P53 Immunostaining Research Articles

Differential immunohistochemical expression of hTERT in lung cancer patients with and without idiopathic pulmonary fibrosis

BackgroundHuman telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase enzyme, which adds nucleotides to telomeres and counteracts their length shortening. The development of a telomere maintenance mechanism represents a hallmark of cancer. On the other hand, idiopathic pulmonary fibrosis (IPF) is associated with mutations in telomerase genes and shorter telomeres. IPF is frequently complicated with lung cancer. AimTo investigate the expression of hTERT in lung cancer with co-existing IPF and to compare with lung cancer without fibrosis. MethodsDiagnostic lung cancerous biopsies were retrieved from 18 patients with lung cancer and concomitant IPF, as well as 18 age and gender matched controls with lung cancer without pulmonary fibrosis. The expression of hTERT was studied with immunohistochemistry. ImajeJ software was used to quantitate subcellular stain intensity. Immunohistochemical investigation of two senescence-associated markers, p16 and p21, was also performed in all 36 cases. ResultsBoth groups highly expressed hTERT, without significant difference (100% vs 95%, p = 0.521). Evaluation of p16 and p21 immunostaining revealed negative to minimal immunoreactivity in both groups. hTERT localization exhibited higher median nuclear intensity in the group of lung cancer with IPF (0.62 vs 0.45, p = 0.016), while cytoplasmic intensity did not differ significantly (0.17 vs 0.15, p = 0.463). Higher median nuclear intensity was also correlated with small cell lung cancer subtype in the whole study sample (0.69 vs 0.45, p = 0.09). ConclusionhTERT is highly expressed in lung cancer with concomitant IPF, but with differential localization compared to lung cancer without IPF, implying differences in pathogenicity and requiring further investigation.

Validation of p16 immunocytochemical staining on alcohol‐fixed aspirate smears: A single‐institutional study with review of the literature

Determining human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma can have a significant impact on treatment and clinical outcomes. Because fine-needle aspiration (FNA) is typically an initial diagnostic modality in a patient workup for primary or suspected metastatic disease, immunostaining for p16 on FNA material is a promising option to determine HPV tumor status, possibly avoiding biopsies or excisions. In this study, the authors investigated the possibility of using alcohol-fixed smears as a reliable alternative for reporting p16 status. Twenty HPV-associated tumors and 20 non-HPV-associated tumors were identified using the gold-standard histologic cutoff for positivity of ≥70% strong nuclear and cytoplasmic staining. Matched FNA specimens were identified for comparison staining, and a positive p16 result was rendered on a single aspirate smear using the same cutoff of ≥70%. On alcohol-fixed cytology smears, 16 of 20 (80%) HPV-associated tumors showed positive p16 staining in ≥70% tumor cells. Four cases showed lower level (30%-60%) nuclear and cytoplasmic staining. Nineteen of 20 (95%) non-HPV-associated tumors showed no or minimal p16 staining (0%-10%), and 1 case had a p16-equivocal cytology result. The authors performed immunocytochemical validation for p16 using alcohol-fixed smears and observed promising results, offering this technique as a potential alternative to formalin-fixed tissue in the appropriate clinical context. By using a positive staining cutoff of ≥70%, this technique offers 80% sensitivity and 95% specificity for detecting HPV-associated tumors. Although it was not performed in the current study, HPV-specific testing on available formalin-fixed, paraffin-embedded tissue should be considered in cases with equivocal or negative p16 staining.

Mesonephric-like Adenocarcinoma of the Ovary: Clinicopathological and Molecular Characteristics.

