P16 Immunohistochemistry Research Articles

A clinicopathologic and immunohistochemical study of primary and secondary breast angiosarcoma.

We aimed to study the clinicopathologic and immunohistochemical (IHC) (CD117, c-Myc, and p53) characteristics, and overall survival of primary and secondary breast angiosarcoma (BAS). This was a retrospective study of BAS cases diagnosed between 1997 and 2020 at our institution. Hematoxylin and eosin-stained slides were reviewed for tumor morphology, margin status, and lymph node metastasis. CD117, p53, D2-40, CD31, and c-Myc IHC stains were performed on 11 viable tissue blocks. Additional clinical information was obtained from the electronic medical records. Seventeen patients with BAS were identified. Of these, five (29%) were primary and 12 (71%) were secondary BAS, respectively. The median age at diagnosis for primary BAS was 36 years. The median age at diagnosis for secondary BAS was 67 years. The median time to secondary BAS development following radiotherapy was 6.5 years (range, 2 to 12 years). There was no significant difference between primary and secondary BAS in several histopathologic parameters examined, including histologic grade, necrosis, mitotic count, lymph node metastasis, and positive tumor margins. There was also no difference in CD117, p53, D2-40, CD31, and c-Myc expression by IHC between primary and secondary BAS. During a median followup of 21 months, primary BAS had two (40%) reported deaths and secondary BAS had three (25%) reported deaths. However, this difference in survival between both groups was not statistically significant (hazard ratio, 0.51; 95% confidence interval, 0.09 to 3.28; p = .450). BAS is a rare and aggressive disease. No histologic, IHC (CD117, c-Myc, and p53), or survival differences were identified between primary and secondary BAS in this study.

Viral Integration Plays a Minor Role in the Development and Prognostication of Oral Squamous Cell Carcinoma.

Viruses are well known drivers of several human malignancies. A causative factor for oral cavity squamous cell carcinoma (OSCC) in patients with limited exposure to traditional risk factors, including tobacco use, is yet to be identified. Our study aimed to comprehensively evaluate the role of viral drivers in OSCC patients with low cumulative exposure to traditional risk factors. Patients under 50 years of age with OSCC, defined using strict anatomic criteria were selected for WGS. The WGS data was interrogated using viral detection tools (Kraken 2 and BLASTN), together examining >700,000 viruses. The findings were further verified using tissue microarrays of OSCC samples using both immunohistochemistry and RNA in situ hybridisation (ISH). 28 patients underwent WGS and comprehensive viral profiling. One 49-year-old male patient with OSCC of the hard palate demonstrated HPV35 integration. 657 cases of OSCC were then evaluated for the presence of HPV integration through immunohistochemistry for p16 and HPV RNA ISH. HPV integration was seen in 8 (1.2%) patients, all middle-aged men with predominant floor of mouth involvement. In summary, a wide-ranging interrogation of >700,000 viruses using OSCC WGS data showed HPV integration in a minority of male OSCC patients and did not carry any prognostic significance.

Prognosis of P16 and HPV Discordant Oropharyngeal Cancers: Natural Language Processing to Extract Data from Free-Text Pathology Reports

