December 2021, were included. SNs were diagnosed based on their location, endoscopic features, and histopathologic findings along with immunohistochemical staining for Ki67 and p53. RESULTS: Of the SNs, 91.5% were diagnosed as adenoma and 8.5% were diagnosed as carcinoma (Tis carcinoma, 3.5%; T1 carcinoma, 5.0%). 61.0% of the SNs were located in the right colon, 31.2% were located in the left colon, and 7.8% were located in the rectum. When classified based on the site of the lesion, 70.9% of SNs occurred outside and 29.1% within the affected area. Of all SNs included, 95.7% were endoscopically resected and 4.3% were surgically resected. Among the 59 patients included, synchronous SNs occurred in 23.7% and metachronous multiple SNs occurred in 40.7% during surveillance. The 5-year cumulative incidence of metachronous multiple SNs was higher in patients with synchronous multiple SNs (54.2%) than in those without synchronous multiple SNs (46.4%). CONCLUSION: Patients with UC with synchronous multiple SNs are at a higher risk of developing metachronous multiple SNs and may require a closer follow-up by total colonoscopy than patients without synchronous SNs.

A descriptive analysis of gastric cancer with an immunohistochemical Study of Ki67 and p53 as prognostic factors.: Bahrain experience.

OBJECTIVES: To describe the increasing number of gastric cancer cases at Bahrain Defense Force Hospital with implementation of immunohistochemistry markers as prognostic factors. METHODS: This study included histologically confirmed malignant gastric tumors diagnosed at Bahrain Defense Force Hospital from January 2009 to June 2019. Various epidemiological and pathological data were abstracted and recorded with immunohistochemical analysis of the proliferation marker Ki67 and cell-cycle regulator p53 as prognostic factors. RESULTS: A total of 53 patients with gastric cancer were included in the study, with mean age of 59.75 ± 12.9 years. The typical histological types were signet ring cell adenocarcinoma (68%) and intestinal type adenocarcinoma (17%). Helicobacter pylori and intestinal metaplasia were significantly associated with gastric cancer (p<0.01). The studied population's mortality was 39 (74%). The Ki67 proliferation index showed a mean and standard deviation of 67.09 ± 16.338, with a higher mortality rate in patients with low Ki67 but no difference in survival time. No statistically significant association was found between clinicopathological findings with p53 immunostaining positivity. CONCLUSION: The common gastric cancers are signet ring cell adenocarcinoma and intestinal type adenocarcinoma, affecting a wide range of age groups (33-91 years), with those over 60 years at greater risk. Interestingly, low Ki67 is associated with a higher mortality rate, whereas p53 has no prognostic significance. Expression of both Ki67 and p53 showed no association with survival time.

Incidence and Clinicopathologic Characteristics of Human Papillomavirus-independent Invasive Squamous Cell Carcinomas of the Cervix: A Morphologic, Immunohistochemical, and Human Papilloma-Virologic Study of 670 Cases.

We aimed to determine the frequency of human papillomavirus-independent (HPVI) cervical squamous cell carcinoma (SCC) and to describe clinicopathologic characteristics. Among 670 patients with surgically treated SCCs in an established multi-institutional cohort, 447 had available tissue. Tissue microarrays were constructed and studied by in situ hybridization (ISH) for high-risk and low-risk human papillomavirus (HPV) mRNA and immunohistochemistry for p16 and p53. Tumors were HPVI if negative by HPV ISH and they failed to show diffuse p16 positivity by immunohistochemistry, and human papillomavirus-associated (HPVA) if positive by HPV ISH. Ten HPVI SCCs and 435 HPVA SCCs were identified; 2 cases were equivocal and excluded from analysis. The overall rate of HPVI SCC was low (2%) but was higher among older patients (7% in patients above 60 y of age and 17% in patients above 70 y of age). Compared with HPVA, patients with HPVI SCC were significantly older (median age, 72 vs. 49, P <0.001) and diagnosed at a higher stage (40% vs. 18% with stage III/IV disease, P =0.055). p53 expression was varied; 2 cases (20%) had null expression and 8 (80%) had wild-type expression. HPVI SCCs were heterogenous, with keratinizing, nonkeratinizing, and warty morphologies observed. Several cases had a precursor lesion reminiscent of differentiated vulvar intraepithelial neoplasia, with prominent basal atypia and hypereosinophilia or a basaloid-like morphology. Two patients (20%) had distant recurrences within 12 months, and 3 (30%) died of disease during follow-up. HPVI SCCs are rare tumors that are more common among older patients with higher stage disease and have important clinical and histologic differences from HPVA SCCs.