Mesonephric-like adenocarcinoma (MLA) arising in the ovary is a rare malignant tumor of the female genital tract. Although the clinicopathological and molecular characteristics of uterine MLA have been accumulated, those of ovarian MLA have not been firmly clarified. In this study, we investigated the clinicopathological, immunohistochemical, and genetic features of five ovarian MLAs. A review of electronic medical records and pathology slides, immunostaining, and targeted sequencing was performed. On imaging, ovarian MLA presented as either a mixed solid and cystic mass or a purely solid mass. One, three, and one patient were diagnosed as having FIGO stage IA, IC, and II MLA, respectively. Four patients with stage IC–II tumor underwent post-operative adjuvant chemotherapy. Three of the four patients whose follow-up information was available did not experience recurrence. In contrast, the remaining patient with stage IA tumor who did not receive any adjuvant treatment developed multiple metastatic recurrences at post-operative 13 months. Histologically, ovarian MLAs characteristically displayed architectural diversity, compactly aggregated small tubules, and eosinophilic intraluminal secretions. Four tumors were found to be associated with endometriotic cysts. Two cases showed some areas of high-grade nuclear atypia, brisk mitotic activity, and necrosis. Immunohistochemically, all cases showed positive immunoreactivities for at least three of the four examined mesonephric markers (GATA3, PAX2, TTF1, and CD10), lack of WT1 expression, non-diffuse p16 immunoreactivity, and wild-type p53 immunostaining pattern. Targeted sequencing analysis revealed that all four examined cases harbored pathogenic KRAS mutations: p.G12V (2/4); p.G12D (1/4); and p.G12C (1/4). In addition, we reviewed the previous literature reporting 60 cases of ovarian MLA. Our findings corroborate those of the previous data regarding the clinical presentation, histological features, immunophenotypes, and molecular alterations. Our observations should encourage pathologists to recognize and accurately diagnose this rare but distinct entity.

Hyperglycemia Induces Meibomian Gland Dysfunction.

PurposePatients diagnosed with diabetes are inclined to have abnormalities on stability of tear film and disorder of meibomian gland (MG). This study aims to explore the pathological change of MG induced by diabetes in a rat model.MethodsSprague-Dawley (SD) rats were intraperitoneally injected with streptozotocin (STZ) to establish a diabetic animal model. Lipid accumulation in MG was detected by Oil Red O staining and LipidTox staining. Cell proliferation status was determined by Ki67 and P63 immunostaining, whereas cell apoptosis was confirmed by TUNEL assay. Gene expression of inflammatory cytokines and adhesion molecules IL-1α, IL-1β, ELAM1, ICAM1, and VCAM1 were detected by RT-PCR. Activation of ERK, NF-κB, and AMPK signaling pathways was determined by Western Blot analysis. Oxidative stress-related factors NOX4, 4HNE, Nrf2, HO-1, and SOD2 were detected by immunostaining or Western Blot analysis. Tom20 and Tim23 immunostaining and transmission electron microscopy were performed to evaluate the mitochondria functional and structure change.ResultsFour months after STZ injection, there was acini dropout in MG of diabetic rats. Evident infiltration of inflammatory cells, increased expression of inflammatory factors, and adhesion molecules, as well as activated ERK and NF-κB signaling pathways were identified. Oxidative stress of MG was evident in 4-month diabetic rats. Phospho-AMPK was downregulated in MG of 2-month diabetic rats and more prominent in 4-month rats. After metformin treatment, phospho-AMPK was upregulated and the morphology of MG was well maintained. Moreover, inflammation and oxidative stress of MG were alleviated after metformin intervention.ConclusionsLong-term diabetes may lead to Meibomian gland dysfunction (MGD). AMPK may be a therapeutic target of MGD induced by diabetes.

Chlorogenic Acid Ameliorates Liver Function in Association with Bax Downregulation, P53 Downregulation and Bcl-2 Upregulation in Diabetic Wistar Rat

Hyperglycemia in Diabetes mellitus (DM) induces oxidative stress and mitochondrial disruptions in the liver, thereby triggering apoptosis through Bcl-2 family signaling. Excessive apoptosis leads to liver dysfunction. Chlorogenic acid (CGA) was elucidated in this study in preventing liver dysfunction as a progression of DM. The diabetic model was conducted in Wistar rats, divided into six groups. Blood examinations were done to measure the levels of blood glucose, SGOT, and SGPT. The liver was harvested for analysis of SOD2, Bax, and Bcl-2 mRNA. A paraffin section was used for p53 immunostaining. Liver dysfunction occurred in the DM 2 months group indicated by higher levels of SGPT and SGOT, higher expression of Bax, and lower expression of Bcl-2 compared to the control group. Giving CGA 12.5 mg/kgBW ameliorated blood glucose and liver enzyme levels, which were associated with lower expression of Bax, lower signaling of p53, and higher expression of Bcl-2 compared to DM 2 months group. Administration of CGA did not affect SOD2 expression in diabetic rats. CGA may attenuate liver dysfunction in diabetic rats through downregulation of Bax and p53, and upregulation of Bcl-2 signaling.