<h3>Purpose/Objective(s)</h3> P16 immunohistochemistry (IHC) is not as specific for detecting human papillomavirus (HPV) related oropharyngeal cancers (OPC) as in situ hybridization (ISH). However, given its high sensitivity and wide availability, P16 IHC is a standard of care requirement for OPC staging and is also used for clinical trial eligibility. As both P16 and HPV testing are not required for routine clinical management, there are varying reports on the prognosis of P16 and HPV discordant tumors. We aimed to assess the prognosis of P16 and HPV discordant OPC tumors, using natural language processing (NLP) techniques to extract results from free-text OPC pathology reports. <h3>Materials/Methods</h3> We reviewed our institutional OPC radiation database and included a series of patients from 7/1994-6/2020 with digitized pathology reports. Patients were excluded if they did not receive curative intent radiation, did not have both P16 IHC and HPV ISH, or if either test was equivocal. 422 patients were included with a median follow-up of 87.8 months (0.3-241.0). We performed text extraction and classification to ascertain P16 and HPV results from free-text pathology reports. All reports were manually evaluated by two independent reviewers as our gold standard. Four groups were identified: P16-negative/HPV-negative (P16-/HPV-, n=60), P16-/HPV-positive (P16-/HPV+, n=9), P16-positive/HPV- (P16+/HPV-, n=49), and P16+/HPV+ (P16+/HPV+, n=304). The Kaplan-Meier method was used to estimate cancer-specific survival (CSS) and overall survival (OS). <h3>Results</h3> 39% of reports could not be processed by our NLP techniques. The algorithm accurately coded 92.6% of P16 and 94.2% of HPV results. Positive predictive value/precision, sensitivity/recall, and F-score for P16/HPV were: 99.5%/99.0%, 91.7%/94.0%, and 95.5%/96.5%. 13.7% of tumors were discordant (P16-/HPV+ or P16+/HPV-). As expected, P16-/HPV- tumors had the worst CSS and OS, and P16+/HPV+ had excellent prognosis (Table 1). P16-/HPV+ constituted only 2.1% of the cohort but outcomes performed favorably with P16+/HPV+. P16+/HPV- tumors had favorable outcomes in the first two years, but with longer follow-up clustered between P16-/HPV- and P16+/HPV+ tumors. <h3>Conclusion</h3> P16 and HPV results were accurately abstracted from the majority of free-text OPC pathology reports. We provide the first example to our knowledge of employing NLP techniques to extract data from head and neck cancer pathology reports and correlate results with clinical outcomes. P16 and HPV discordant tumors constitute a minority of patients, but their divergent prognoses suggest that performing both tests may be of utility—especially for clinical trial eligibility or treatment de-escalation.

P53, Pirh2, and L1CAM as Promising Prognostic Biomarkers of Endometrial Carcinoma: An Immunohistochemical and Genetic Study.

Endometrial cancer (EC) is the most common gynecologic cancer and the current methods for the prediction of its prognosis and treatment response are unfortunately suboptimal. In this study, we evaluated the prognostic value of p53, Pirh2, and L1CAM in 60 cases of EC using immunohistochemistry (IHC) and polymerase chain reaction. TP53 missense mutations result in nuclear accumulation of p53 protein that can be detected as overexpression by IHC. This is in the form of diffuse strong nuclear positivity involving at least at least >50% of the tumor cells as a whole or if >50% of the tumor cells of a discrete geographical areas. Abnormal p53 IHC expression was expressed in 33.3% of the cases and significantly associated with the tumor grade, myometrial invasion (MI), lymphovascular invasion (LVSI), nodal metastasis, and FIGO stage, and the advanced European Society for Medical Oncology (ESMO) risk groups ( P <0.001 for each). High IHC Pirh2 expression was noted in 58.3% of the cases, and significantly associated with MI, LVSI, nodal metastasis, FIGO stage, and high-risk group ( P <0.001, P =0.011, P =0.010, P =0.024, P =0.005, respectively). There was a significant upregulation of Pirh2 mRNA expression in EC specimens as compared with the control adjacent tissues ( P =0.001). Upregulated Pirh2 mRNA expression had a significant association with Pirh2 immunostaining, tumor grade, tumor stage, MI, lymph node involvement, LVSI, and relapse ( P <0.001 for each). Positive L1CAM immunoexpression was noted in 26.7% and was significantly associated with grade, MI, LVSI, nodal metastasis, FIGO stage, and high-risk group ( P =0.003, P =0.023, P =0.003, P <0.001, P <0.001, P =0.002, respectively). Analysis of follow-up period revealed that EC with abnormal p53 IHC expression, high pirh2 and positive L1CAM expression exhibited a potent relation with tumor relapse, shorter overall survival and disease-specific survival ( P <0.001 for each). Mutant p53, high Pirh2, and L1CAM-positive EC are highly aggressive tumors with a shortened survival rate, dismal outcome, and high risk of relapse after the standard protocol of therapy.

Molecular classification and clinicopathological features of endometrial carcinoma