Effects of high-risk human papillomavirus infection on P53, pRb, and survivin in lung adenocarcinoma-a retrospective study.

Objective: To observe the effects of high-risk human papillomavirus (HR-HPV) infection on P53, pRb, and survivin in lung adenocarcinoma (LUAD). Methods: The cancerous and cancer-adjacent tissues of 102 patients with LUAD from January 2020 to April 2022 were selected for the study. HR-HPV infection was detected by flow fluorescence method, and P53, pRb, and survivin protein expression was detected by immunohistochemical staining method. Statistical analysis was performed to determine the differences in the HR-HPV infection and the expression of P53, pRb, and survivin proteins between LUAD tissues and cancer-adjacent tissues; the correlation between HR-HPV infection and P53, pRb, and survivin protein expression in cancer tissues; and the correlation between HR-HPV infection and clinicopathological features of LUAD. Results: The infection rate of HR-HPV was higher in the LUAD tissues (28.43%) than in cancer-adjacent tissues (7.84%), and the difference was statistically significant (P < 0.05). The positive rates of P53 and survivin protein were higher in the LUAD group (33.33% and 67.16%, respectively) than in the cancer-adjacent group (3.92% and 11.73%, respectively), and the difference was statistically significant (P < 0.05). The positive rate of pRb protein was lower in the LUAD group (58.82%) than in the cancer-adjacent group (92.14%), and the difference was statistically significant (P < 0.05). The positive rates of P53 and survivin proteins were significantly higher in the HR-HPV LUAD group (58.62% and 86.21%, respectively) than in the non-HR-HPV LUAD group (41.38% and 67.12%, respectively), and the difference was statistically significant (P < 0.05). The expression rate of pRb protein was significantly lower in the HR-HPV LUAD group (37.93%) than in the non-HR-HPV LUAD group (67.12%), and the difference was statistically significant (P < 0.05). The expression of p53 and survivin protein was positively correlated with HR-HPV infection (r = 0.338 and 0.444, P < 0.05), whereas the expression of pRb protein was negatively correlated with HR-HPV infection (r =  - 0.268, P < 0.05). HR-HPV infection was not associated with gender, age, and smoking in patients with LUAD (P > 0.05). HR-HPV infection was associated with lymph node metastasis and clinical stage of LUAD (P < 0.05). Conclusions: HR-HPV infection was associated with lymph node metastasis and clinical stage of LUAD, which may be achieved by up-regulating p53 and survivin protein expression and down-regulating pRb protein expression.

Amonafide Induces HUVEC Senescence by Inhibiting Autophagy.

BACKGROUND: Amonafide (Amo), due to hematotoxicity and digestive tract symptoms, the clinical application of which is limited. Several studies have reported that chemotherapy side effects are closely related to cellular senescence accumulation. Our study aims to examine whether amonafide causes senescence in human umbilical vein endothelial cell (HUVEC) lines and investigate its mechanisms associated with senescence. METHODS: The experiments of expression of genes and proteins associated with aging were carried out with HUVEC cell lines. The experiments were divided into a control group and an amonafide group with different days. The HUVEC senescence cells were detected by SA-ß-Gal staining, Western blotting detected the protein levels of p16, p53, AMPK (Adenosine 5'-Monophosphate (AMP)-Activated Protein Kinase), mTOR (mechanistic Target of Rapamycin), p62, and LC3 (microtubule-associated protein1 light chain 3, MAP1LC3). Fluorescence detected the expression of mRFP (monomeric Red Fluorescent Protein)-GFP (Green Fluorescent Protein)-LC3 and LC3 puncta of HUVEC cells. RT-qPCR (Real-Time Quantitative Polymerase Chain Reaction) tested the expressions of p53, p21, IL (Interleukin)-1ß, IL-6 (Interleukin-6), IL-8 (Interleukin-8), and MCP-1 (Monocyte Chemoattractant Protein-1). CCK-8 (Cell Counting Kit-8) assessed the HUVEC cell viability. RESULTS: Here, we reported that amonafide resulted in an increased proportion of SA-ß-Gal positive cells, high expression of aging-related proteins (p53 p < 0.05; p16 p < 0.05), and aging-related genes (p53 p < 0.05; p21 p < 0.05; IL-1ß p < 0.05; IL-6 p < 0.05; IL-8 p < 0.05; MCP-1 p < 0.05) on the 3rd day. Mechanistically, amonafide could cause an increase in the levels of the mTOR (p < 0.05) on days 1 and 3, and p62 protein (p < 0.05) on day 1, and a decline in LC3II (microtubule-associated protein1 light chain 3Ⅱ)/LC3I levels (p < 0.05) on day 3, which is associated with the regulation of senescence. Additionally, the viability of HUVECs (human umbilical vein endothelial cells) was significantly inhibited by amonafide starting with a concentration of 0.8 µm (p < 0.05). CONCLUSIONS: We first discovered that amonafide caused normal cellular senescence in our experiments. Amonafide-induced cellular aging by inhibiting autophagy and activating the mTOR pathway. The findings may offer new strategies for managing adverse reactions to amonafide.