Significance of p53 immunostaining in mesothelial proliferations and correlation with TP53 mutation status

p53 immunohistochemistry has long been proposed for the separation of benign from malignant mesothelial proliferations, with the older literature suggesting that any degree of positivity supported a diagnosis of mesothelioma. However, using modern immunohistochemistry platforms in other organ systems, notably gynecologic tumors, it has become clear that p53 staining can represent wild-type protein, and only specific staining patterns (absent, overexpression, or cytoplasmic expression) are indicative of a TP53 mutation. We applied these principles to two tissue microarrays containing 94 mesotheliomas and 66 reactive mesothelial proliferations. Seven/65 (11%) epithelioid mesotheliomas showed aberrant staining (four absent and three overexpression patterns) as did 5/29 (17%) of sarcomatoid mesotheliomas (all overexpression patterns). We sequenced the TP53 gene (exons 2–11) in five of the epithelioid and three of the sarcomatoid cases with aberrant staining as well as 12 epithelioid and eight sarcomatoid mesotheliomas with wild-type staining. All three sarcomatoid cases with aberrant staining showed mutated TP53, as did three of the epithelioid cases; in two of the epithelioid cases no mutation was detected, most likely because of large deletions not detected by this assay. In contrast, none of the 20 mesotheliomas with wild-type staining contained mutated TP53. We conclude that absent or overexpression p53 staining patterns can be used as a marker of a malignant vs. a benign mesothelial proliferation. The sensitivity of p53 staining by itself is low, but here addition of p53 to BAP1/MTAP staining increased sensitivity from 72 to 81% for epithelioid and 38 to 50% for sarcomatoid mesotheliomas.

Role of Immunohistochemical Expression of p53 in Intestinal Epithelial Cells to Detect Dysplasia in Patients with Inflammatory Bowel Disease

Introduction: Inflammatory Bowel Disease (IBD) has a genetic predisposition, in which patients are more prone to develop colorectal carcinoma. Dysplasia, a precursor of malignancy, can be very difficult to detect based on histopathological features alone. Tumour suppressor gene, p53, is overexpressed in IBD even when there is no histological evidence of dysplasia. Therefore, p53 can be used as a tissue biomarker for routine surveillance to initiate treatment for prevention of carcinoma. Aim: To identify the diagnostic utility of p53 immunohistochemistry in the detection of dysplasia in patients with IBD. Materials and Methods: This cross-sectional diagnostic study was conducted in the Department of Pathology at Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla, Kerala, India, from June 2016 to June 2018. Total 31 cases of intestinal biopsies in patients with chronic bloody diarrhoea were selected for the study. Immunohistochemical (IHC) staining using anti p53 antibody was done in all the cases to detect the expressed protein. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) version 20.0. Significance of p53 immunostains was done using Fischer’sexact test. Results: The age group of patients with IBD ranged from 15 years to 73 years with a bimodal age peak between 20-29 years and 60-69 years. Male predominance was seen among 20 cases while 11 cases were among females. Negative staining for p53 was seen in 28 cases which were negative for dysplasia histopathologically. Only one case which was histopathologically positive for dysplasia and one of the negative cases of dysplasia showed p53 positivity. Another case which was histopathologically positive for dysplasia showed negative staining for p53. Sensitivity of p53 in this study was 50% while the specificity was 96.55%. Conclusion: The finding of positive p53 expression in dysplasia positive case follows the fact that p53 mutations found in dysplastic mucosa precedes progression to neoplasia. In contrast, positive p53 expression in a case that was negative for dysplasia histopathologically, should be handled cautiously and warrants regular follow-up in order to prevent neoplastic progression. However, diagnosis of dysplasia should not be made in areas of active inflammation as inflammatory changes can lead to regenerative atypia which could result in over interpretation of histological features and attributing it to dysplasia.

Low frequency of HPV positivity in breast tumors among patients from south-central Poland

BackgroundSome studies suggest that Human Papilloma Virus (HPV) infection is important factor in carcinogenesis of breast tumors. This study’ objective was to analyze HPV prevalence in breast cancers of patients from south-central Poland.Materials and methodsThe study was performed based on archival paraffin embebbed and formalin fixed blocks in the group of 383 patients with breast cancer. HPV prevalence and its genotype were assessed, respectively by: nested PCR (with two groups of primers: PGMY09/PGMY11 and GP5+/GP6+), quantitative PCR (qPCR). Tumors were classified as HPV positive in case of at least one positive result in nested PCR and positive results in genotyping procedure. For all HPV positive tissues P16 immunostaining was applied in order to confirm active viral infection.ResultsIn the group of 383 breast cancers, HPV positivity was found in 17 samples (4.4%) in nested PCR. All these samples were subjected to HPV genotyping. This analysis revealed presence of HPV type 16 into two tumors (0.5%). In these two cancers, P16 overexpression was reported.ConclusionIn breast tumors of patients from south-central Poland in Poland, HPV positivity is demonstrated in very low percentage of cases.