Objective: To investigate the molecular classification and clinicopathological features of endometrial carcinoma(EC). Methods: One hundred cases of EC diagnosed in the Department of Pathology, Tianjin Central Hospital of Gynecology and Obstetrics from November 2020 to November 2021 were selected. Sanger sequencing and immunohistochemical staining were used for molecular classification according to the 5th WHO classification. The clinicopathological characteristics of each molecular subtype was analyzed. Results: The 100 EC patients had a mean age of 53 years (range 26 to 72 years). There were 10 cases of POLE mutation (POLE mut), including two cases (2/10) of "binary-classifier" EC, two cases (2/10) of FIGO Grade 3 endometrioid endometrial carcinoma (G3-EEC), and three cases (3/10) of other high-grade subtypes. There were 38 cases of mismatch repair deficiency (dMMR), including one case (1/38, 2.6%) of "binary-classifier" EC and 36 cases (36/38, 94.7%) were EEC. Twenty-one cases (21/38, 55.3%) showed simultaneous loss of expression of MLH1 and PMS2, and 20 cases (20/21, 95.2%) were positive for MLH1 methylation, indicating that they were sporadic EC. Six patients (6/38, 15.8%) were tested for germline detection of Lynch syndrome (LS) related genes, and one patient was LS-related EC. There were 44 cases of non-specific molecular profile (NSMP), including 34 cases (34/44, 77.3%) of G1-2 EEC and seven cases (7/44, 15.9%) of G3-EEC. There were eight cases of p53 abnormality (p53 abn), including four cases (4/8) of G3-EEC, two cases (2/8) of other high-grade subtypes, and one patient had hereditary breast cancer and ovarian cancer syndrome. Conclusions: Correct interpretation of POLE mutation, MMR and p53 immunohistochemistry is the key of molecular classification. The interpretation must strictly follow standard diagnostic procedures and specifications to ensure the accuracy of molecular classification.

P16 and P21 are involved in the pathogenesis of endometrial thinning: A cross-sectional study.

P16 plays a role in the negative regulation of cell proliferation, regulating cell apoptosis to control the growth of tumor cells. P21 is a nuclear protein that suppresses DNA synthesis and inhibits cell division. This study aimed to examine the expression and roles of P16 and P21 in endometrial thinning. Thirty cases of endometrial biopsy diagnosed as endometrial thinning were assessed by p16 and p21 immunohistochemistry from March 2014 to August 2020 in Huazhong University of Science and Technology Union Shenzhen Hospital. Another thirty cases of normal endometrium in the same period were assessed as controls. The specimens underwent histological analysis, and P16 and P21 were assessed by immunohistochemistry. There were no statistically significant differences in age, menstrual cycle, BMI, sex hormone levels, gravidity and parity between the two groups (all P > .05). In the endometrial thinning group, P16 was expressed in the endometrial adenoid nucleus, cytolymph and interstitial cell nucleus. In the normal group, P16 was mainly expressed in the endometrial adenoid nucleus, with some P16 signals detected in the endometrial interstitial nucleus. P21 expression was mainly detected in the endometrial adenoid nucleus. P16 and P21 amounts in endometrial thinning cases were significantly lower than those of the normal endometrial group. There was no correlation between p16 and p21 amounts. This study revealed aberrant expression of P16 and P21 in the endometrium might be due to a compensatory effect of the thin endometrium to increase cell proliferation and suppress cell apoptosis. However, the pathological roles of P16 and P21 in endometrial thinning and the contribution of cell senescence deserve further investigation.

Targeted Molecular Testing in Endometrial Carcinoma: Validation of a Clinically Driven Selective ProMisE Testing Protocol.

Incorporation of molecular classification into clinicopathologic assessment of endometrial carcinoma (EC) improves risk stratification. Four EC molecular subtypes, as identified by The Cancer Genome Atlas, can be diagnosed through a validated algorithm Pro active M olecular R is k Classifier for E ndometrial Cancer (ProMisE) using p53 and mismatch repair (MMR) protein immunohistochemistry (IHC), and DNA polymerase epsilon ( POLE) mutational testing. Cost and access are major barriers to universal testing, particularly POLE analysis. We assessed a selective ProMisE algorithm (ProMisE-S): p53 and MMR IHC on all EC's with POLE testing restricted to those with abnormal MMR or p53 IHC (to identify POLEmut EC with secondary abnormalities in MMR and/or p53) and those with high-grade or non-endometrioid morphology, stage >IA or presence of lymphovascular space invasion (so as to avoid testing on the lowest risk tumors). We retrospectively compared the known ProMisE molecular classification to ProMisE-S in 912 EC. We defined a group of "very low-risk" EC (G1/G2, endometrioid, MMR-proficient, p53 wild-type, stage IA, no lymphovascular space invasion) in whom POLE testing will not impact on patient care; using ProMisE-S, POLE testing would not be required in 55% of biopsies and 38% of all EC's, after evaluation of the hysterectomy specimen, in a population-based cohort. "Very low-risk" endometrioid EC with unknown POLE status showed excellent clinical outcomes. Fifteen of 166 (9%) of all p53abn EC showed G1/G2 endometrioid morphology, supporting the potential value of universal p53 IHC. The addition of molecular testing changed the risk category in 89/896 (10%) EC's. In routine practice, POLE testing could be further restricted to only those patients in whom this would alter adjuvant therapy recommendations.