Human papillomavirus and p53 status define three types of vulvar squamous cell carcinomas with distinct clinical, pathological, and prognostic features.

INTRODUCTION: Based on their etiological relationship with human papillomavirus (HPV), the 2020 WHO classification has divided vulvar squamous cell carcinomas (VSCC) into two distinct types, HPV-associated and HPV-independent, and HPV-independent tumours have recently been divided according to p53 status. Nevertheless, the clinical and prognostic significance of this classification has not been clearly established. We analysed the differential clinical, pathological, and behavioural characteristics of these three types of VSCC in a large series of patients. METHODS AND RESULTS: VSCC samples from patients who underwent primary surgery at the Hospital Clinic of Barcelona, Spain, during a 47-year period (January 1975 to January 2022) were analysed (n = 190). HPV detection, p16, and p53 immunohistochemical staining were evaluated. We also analysed recurrence-free survival (RFS) and disease-specific survival (DSS). Thirty-three tumours (17.4%) were HPV-associated and 157 (82.6%) HPV-independent. Of these, 20 showed normal and 137 abnormal p53 expression. The two types of HPV-independent tumours showed worse RFS in the multivariate analysis (hazard ratio [HR] = 3.63; P = 0.023 for the HPV-independent p53 normal VSCC and HR = 2.78; P = 0.028 for the HPV-independent p53 abnormal VSCC). Although the differences were not significant, HPV-independent VSCC had worse DSS than HPV-associated VSCC. Although patients with HPV-independent p53 normal tumours had worse RFS than patients with HPV-independent p53 abnormal tumours, the DSS was better for the former group. Only advanced FIGO stage was associated with worse DSS in multivariate analysis (HR = 2.83; P = 0.010). CONCLUSION: The association of HPV and p53 status have prognostic implications, reinforcing a three-tier molecular classification of VSCC (HPV-associated VSCC, HPV-independent VSCC with normal p53, HPV-independent VSCC with abnormal p53).

Urothelial carcinoma in situ with "overriding" features can evade detection by mimicking umbrella cells.

Urothelial carcinoma in situ (uCIS) is typically recognized by overtly malignant cells with characteristic nuclear features; multiple histologic patterns have been described. A rare "overriding" pattern, in which uCIS tumor cells extend on top of normal urothelium, has previously been mentioned in the literature, but not well described. Herein, we report 3 cases of uCIS with "overriding" features. Detailed morphologic evaluation revealed somewhat subtle cytologic atypia: variably enlarged hyperchromatic nuclei and scattered mitotic figures but with abundant cytoplasm and limited to superficial urothelium. Immunohistochemical (IHC) analysis showed a distinctive diffuse positive aberrant p53 pattern, limited to the atypical surface urothelial cells; these cells also showed CK20+, CD44-, and increased Ki-67. In 2 cases, there was a history of urothelial carcinoma and adjacent conventional uCIS. In the third case, the "overriding" pattern was the first presentation of urothelial carcinoma; therefore, next-generation sequencing molecular testing was also performed, revealing pathogenic mutations in TERTp, TP53, and CDKN1a to further support neoplasia. Notably, the "overriding" pattern mimicked umbrella cells, which normally line surface urothelium, can have abundant cytoplasm and more variation in nuclear and cell size and shape, and show CK20+ IHC. We therefore also evaluated umbrella cell IHC patterns in adjacent benign/reactive urothelium, which showed CK20+, CD44-, p53 wild-type, and very low Ki-67 (3/3). We also reviewed 32 cases of normal/reactive urothelium: all showed p53 wild-type IHC in the umbrella cell layer (32/32). In conclusion, caution is warranted to avoid overdiagnosis of usual umbrella cells as CIS; however, "overriding" uCIS should be recognized, may have morphologic features that fall short of the diagnostic threshold of conventional CIS, and requires further study.