P53 expression in repair/reactive renal tubular cells: A potential pitfall leading to a false-positive diagnosis of urine cytology.

Backgroundp53 immunostaining is routinely used as a surrogate marker for TP53 mutational status. In urine cytology, p53 immunocytochemistry is reportedly useful in detecting urothelial carcinoma cells as well as in improving the detection sensitivity and specificity. However, to the best of our knowledge, p53 expression in repair/reactive renal tubular cells (RRTCs) from urine cytologic specimens has not been assessed to date.MethodsWe evaluated the immunoexpression of p53 and homogentisate 1,2‐dioxygenase (HGD) antibody, a renal tubular cells marker, in RRTCs using voided urine and renal biopsy samples from 80 patients who were histologically diagnosed with glomerular disease.ResultsRepair/reactive renal tubular cells were detected in 68 (68/80, 85%) samples at a mean count of 141.1 cells per sample (range, 5–4220). Immunocytochemical analysis found p53‐positive RRTCs in all the samples (68/68, 100%) with an average p53 positivity rate of RRTCs per sample at 47.7% (range, 3.8%–96.5%). Of the 68 p53‐positive RRTC samples, 38 (55.9%) included cells that were HGD positive for cytoplasm. Similarly, renal biopsy analysis revealed p53‐positive RRTCs in all the specimens (68/68, 100%). All 68 (100%) cases showed RRTCs that were positive for both p53 and HGD.ConclusionTo avoid false positives of p53 immunocytochemistry, cytologists must consider the fact that RRTCs from patients with glomerular disease are positive for p53.

P16 immunostaining in fine-needle aspirations of the head and neck: determining the optimal positivity threshold in HPV-related squamous cell cancer.

There is no consensus for interpretation of p16 immunohistochemistry (IHC) in cytology preparations. Our study aims to assess p16 IHC staining in formalin-fixed cytology cell blocks (CBs) from head and neck squamous cell carcinoma (HNSCC) fine-needle aspiration (FNA) specimens in comparison with surgical pathology p16 staining and to determine the reproducibility of p16 IHC scoring in CBs. A total of 40 FNAs from 2014 to 2019 of HNSCC with p16 IHC were obtained. CB p16 staining was scored independently by 5 cytopathologists as interval percentages of tumor cell positivity. Receiver operating characteristic (ROC) curves were examined to determine optimal cutoffs for each pathologist based on sensitivity and specificity values. Gwet's coefficient (AC1) was calculated to assess inter-rater reliability. Greater than 10% was the lowest threshold to reach 100% specificity with high sensitivity (55%-84%) in all 5 raters. Rater performances were similar, with areas under the curve (AUCs) ranging from 0.89 to 0.95. Using the >10% threshold, Gwet's AC1 = 0.72 (95% CI: 0.56-0.89). Diagnostic performance improved further when low-cellularity cases were excluded, with AUC ranging from 0.94 to 0.99 and Gwet's AC1 = 0.79 (95% CI: 0.61-0.98). p16 IHC performed on cytology CBs can serve as a surrogate marker for the detection of HPV with high sensitivity and specificity levels. Using a threshold lower than that recommended for surgical pathology for the interpretation of p16 positivity may be appropriate for FNA cytology CB preparations. All cytopathologists in our study displayed reproducible high sensitivity and specificity values at the >10% threshold.

HPV-Related Multi-Phenotypic Sinonasal Carcinoma with Aggressive Clinical Behavior; A Rare Case