Clinical and molecular validation of BAP1, MTAP, P53, and Merlin immunohistochemistry in diagnosis of pleural mesothelioma

AbstractBAP1 and MTAP immunostains play an important role in diagnosis of mesothelioma, but additional markers are needed to increase sensitivity. We analyzed 84 pleural mesotheliomas (51 epithelioid, 27 biphasic, 6 sarcomatoid) by a hybrid-capture next-generation sequencing (NGS) panel including complete coverage of coding and splicing regions for BAP1, CDKN2A/MTAP, NF2, and TP53 and correlated molecular findings with diagnostic immunostains for BAP1, MTAP, Merlin, and p53, respectively. Fifty-seven reactive mesothelial proliferations served as benign comparators. Loss of BAP1, MTAP, and Merlin protein expression were, respectively, 54%, 46%, and 52% sensitive and 100% specific for mesothelioma. Two-marker immunopanels of BAP1 + MTAP, BAP1 + Merlin, and MTAP + Merlin were 79%, 85%, and 71% sensitive for mesothelioma, while a three-marker immunopanel of BAP1 + MTAP + Merlin was 90% sensitive. Diffuse (mutant-pattern) p53 immunostaining was seen in only 6 (7%) tumors but represented the only immunohistochemical abnormality in 2 cases. Null-pattern p53 was not specific for malignancy. An immunopanel of BAP1 + MTAP + Merlin + p53 was 93% sensitive for mesothelioma, and panel NGS detected a pathogenic alteration in BAP1, MTAP, NF2, and/or TP53 in 95%. Together, 83 (99%) of 84 tumors showed a diagnostic alteration by either immunohistochemistry or panel NGS. Adding Merlin to the standard BAP1 + MTAP immunopanel increases sensitivity for mesothelioma without sacrificing specificity. p53 immunohistochemistry and panel NGS with complete coverage of BAP1, CDKN2A/MTAP, TP53, and NF2 may be useful in diagnostically challenging cases.

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

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

Comparison of p63 immunohistochemistry and shear wave elastography in the diagnosis of indeterminate breast lesions: A prospective study.

To evaluate the accuracy of breast shear wave elastography (SWE) and p63 immunohistochemistry (IHC) in the diagnosis of indeterminate breast lesions. Based on detailed clinical examination and a combination of X-ray mammography/B-mode ultrasound with SWE, a total of 40 patients with breast lumps (BI-RADS 4) were included. Patients with previous diagnosis of breast cancer and a previous history of surgery, chemotherapy, or radiotherapy in the same breast as the present lesion were excluded. Core needle biopsy of the breast lesion was performed, and p63 IHC staining was performed. A final histopathological report of the definitive procedure was considered as the gold standard. The sensitivity, specificity, positive (PPV) and negative predictive values (NPV), and accuracy were calculated for each modality. The mean age of the patients included in the study was 50.85 ± 13.53 years. Of the 40 patients recruited, 23 were clinically malignant and 17 were benign. The sensitivity, specificity, PPV, NPV, and accuracy of SWE were 91.3%, 94.1%, 95.5%, 88.9%, and 92.5% and those of p63 IHC were 95.7%, 100%, 100%, 94.4%, and 97.5%, respectively. Overall, the parametric values were higher for p63 IHC as compared to clinical examination and elastography. The area under the ROC curve (AUC) for p63 IHC (.978) was higher than those for SWE (.927) and clinical examination (.898). SWE and p63 IHC are highly reliable novel modalities that demonstrate enhanced diagnostic accuracy of indeterminate breast lesions aiding in the early initiation of appropriate treatment and reducing the number of women subjected to biopsy or short-term follow-up for benign-appearing solid breast lesions.