CMTM6 recruits T cells within the endocervical adenocarcinoma microenvironment and suppresses cell proliferation via the p53 pathway.

Endocervical adenocarcinoma (ECA), harboring poor prognosis, is divided into human papilloma virus (HPV)-associated adenocarcinoma (HPVA) and non-HPVA (NHPVA), each consisting of a heterogeneous immune microenvironment. We aim to examine the effect of CKLF-like MARVEL transmembrane domain 6 (CMTM6), a key regulator of PD-L1, on ECA. Immunohistochemistry and RNA-sequencing (RNA-seq) were used to detect CMTM6, Programmed death ligand 1 (PD-L1), and immune cells biomarkers levels in tumors. RT-qPCR and Western Blotting were used to detect the mRNA and protein level changed in cells. The expression of CMTM6 in ECA is upregulated compared to cervical squamous cell carcinoma tissues. More infiltrating T cells were observed in CMTM6high ECA tissues, especially in CMTM6high HPVA. Higher expression of CMTM6 is associated with a higher rate of infiltrating CD8+ T cells in HPVA, but not in NHPVA. ECA patients were divided into three groups according to the co-expression status of CMTM6 and PD-L1(CPS) . Patients with CMTM6high /PD-L1(CPS+) had the longest OS and DFS, especially in NHPVA patients. Moreover, knock down of CMTM6 promotes ECA cell proliferation via the p53 pathway. CMTM6 recruits T cells, suppresses ECA cell proliferation via the p53 pathway and can be used as a novel prognostic indicator for ECA patients.

Quantitative p53 immunostaining aids in the detection of prevalent dysplasia.

AIMS: The lack of accepted scoring criteria has precluded the use of p53 in routine practice. We evaluate the utility of automated quantitative p53 analysis in risk stratifying Barrett's oesophagus (BE) patients using non-dysplastic BE (NDBE) biopsies in a multicentric cohort of BE progressor (P) and non-progressor (NP) patients. METHODS: NDBE biopsies prior to the diagnosis of advanced neoplasia from 75 BE-P, and index and last surveillance biopsies from 148 BE-NP were stained for p53, and scored digitally as 1+, 2+ and 3+. A secondary cohort of 30 BE-P was evaluated. RESULTS: Compared with BE-NP, BE-P was predominantly men (p=0.001), ≥55 years of age (p=0.008), with longer BE segments (71% vs 33%; p<0.001). The mean number of 3+p53 positive cells and 3+ positive glands were significantly more in BE-P versus BE-NP NDBE biopsies (175 vs 9.7, p<0.001; 9.8 vs 0.1; p<0.001, respectively). At a cut-off of ≥10 p53 (3+) positive cells, the sensitivity and specificity of the assay to identify BE-P were 39% and 93%. On multivariate analysis, scoring p53 in NDBE biopsies, age, gender and length of BE were significantly associated with neoplastic progression. 54% of patients classified as prevalent dysplasia showed an abnormal p53 immunohistochemical stain. These findings were validated in the secondary cohort. CONCLUSIONS: Automated p53 analysis in NDBE biopsies serves as a promising tool for assessing BE neoplastic progression and risk stratification. Our study highlights the practical applicability of p53 assay to routine surveillance practice and its ability to detect prevalent dysplasia.

Mass Spectrometry Proteomic Analysis of Four p53 Patterns in Differentiated Vulvar Intraepithelial Neoplasia.

OBJECTIVE: The histopathologic diagnostic criteria of differentiated vulvar intraepithelial neoplasia (dVIN), the precursor of human papillomavirus-independent squamous cell carcinoma, are basal atypia, a negative or non-block-positive p16, and a supportive p53 immunohistochemistry (IHC). Several different patterns of supportive p53 IHC have been described. This study aims to determine the relationship between p53 IHC patterns and mass spectrometry analysis of cellular proteins in dVIN. METHODS: Four patterns of p53 IHC were studied: overexpression, cytoplasmic, wild type, and intermediate expression between wild type and overexpression. For each pattern, tissue samples of 4 examples were subjected to mass spectrometry. RESULTS: The protein profile within each p53 IHC pattern shared common features. Each of the 4 p53 patterns had a distinguishable protein profile when compared with the other 3 patterns. CONCLUSIONS: The distinguishable protein profiles in different p53 IHC patterns suggest diverse mechanisms of TP53 dysfunction. Subtyping dVIN by p53 IHC is worthy of further study because varied protein expression profiles may translate into different clinical behavior.