Abstract Introduction/Objective Human Papilloma Virus (HPV)-related multiphenotypic sinonasal carcinoma, previously known as HPV-related sinonasal carcinoma with adenoid cystic carcinoma-like features, is a rare type of sinonasal carcinoma with both epithelial-derived and salivary gland-type morphologic features. It is associated with high-risk HPV, but lacks MYB gene rearrangements. Methods/Case Report We report a case of a 59-year-old male who presented with a rapidly growing sinonasal mass. On MRI, a left nasal cavity lesion was identified growing laterally along the frontal process of the maxilla, extending into the middle meatus and into the maxillary sinus. Patient underwent a complex left medial maxillectomy, spheno- ethmoidectomy, and sinusotomy. On gross evaluation, the left inferior turbinate and sidewall demonstrated a 4 cm unremarkable turbinate with attached friable soft tissue. Microscopic examination revealed sections of carcinoma with various architectural patterns comprised of foci with adenoid cystic carcinoma-like morphology, basaloid squamous cell carcinoma and adenocarcinoma. The tumor showed positive immunostaining for P40, but focal reactivity to S100 and rare scattered reactivity with CD117. INI-1 immunostain was retained in tumor cells. P16 immunostain was strong and diffuse and high-risk cocktail HPV RNA ISH was positive. However, MYB FISH testing was equivocal. Morphologic and immunophenotypic findings were consistent with HPV-related multiphenotypic sinonasal carcinoma. The tumor involved the olfactory nerve fibers requiring a skull base resection and showed extension into the dura mater. Results (if a Case Study enter NA) NA Conclusion HPV related multiphenotypic sinonasal carcinoma is a recently described entity that can pose significant diagnostic challenge. It typically has an indolent clinical course with potential for late recurrences. This case study highlights the potential aggressive nature of this type of sinonasal carcinoma, despite association with high-risk HPV, and use of ancillary testing in aiding diagnosis.

Predictive Role of the p16 Immunostaining Pattern in Atypical Cervical Biopsies with Less Common High Risk HPV Genotypes.

P16 immunostaining is considered a useful surrogate of transcriptionally active high-risk (hr) HPV infection. Only strong and widespread “block-like” immunoreactivity is considered specific, whereas weak/focal p16 positive immunostaining is considered not specific, and follow-up and HPV molecular detection is not indicated. The aim of the study was to evaluate the presence of HPV DNA and Ki67 immunostaining in 40 cervical atypical biopsies (CALs) with mild and focal histological features suggestive of HPV infection—20 cases with weak/focal p16 positive immunoreactivity and 20 cases negative for p16 expression. In 16/20 weak/focal p16 positive CALs (80%), the INNO-LiPA HPV genotyping detected hrHPV genotypes (HPV 31, 51, 56, 59, 26, 53, 66, 73, and 82). Co-infection of two or more hrHPV genotypes was often evidenced. HPV16 and 18 genotypes were never detected. Ki67 immunostaining was increased in 10/20 cases (50%). In 19/20 p16 negative CALs, hrHPV infection was absent and Ki67 was not increased. These results suggest that weak/focal p16 immunostaining represents the early stage of transcriptionally active infection, strongly related to the presence of less common hrHPV genotypes, probably with a slower transforming power, but with a potential risk of progression if the infection persists. HPV DNA genotyping and follow-up could be useful in these cases to verify if they are able to evolve into overt dysplastic changes and to improve knowledge of less common hrHPV genotypes.

Expression of p16 Within Myenteric Neurons of the Aged Colon: A Potential Marker of Declining Function.

Human colonic neuromuscular functions decline among the elderly. The aim was to explore the involvement of senescence. A preliminary PCR study looked for age-dependent differences in expression of CDKN1A (encoding the senescence-related p21 protein) and CDKN2A (encoding p16 and p14) in human ascending and descending colon (without mucosa) from 39 (approximately 50: 50 male: female) adult (aged 27–60 years) and elderly donors (70–89 years). Other genes from different aging pathways (e.g., inflammation, oxidative stress, autophagy) and cell-types (e.g., neurons, neuron axonal transport) were also examined. Unlike CDKN1A, CDKN2A (using primers for p16 and p14 but not when using p14-specific primers) was upregulated in both regions of colon. Compared with the number of genes appearing to upregulate in association with temporal age, more genes positively associated with increased CDKN2A expression (respectively, 16 and five of 44 genes studied for ascending and descending colon). Confirmation of increased expression of CDKN2A was sought by immunostaining for p16 in the myenteric plexus of colon from 52 patients, using a semi-automated software protocol. The results showed increased staining not within the glial cells (S100 stained), but in the cytoplasm of myenteric nerve cell bodies (MAP2 stained, with identified nucleus) of ascending, but not descending colon of the elderly, and not in the cell nucleus of either region or age group (5,710 neurons analyzed: n = 12–14 for each group). It was concluded that increased p16 staining within the cytoplasm of myenteric nerve cell bodies of elderly ascending (but not descending) colon, suggests a region-dependent, post-mitotic cellular senescence-like activity, perhaps involved with aging of enteric neurons within the colon.