Immunohistochemical expression of proliferation markers in canine osteosarcoma

Canine osteosarcoma is an extremely malignant bone tumor that often arises in the bones of the limbs. It is a highly metastatic disease distinguished by proliferative bone lesions and a tendency for pulmonary metastasis. Overexpression of proliferative proteins are associated with bad prognosis in human osteosarcoma. Here, we tested the expression of the different proliferative proteins (p53, p16, vimentin, and mdm2) in nine archival samples with canine osteosarcoma. Paraffin-embedded tissue sections were confirmed by histopathology and stained by immunohistochemistry for p53, p16, vimentin and mdm2. Positive expression of these proteins was evaluated as the ratio of positive cancer cells and the intensity staining was assessed in several areas. Histopathologically, 95% of samples were grade II and III. All high-grade osteosarcomas were particularly cellular. The cancer cells were generally large spindle-shaped and large nucleus with distribution of osteoid between the cancer cells. Immunohistochemical detection of p53, p16, vimentin and mdm2 was 89%, 56%, 78%, and 89% of samples respectively. The staining intensity for p53, p16, vimentin and mdm2 was particularly nuclear in 81%, 66%, 78%, and 79% of the cancer cells respectively. Our present work suggests that p53, p16, vimentin, and mdm2 were detected in grade III canine osteosarcomas samples. In addition, these proliferative markers are the significant biomarkers in canine osteosarcomas and can be used as a predictor for diagnostic and prognostic value and allowing cancer differentiation. This primary data supports that both canine and human osteosarcomas share same molecular characters which are approved by expression of proliferative genes.

Sinonasal mass lesions: A clinicopathological study with p63 and p16 immunohistochemical expressions.

The worldwide annual incidence of carcinomas of the sinonasal tract is 0.5 to 1.0 patients per 100,000 per year. P63 plays a role in epithelial development and is used as a marker for basal and myoepithelial cells. Expression of p16 occurs as a result of functional inactivation of the retinoblastoma protein (pRb) by the human papilloma virus (HPV) E7 protein. This study aims to study the histological spectrum of benign and malignant sinonasal mass lesions and to study the immunohistochemical expression of p63 in different type of sinonasal mass lesions. It also aims to ascertain the incidence of high-risk HPV in primary sinonasal mass lesions with p16 immunohistochemistry and delineate the histological spectrum of HPV-related sinonasal lesions. This cross-sectional study was conducted on 80 cases from June 2018 to June 2020 at a tertiary care hospital. Clinical history including demographic parameters were collected in the study proforma. The gross findings of the specimens noted and histopathological examination by H&E staining done. Immunohistochemistry staining for p63 and p16 expression was performed on all cases. Most common age group affected was 41-60 years with male:female ratio of 1.67:1. Nonneoplastic lesions (38.7%) comprised majority of the cases followed by benign neoplastic lesions (31.3%) and malignant neoplastic lesions (30%). Among the malignant neoplastic lesions, p63 showed positive expression in 75% (p = 0.005) and p16 showed positive expression in 41.7% (p = 0.023). Among benign and nonneoplastic lesions, p63 showed positivity in 21.4% (p = 0.000) and p16 showed positivity in 44.6% (p = 0.040). We analyzed p63 and p16 expression in varied lineages like carcinomas, papillomas, and neuroectodermal differentiation arising from the sinonasal tract and also in relation to other clinicopathological parameters. This study revealed p63 expression was associated more with the squamous cell carcinomas and nasopharyngeal carcinomas. Sinonasal tract malignancies are also associated with HPV infections that are identifiable by p16 immunostaining and, thus, could provide new prospects in identifying any definite biological and clinical characteristics associated with HPV as well as advancement in the targeted therapies for this patient population.

Human papillomavirus-related neoplasia of the ocular adnexa.