The association between ambient particulate matter exposure and the telomere-mitochondrial axis of aging in newborns.

BACKGROUND: Particulate matter (PM) is associated with aging markers at birth, including telomeres and mitochondria. It is unclear whether markers of the core-axis of aging, i.e. tumor suppressor p53 (p53) and peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), are associated with prenatal air pollution and whether there are underlying mechanisms. METHODS: 556 mother-newborn pairs from the ENVIRONAGE birth cohort were recruited at the East Limburg Hospital in Genk (Belgium). In placenta and cord blood, telomere length (TL) and mitochondrial DNA content (mtDNAc) were measured using quantitative real-time polymerase chain reaction (qPCR). In cord plasma, p53 and PGC-1α protein levels were measured using ELISA. Daily ambient PM2.5 concentrations during gestation were calculated using a spatial temporal interpolation model. Distributed lag models (DLMs) were applied to assess the association between prenatal PM2.5 exposure and each molecular marker. Mediation analysis was performed to test for underlying mechanisms. RESULTS: A 5 µg/m3 increment in PM2.5 exposure was associated with -11.23 % (95 % CI: -17.36 % to -4.65 %, p = 0.0012) and -7.34 % (95 % CI: -11.56 % to -2.92 %, p = 0.0014) lower placental TL during the entire pregnancy and second trimester respectively, and with -12.96 % (95 % CI: -18.84 % to -6.64 %, p < 0.001) lower placental mtDNAc during the third trimester. Furthermore, PM2.5 exposure was associated with a 12.42 % (95 % CI: -1.07 % to 27.74 %, p = 0.059) higher cord plasma p53 protein level and a -3.69 % (95 % CI: -6.97 % to -0.31 %, p = 0.033) lower cord plasma PGC-1α protein level during the third trimester. Placental TL mediated 65 % of the negative and 17 % of the positive association between PM2.5 and placental mtDNAc and cord plasma p53 protein levels, respectively. CONCLUSION: Ambient PM2.5 exposure during pregnancy is associated with markers of the core-axis of aging, with TL as a mediating factor. This study strengthens the hypothesis of the air pollution induced core-axis of aging, and may unravel a possible underlying mediating mechanism in an early-life epidemiological context.

Defining an abnormal p53 immunohistochemical stain in Barrett's oesophagus-related dysplasia: a single-positive crypt is a sensitive and specific marker of dysplasia.

AIMS: p53 is an independent risk stratification marker in Barrett's oesophagus (BE), but no universally accepted definition exists for abnormal p53 staining. Herein, we assess p53 stains in two cohorts to: (1) define abnormal p53 staining in BE-related dysplasia (BERD) and (2) assess the specificity and sensitivity of this cut-point for the diagnosis of dysplasia. METHODS: Cohort 1 (n = 313) included (1) dysplastic BE biopsies, (2) prior non-dysplastic BE (NDBE) biopsies from the same patients and (3) NDBE biopsies from patients who never progressed to dysplasia. Cohort 2 (n = 191) consisted of BE biopsies in which p53 staining aided in diagnosing dysplasia. Automated p53 staining quantification was performed on cohort 1. A semiquantitative p53 analysis, performed on both cohorts, included: (1) number of strongly positive glands, (2) strong glandular surface staining, (3) percentage of strongly positive glands and (4) null phenotype. RESULTS: NDBE biopsies from cohort 1 patients who progressed to dysplasia were more likely to show p53 positivity than non-progressors (16.9 versus 0.6%) (P = 0.0001). The optimal quantitative cut-point for distinguishing dysplastic from never-dysplasia biopsies was 10 strongly positive cells. By semiquantitative analysis, a single strongly p53-positive gland distinguished dysplastic from never-dysplasia BE (sensitivity 98.6%, specificity 99.4%). The semiquantitative and quantitative analyses correlated (P = 0.0001). In cohort 2, the sensitivity and specificity for BERD of ≥ 1 strongly positive p53 gland were 86.0 and 88.6%. CONCLUSIONS: A single strongly positive p53 gland is sensitive and specific for BERD. Automated p53 analysis may reduce subjectivity associated with the diagnosis of BERD.

Application of NGS molecular classification in the diagnosis of endometrial carcinoma: A supplement to traditional pathological diagnosis.