MULTIFOCAL PULMONARY GRANULAR CELL TUMOR

TOPIC: Lung Cancer TYPE: Medical Student/Resident Case Reports INTRODUCTION: Granular cell tumors, also known as Abrikossoff tumors, are a rare group of Schwannian neoplasms typically found in the skin and tongue, but also occur in the lung. Granularity of the cells is due to accumulation of secondary lysosomes in the cytoplasm. Only 0.5-2% of pulmonary granular cell tumors (pGCT) are malignant, but some benign cases show similar behavior. Here, we describe a diagnostically challenging case of multifocal pGCT. CASE PRESENTATION: A 48-year old male with history of asthma, chronic obstructive pulmonary disease, hypertension, and alcoholic steatohepatitis presented for evaluation of a right lung mass found on chest X-ray 5 years ago. He endorsed progressive dyspnea poorly controlled with beclomethasone and albuterol inhalers. He had history of tobacco smoking with 31 pack-years, as well as alcohol abuse. He worked in a shipyard for 3 years with exposure to asbestos and welding fumes. Computed tomography of the chest showed a 4.1-cm solid mass in the right lower lobe, as well as a 1.1-cm endobronchial lesion in the right bronchus intermedius, multiple left lower lobe lesions, and mediastinal lymphadenopathy. Spirometry showed moderate obstructive disease with bronchodilator response. Biopsies of lower lobe and tracheal mucosal lesions obtained by bronchoscopy revealed sheets of epithelioid cells with abundant eosinophilic granular cytoplasm and small uniform nuclei consistent with pGCT. There was no evidence of necrosis or increased mitotic figures to suggest malignancy. Immunostaining was positive for S100, CD68, SOX10, inhibin, and PAS. He was referred to Interventional Pulmonology for laser debulking and endobronchial stenting. DISCUSSION: Determining whether pGCTs are malignant or benign can be challenging. Both types occur in all age groups. Both can cause dyspnea, cough, and pleuritic chest pain. Large tumor size is not predictive, as many malignant pGCT's are <4 cm at time of diagnosis. Additionally, 20% of benign pGCTs have multiple lung lesions. However, the presence of extrapulmonary metastases is diagnostic of malignancy. Malignant pGCTs also show 3 or more histologic features of the Fanburg-Smith criteria, including necrosis, spindling, pleomorphism, vesicular nuclei with large nucleoli, high nuclear/cytoplasmic ratio, and increased mitotic activity. Positive p53 or Ki67 immunostaining can indicate malignancy in indeterminate cases. Management of benign pGCTs is surgical. Small tumors <1.0 cm may be removed by bronchoscopic extirpation, laser therapy, or sleeve resection. Larger tumors are resected surgically. Prognosis for benign pGCT is good (2-8% recurrence) if complete margin-free removal is achieved. CONCLUSIONS: Malignant and benign pGCTs share many features that make them difficult to differentiate. Absence of extrapulmonary metastases, Fanburg-Smith criteria, and specific immunostaining helps rule out malignancy in cases with multifocal lung lesions. REFERENCE #1: Jiang M, Anderson T, Nwogu C, Tan D. Pulmonary malignant granular cell tumor. World J Surg Oncol. 2003;1(1):22. Published 2003 Oct 21. doi:10.1186/1477-7819-1-22. REFERENCE #2: Fanburg-Smith JC, Meis-Kindblom JM, Fante R, Kindblom LG. Malignant granular cell tumor of soft tissue. Diagnostic criteria and clinicopathologic correlation. Am J Surg Pathol. 1998;22:779–794. doi: 10.1097/00000478-199807000-00001. REFERENCE #3: Khansur T, Balducci L, Tavassoli M. Granular cell tumor: clinical spectrum of the benign and malignant entity. Cancer. 1987;60:220–222. DISCLOSURES: No relevant relationships by Ramiz Fargo, source=Web Response No relevant relationships by Joseph Mak, source=Admin input No relevant relationships by Bryce Mikel, source=Web Response

Endocervical adenocarcinomas classified by International Endocervical Adenocarcinoma Criteria and Classification: a clinicopathological and prognostic analysis of 286 cases