Human papillomaviruses (HPV) are involved in approximately 5% of solid cancers worldwide. The mucosotropic genotypes infect the stratified epithelium of various locations, where persistent infection may lead to invasive carcinomas. While the causative role of HPV in certain anogenital and head and neck carcinomas is well established, the role of HPV in carcinomas arising in the mucosal membranes of the ocular adnexal tissue (the lacrimal drainage system and the conjunctiva) has been a topic of great uncertainty. Therefore, we conducted a series of studies to assess the correlation between HPV and carcinomas arising in the mucosa of the ocular adnexal tissue and characterize the clinical, histopathological, and genomic features of the tumors in the context of HPV status in a Danish nationwide cohort. We collected clinical and histopathological data and tumor specimens from patients with carcinomas of the conjunctiva and the lacrimal drainage system, and their potential precursors, identified in Danish nationwide registries. The HPV status of the tumors was determined by the combined use of HPV DNA polymerase chain reaction (PCR), HPV E6/E7 mRNA in-situ hybridization, and p16 immunohistochemistry. The genomic profile was investigated by high-throughput DNA sequencing targeting 523 cancer-relevant genes. The literature to date on carcinomas of the lacrimal drainage system and the conjunctiva was summarized. In the Danish cohort, 67% of all carcinomas of the lacrimal drainage system and 21% of all conjunctival carcinomas were HPV-positive. HPV16 was the most frequently implicated genotype. A full-thickness expression of the viral oncogenes E6 and E7 was evident in almost all HPV DNA-positive cases. The HPV-positive carcinomas of the conjunctiva and the lacrimal drainage system shared histopathological and genomic features distinct from their HPV-negative counterparts. The HPV-positive carcinomas were characterized by a non-keratinizing morphology, p16 overexpression, high transcriptional activity of HPV E6/E7, and frequent pathogenic variants in the PI3K-AKT signaling cascade. In contrast, the HPV-negative carcinomas were characterized by a keratinizing morphology, lack of p16 and E6/E7 expression, and frequent somatic pathogenic variants in TP53, CDKN2A, and RB1. Among the patients with conjunctival tumors, HPV positivity was associated with a younger age at diagnosis and a higher risk of recurrence. In conclusion, the results support an etiological role of HPV in a subset of conjunctival and LDS carcinomas and their precursor lesions. Our investigations have shown that the HPV-positive carcinomas of the ocular adnexa share genomic and phenotypic characteristics with HPV-positive carcinomas of other anatomical locations. Therefore, these patients may be eligible for inclusion in future basket trials and future treatment regimens tailored to the more frequently occurring HPV-positive carcinomas of other locations. Future research will further elucidate the diagnostic, prognostic, and predictive role of HPV in these carcinomas.

New progress in diagnosis and treatment of HPV-positive oropharyngeal carcinoma

In recent years, the incidence of oropharyngeal carcinoma （OPC） is increasing, while the better prognosis of patients with Human papillomavirus （HPV） positive oropharyngeal carcinoma has been confirmed in a number of studies. There are a variety of detection methods for HPV-associated oropharyngeal carcinoma. Including P16 immunohistochemistry, Polymerase Chain Reaction （PCR） or In situ hybridization （ISH） detection of HPV DNA, HPV RNA, Revers transcriptase Polymerase Chain Reaction （RT PCR） was used to detect HPV RNA. The better prognosis of patients with HPV-positive oropharyngeal carcinoma has led to the emergence of a large number of degraded treatment trials. The traditional P16 test has certain limitations in the diagnosis of patients with HPV-positive oropharyngeal carcinoma. It is necessary to combine with other detection methods to accurately screen out patients with HPV-positive oropharyngeal carcinoma and better apply to degraded therapy. In this article, we will briefly introduce the trend of HPV-associated oropharyngeal carcinoma, the detection methods and the new progress of degraded treatment trials.

Adjunctive use of p16 immunohistochemistry for optimizing management of CIN lesions in a high-risk human papillomavirus-positive population.

Immunostaining with p16INK4a (p16), a tumor-suppressor surrogate protein biomarker for high-risk human papillomavirus (hrHPV) oncogenic activity, may complement standard hematoxylin and eosin (H&E) histology review, and provide more objective criteria to support the cervical intraepithelial neoplasia (CIN) diagnosis. With this study we assessed the impact of p16 immunohistochemistry on CIN grading in an hrHPV-based screening setting. In this post-hoc analysis, 326 histology follow-up samples from a group of hrHPV-positive women were stained with p16 immunohistochemistry. All H&E samples were centrally revised. The pathologists reported their level of confidence in classifying the CIN lesion. Combining H&E and p16 staining resulted in a change of diagnosis in 27.3% (n=89) of cases compared with the revised H&E samples, with a decrease of 34.5% (n=18) in CIN1 and 22.7% (n=15) in CIN2 classifications, and an increase of 18.3% (n=19) in no CIN and 20.7% (n=19) in CIN3 diagnoses. The level of confidence in CIN grading by the pathologist increased with adjunctive use of p16 immunohistochemistry to standard H&E. This study shows that adjunctive use of p16 immunohistochemistry to H&E morphology reduces the number of CIN1 and CIN2 classifications with a proportional increase in no CIN and CIN3 diagnoses, compared with standard H&E-based CIN diagnosis alone. The pathologists felt more confident in classifying the material with H&E and p16 immunohistochemistry than by using H&E alone, particularly during assessment of small biopsies. Adjunctive use of p16 immunohistochemistry to standard H&E assessment of CIN would be valuable for the diagnostic accuracy, thereby optimizing CIN management and possibly decreasing overtreatment.