OBJECTIVE: This study aims to demonstrate the advantages of NGS molecular classification in EC diagnosis and to assess whether molecular classification could be performed on curettage specimens and its concordance with subsequent hysterectomy specimens. METHODS: 80 patients with hysterectomy specimens and 35/80 patients with paired curettage specimens were stratified as POLE mut, MSI-H, TP53 wt, or TP53 abn group by NGS panel. Histotype, tumor grade, IHC results, and other pathological details were taken from original pathological reports. RESULTS: The correlation analysis of 80 patients with hysterectomy specimens between NGS molecular classification and clinicopathological characters displayed that the POLE mut group was associated with EEC (87.5%) and TP53 abn subtype was correlated to a later stage (Stage II-IV, 47.6%), G3 (76.2%), serous histology (61.9%) and myometrial invasion ≥50% (47.6%). A favorable concordance (31/32, 96.9%) was shown in MSI analysis and MMR IHC results, and the agreement rate of p53 IHC and TP53 mutation was 81.5% (53/65). Compared with the p53 IHC abnormal group, the TP53 mutation group had a higher correlation with high-risk factors. A high level of concordance (31/35, 88.0%) of NGS molecular classification was achieved between curettage specimens and hysterectomy specimens while grade and histotype (including unclassified group) from curettage specimens and hysterectomy specimens showed only moderate levels of agreement, 54.3% (19/35) and 68.6% (24/35), respectively. CONCLUSION: NGS molecular classification achieved on curettage samples showed high concordance with the final hysterectomy specimens, demonstrating superior to the conventional pathological assessment of grade and histotype and potential utilization in clinical practice.

A systematic review and meta-analysis of CK20, CD44, Ki67 and p53 as immunohistochemical markers in bladder carcinoma in situ.

BACKGROUND: Urothelial dysplasia and carcinoma in situ (CIS) are related to recurrence and progression of urothelial carcinoma. Differentiating CIS and dysplasia from reactive atypia is often difficult based only on histological features. The integration of histological findings with immunohistochemistry is used in routine practice to make a diagnosis of CIS and, for this purpose, the immunohistochemical markers CK20, CD44, Ki67 and p53 are used to supplement histology. In this work, we aimed to assess CK20, CD44, Ki67 and p53 as immunohistochemical markers in patients with CIS through a systematic review and meta-analysis. MATERIALS AND METHODS: A systematic review was performed by searching electronic databases for English-language studies published from January 2010 to April 2021. Studies were considered eligible if they evaluated the CK20, CD44, Ki67 and p53 expression in CIS. RESULTS: In total, 15 references were suitable for quantitative review. The overall rate of CK20, CD44, Ki67 and p53 expression in CIS was 43%, 31%, 44%, 38%, respectively. CONCLUSIONS: Our study supports the 2014 International Society of Urologic Pathology consensus that histological assessment remains the gold standard to diagnose urothelial CIS and suggests that a very close correlation between morphological, immunohistochemical and clinical data is essential to provide the best management for patients with bladder carcinoma.

Evaluation of utility of immunohistochemistry markers as a tool for objective diagnosis of low-grade myelodysplastic syndrome in routine reporting: Prospective observational study.

Purpose: Diagnosis of myelodysplastic syndrome (MDS) primarily relies on the detection of morphological dysplasia in bone marrow. It is subjective and many studies have reported lack of interobserver agreement in reporting. Biopsy is preferred specimen for megakaryocyte assessment. We studied 43 bone marrow biopsies from 40 suspected MDS patient having persistent undiagnosed cytopenia. Utility of immunohistochemistry (IHC) with CD61 and p53 in detecting low-grade MDS was analyzed over routine morphology. Method and Results: Total number of megakaryocytes and number of dysplastic megakaryocytes seen on CD61 IHC was significantly higher than that on H and E stain (P value < 0.05) Out of total 43 biopsies, 13 [30.2%] cases showed dysplastic megakaryocytes that were confirmed by interobserver agreement after IHC. From 30 cases with no significant dysplasia on morphology, 21/43 [48.8%] cases showed >10% dysplastic megakaryocytes on CD61 (P value 0.0001). Nine cases showed no significant dysmegakaryopoiesis with either H and E or CD61 IHC. Fourteen cases could meet higher cut off (30%) of dysmegakaryopoiesis with CD 61 IHC. Out of total 34 cases showing significant dysplasia 7 cases (20.6%) showed positivity for p53 on IHC, which is little less than that reported in low-grade MDS. Conclusion: CD61 IHC is helpful in making correct diagnosis of MDS in cases with minimal dysplasia and should be performed before excluding possibility of MDS on morphology in a patient with undiagnosed cytopenia. IHC is cost effective tool for MDS diagnosis in developing world where access to extensive flow cytometery and molecular testing is limited.