Objectives: To investigate the clinicopathological and prognostic significance of International Endocervical Adenocarcinoma Criteria and Classification (IECC) in classifying endocervical adenocarcinomas among Chinese women. Methods: A total of 286 endocervical adenocarcinomas diagnosed from January 2013 to December 2019 at the Women's Hospital, Zhejiang University School of Medicine were identified and included. The cases were reviewed and reclassified based on IECC. The histological types were correlated with p16 immunostaining, human papilloma virus (HPV) mRNA status, the clinicopathological parameters including the International Federation of Gynecologic Oncology (FIGO) stage, and clinical follow-up data. Results: The patients aged from 19 to 77 (median 47) years. There were 223 patients at FIGO stage Ⅰ, 22 at stage Ⅱ, 38 at stage Ⅲ and 3 at stage Ⅳ. The IECC types included 213 (74.5%) HPV-related adenocarcinomas (HPVA), 60 (21%) non-HPV-related adenocarcinomas (NHPVA), and 13 (4.5%) adenocarcinomas, no other specified (NOS). The major histological subtypes in HPVA and NHPVA were common type (n=156, 54.5%) and gastric type (GAC, n=46, 15.9%), respectively. The p16 positive rates in HPVA, NHPVA and adenocarcinoma, NOS were 92% (173/188), 26.6% (17/64) and 61.5% (8/13), respectively, and those of HPV mRNA hybridization in situ were 89.4% (144/161), 0/18 and 7/13, respectively. Compared to HPVA, NHPVA was more frequently associated with older age, FIGO stage Ⅱ-Ⅳ, neural involvement, lymphovascular invasion and aberrant p53 expression (P<0.05). Univariate survival analysis showed that age (>47 years), NHPVA, GAC, FIGO stage Ⅱ-Ⅳ, neural involvement, lymphovascular invasion and aberrant p53 expression were indicators for a poorer overall survival and tumor recurrence (P<0.05). Mucinous HPVA showed worse clinical outcomes compared to usual-type HPVA (P<0.01). Multivariate survival analysis demonstrated that FIGO stage Ⅱ-Ⅳ, NHPVA and aberrant p53 expression were independent indicators for poor overall survival while FIGO stage Ⅱ-Ⅳ and GAC were independently associated with tumor recurrence (P<0.05). Conclusions: The two broad IECC categories, HPVA and NHPVA, not only provide morphological links to the etiology (HPV infection), but also have significant clinicopathological and prognostic relevance.

Diagnostic value of IMP3 and p53 immunohistochemical staining in EUS-guided fine-needle aspiration for solid pancreatic tumors

We previously identified insulin-like growth factor-II messenger ribonucleic acid-binding protein 3 (IMP3) as a valuable marker to distinguish malignant from benign lesions in pancreatic solid masses. The aim of this prospective study was to evaluate the usefulness of IMP3 and p53 immunohistochemical staining in endoscopic ultrasound-guided fine-needle aspiration (EUS–FNA) samples for pancreatic solid masses. The study recruited 90 consecutive patients with pancreatic masses, including 62 pancreatic ductal adenocarcinomas (PDACs), 11 benign tumors, and 17 other tumors, who underwent EUS–FNA, and conducted IMP3 and p53 immunohistochemical staining. The main outcome measurement was improved diagnostic utility using IMP3 and p53 immunohistochemical staining. IMP3 and p53 expressions were detected in 60.8% and 49.4% of malignant lesions, 69.4% and 58.1% of PDACs, and 0% of benign lesions, respectively. In PDAC and benign tumors, the use of IMP3 and/or p53 immunostaining increased the sensitivity of cytohistological analysis from 88.7 to 93.5%, although the difference was not statistically significant. The sensitivity of histological analysis combined with that of IMP3 staining was 91.9%, which was significantly greater than that of histology alone (80.6%). The use of IMP3 and p53 immunohistochemical staining did not significantly improve the sensitivity of cytohistological analysis; however, IMP3 staining may be helpful for the histological analysis of malignant pancreatic tumors.

A Novel Treatment Strategy for Advanced Lacrimal Sac Carcinomas Confirmed by p16 Immunostaining