435. A DIAGNOSTIC PITFALL; SMALL CELL CARCINOMA-LIKE FEATURES IN BASALOID SQUAMOUS CELL CARCINOMA OF THE ESOPHAGUS

Abstract Esophageal basaloid squamous cell carcinoma may resemble small cell carcinoma biopsy specimens and cause difficulties in pathology diagnosis. We aimed to explore the pathological features of basaloid squamous cell carcinomas in biopsy specimens that mimic small cell carcinoma and to clarify their diagnostic and prognostic features. This study will contribute to draw attention to an important pitfall in pathology diagnosis in endoscopic biopsy specimens of the esophagus. Thirty biopsy specimens of esophageal basaloid squamous cell carcinoma were reviewed and compared with 13 matched surgical specimens. A small cell carcinoma-like feature was defined as, when at least one of the three following findings was observed: 1) diffuse growth pattern, 2) nuclear molding and 3) nuclear crush artifact. Immunohistochemistry of synaptophysin, chromogranin A, CD56 (neuroendocrine markers) and p16 were performed in all cases. Small cell carcinoma-like features, such as diffuse growth, nuclear molding, or nuclear crush artifact, were identified in 80% (24/30) of the biopsies and in 77% (10/13) of the surgery specimens, but in a proportionally much smaller area in the surgical specimens than in the biopsy samples. The presence of a small cell carcinoma-like feature had no impact on patients´ outcome. Immunohistochemically, synaptophysin and chromogranin A were consistently negative, while CD56 was expressed in 42% (10/24) basaloid squamous cell carcinomas with small cell carcinoma-like features. p16, a highly sensitive marker for small cell carcinoma, was also expressed in 8% (2/24). In conclusion, small cell carcinoma-like features are frequent and conspicuous in biopsies, which may probably be caused by exogenous factors such as friction and external pressure that occur in biopsy procedure and in the tumor environment. Small cell carcinoma-like features may lead to a misinterpretation of a true small cell carcinoma, if CD56 is an only neuroendocrine marker expressed. p16 expression may be also detected in basaloid squamous cell carcinoma.

Reclassification of atypical immature metaplasia of the uterine cervix by combination of nuclear features on hematoxylin and eosin–stained sections without auxiliary immunohistochemistry

Reclassification of endocervical atypical immature metaplasia (AIM) into reactive changes and neoplastic lesion is often challenging. We aimed to accurately reclassify AIM on hematoxylin and eosin (HE)-stained sections without auxiliary immunohistochemistry (IHC). A total of 133 AIM diagnosed by punch biopsy were reclassified by IHC for p16 and Ki-67 into high-grade squamous intraepithelial lesion (HSIL) or negative for intraepithelial lesion or malignancy or low-grade squamous intraepithelial lesion (NILM/LSIL) as a reference. Nuclear features significantly associated with HSIL on HE-stained sections were extracted by multivariate logistic regression analysis. Propensity score (PS) of HSIL was calculated in each case and cut-off was determined by receiver operation characteristic (ROC) curve analysis. As a result, AIM was reclassified into 104 NILM/LSIL and 29 HSIL by IHC. Compared with reference diagnosis, accuracy of pathologists' subjective diagnosis was 54.9% (kappa coefficient, 0.208). Three nuclear features on HE-stained sections, ie, nuclear enlargement with anisokaryosis, nuclear hyperchromasia, and mitosis, were significantly associated with HSIL. The ROC curve analyses revealed that PS and number of nuclear features were significant predictors of HSIL. Diagnostic accuracy of PS-based diagnosis was 76.7% (kappa, 0.447). When AIM with 2 or more of the 3 nuclear features was diagnosed with HSIL, diagnostic accuracy was 77.4% (kappa, 0.448). Nuclear feature-based diagnosis significantly improved diagnostic accuracy on HE-stained sections compared with subjective diagnosis and may be useful when IHC is not available. However, a considerable proportion of AIM would still remain misdiagnosed and IHC for p16 and Ki-67 should be mandatory for accurate reclassification.

Cobas HPV Genotyping of FNA Sample Supernatant and Frozen Section Scrapings of Suspected Head and Neck Cancer.