ABSTRACT This article aims to describe the two cases in which chemotherapy and chemoradiotherapy were effective for advanced HPV-related lacrimal sac squamous cell carcinoma and avoided the need for radical surgery. This was an interventional study of two patients with advanced lacrimal sac squamous cell carcinoma. Two patients with advanced lacrimal sac squamous cell carcinoma were treated at our University Hospital between January 2020 and February 2021. Diagnosis of HPV-related lacrimal sac carcinoma was done by p16 immunostaining and RNA in situ hybridization. Received neoadjuvant chemotherapy and chemoradiotherapy, also minimally invasive surgery to remove any residual tumor if the final response, were unfavorable. HPV-related carcinoma was decided by checking p16 and RNA status. Response was assessed by computed tomography, magnetic resonance imaging, positron emission tomography-computed tomography, and endoscopic images. Both patients had positive p16 staining also HPV RNA in situ hybridization. Received definitive chemoradiotherapy instead of radical surgery after showing a partial response to neoadjuvant chemotherapy. A complete response was achieved in one patient and the other had a partial response, leaving a small residual tumor in the nose that was successfully removed by endonasal endoscopic surgery. Cure was achieved in two patients with HPV-related lacrimal sac squamous cell carcinoma by neoadjuvant chemotherapy followed by definitive chemoradiotherapy, with only one requiring minimally invasive surgery. This is a new direction in the treatment of p16-positive lacrimal sac carcinoma, especially for advanced cases, whereby molecular biological indicators can be used to avoid highly invasive surgery and preserve quality of life without compromising prognosis.

A "Null" Pattern of p16 Immunostaining in Endometrial Serous Carcinoma: An Under-recognized and Important Aberrant Staining Pattern.

The ability to distinguish endometrial serous carcinoma (SC) from high-grade endometrioid adenocarcinoma is of great importance given their differences in prognosis and management. In practice, this distinction typically relies upon the use of a focused immunohistochemical panel including p53, p16, and mismatch repair proteins. The expression of p16 is characteristically strong and diffuse in SCs, and weak and/or patchy in many high-grade endometrioid adenocarcinomas. Here, we report a subset of SCs that are entirely negative for p16 immunostaining, a pattern we refer to as "p16 null." This pattern was identified in 2 of 63 cases of SC diagnosed at our institution-1 with histologically classic features and 1 with ambiguous high-grade histologic features. These tumors otherwise showed a SC signature by immunohistochemical and demonstrated an SC pattern of genetic mutations. No mutation in the gene for p16, cyclin-dependent kinase inhibitor 2A (CDKN2A), was identified in either case. However, molecular correlates for the absent p16 expression were present, including homozygous deletion of CDKN2A in one case and hemizygous deletion of CDKN2A with promotor hypermethylation of the remaining allele in the other case. To our knowledge, this constitutes the first report conclusively demonstrating the existence of a small subset of SCs that are completely negative by p16 immunohistochemistry, and the molecular lesions responsible for this pattern. In the context of an otherwise clinically and histologically classic example of SC, we endorse this "null" p16 staining pattern as an alternative aberrant staining pattern that should not deter one from committing to this diagnosis.

Updates on the Immunopathology in Idiopathic Inflammatory Myopathies.

To review recent advances in immunopathology for idiopathic inflammatory myopathies, focusing on widely available immunohistochemical analyses. Sarcoplasmic expression of myxovirus resistance protein A (MxA) is specifically observed in all types of dermatomyositis and informs that type I interferons are crucially involved in its pathogenesis. It is a more sensitive diagnostic marker than perifascicular atrophy. Diffuse tiny dots in the sarcoplasm highlighted by p62 immunostaining are characteristically seen in immune-mediated necrotizing myopathy. This feature is linked to a chaperone-assisted selective autophagy pathway. Myofiber invasion by highly differentiated T cells, a marker of which is KLRG1, is specific to inclusion body myositis and has a crucial role in its pathogenesis. The recent advances in immunopathology contribute to increased diagnostic accuracy and a better understanding of the underlying pathophysiology in different types of idiopathic inflammatory myopathies.

In vivo autophagy quantification: Measuring LC3 and P62 puncta in 3D image system from zebrafish larvae.

Autophagy is a central pathway in maintaining cellular homeostasis through the recycling of damaged proteins and organelles. Detection of LC3 protein levels by immunofluorescence or western blot analysis is one of the most common ways to measure autophagy. For quantitative autophagy analysis, LC3 western blot analysis is commonly used, whereas immunostaining is used for qualitative autophagy analysis. However, zebrafish larvae have a lot of proteases that rapidly degrade LC3 protein in samples. P62 is another autophagy marker that bind to damaged proteins and can reflects autophagic status. This study demonstrates a fast and accurate way to quantify autophagy from LC3 and/or P62 immunostaining images. We used a three-dimensional analysis of whole-mount LC3 immunostaining images of zebrafish larvae. Counting LC3 and P62 punctate by two dimensions can be used as a qualitative method for the analysis of autophagy. However, here we demonstrate that 3D image analysis can be used as a quantitative, rapid tool for monitoring autophagy in zebrafish larvae and avoiding drawbacks of LC3 western blot analysis.