The human papillomavirus (HPV) status of squamous cell carcinomas (SCCs) of the head and neck is relevant for therapy planning, staging, and follow-up. Immunohistochemistry (IHC) for p16 is a surrogate marker of HPV status in oropharyngeal SCC, but not at other head and neck sites. We tested if the cobas HPV test was feasible and superior to p16-IHC on fine-needle aspiration (FNA) supernatants and frozen section (FS) scrapings of suspected SCC. A 500μL aliquots of postcentrifugation supernatant CytoRich Red media of FNA cellblock specimens and scrapings of FS suspended in SurePath media vials were tested with the cobas HPV test and compared with p16-IHC and/or HPV in situ hybridization (ISH) performed on cellblock and/or resections. Twenty-nine (n=29) FNAs were tested for a cobas HPV test, p16, and/or HPV-ISH. The mean collection to testing time was 6.3 days (range: 0 to 24d). Cobas yielded valid results in all cases; p16-IHC could not be interpreted in 4 (13%) cellblocks; correlation was performed on subsequent resections. Cohen κ correlation for cobas versus p16-IHC/HPV-ISH on FNA samples was 0.9, perfect agreement, sensitivity 100%, specificity 92.3%, positive predictive value 94.1%, negative predictive value 100%. Thirty-four (n=34) scrapings from FS were tested for cobas, p16, and/or HPV-ISH. The mean collection to testing time was 10.4 days (range: 1 to 28d). Cohen κ correlation for cobas versus p16-IHC/HPV-ISH on FS scrapings was 1, perfect agreement. Sensitivity, specificity, positive predictive value, and negative predictive value was 100%. Cobas genotype was HPV-16 in 87%, HPV-18 in 3%, and HPV-other in 10%. Cobas HPV test in FNA supernatant and FS scrapings outperformed or was equivalent to p16-IHC and provided genotyping information.

Lymph node metastasis in level IIb in oropharyngeal squamous cell carcinoma: a multicentric, longitudinal, retrospective analysis.

Nowadays, 70% of patients in Europe and the USA are affected by a p16 + , potentially HPV driven oropharyngeal squamous cell carcinoma. However, despite the improved survival rate in this group, the quality-of-life remains low in cases which neck dissection took place. In this vein, in recent years, some surgeons have considered to avoid dissection of level IIB, proposing a supra-selective non-IIb neck dissection. A retrospective, longitudinal, multicentric study was conducted, including patients with pathologically confirmed primary HPV + or HPV - OPSCC who went through surgical treatment for the primary lesion and neck dissection. 141 patients were included. Among them, 99 (70.2%) were male and 42 (29.8%) were female. The mean age was 62 ± 9years (range 36-81). The most frequent anatomical location was the tonsil in 63 (44.7%) of patients. The most common approach was the classic transoral oropharyngectomy in 51 (36.2%) patients. Immunohistochemistry for p16 was positive in 62 (44%) patients. One-hundred and five (74.5%) patients received a unilateral ND, and a 36 (25.5%) a bilateral ND. Of those, a 12.8% (18/141) of patients were level IIb LN + . According to our results, level IIb ND should be considered in patients underwent therapeutic ND with positive LN metastasis in level IIa (OR = 9.83; 95% CI 3.463-27.917) or III (OR = 6.25; 95% CI 2.158-18.143), advanced (T3/T4) oropharyngeal primary tumors (OR = 3.38; 95% CI 1.366-8.405), and patients with ENE (OR = 6.56; 95% CI 2.182-19.770), regardless of p16 status. According to our results, level IIb ND should be considered in patients who underwent therapeutic ND with positive LN metastasis in level IIa or III, advanced oropharyngeal primary tumors, and patients with ENE, independently of p16 status. Prospective data are necessary to definitively ensure the safety of omitting ipsilateral or contralateral level IIb ND in cN - patients with early stage disease.

Clinical features and prognosis of 166 cases of MYC/BCL2 double-expression diffuse large B-cell lymphoma

Objective: To investigate the clinical features and prognosis of MYC/BCL2 double-expression diffuse large B-cell lymphoma (DEL) . Methods: The clinical data, including clinical characteristics, survival, and prognostic factors, of 166 patients with DEL treated at Peking University Third Hospital from January 2016 to December 2020 were retrospectively analyzed. Results: A total of 410 patients with diffuse large B-cell lymphoma were collected, including 166 cases (40.5%) of DEL. There were 82 males and 84 females with a median age of 63.5 (21-95) years at diagnosis. A total of 110 patients (66.3%) were aged over 60 years at initial diagnosis, 106 patients (106/163, 65.0%) had elevated lactate dehydrogenase (LDH) at diagnosis, 74 patients (74/160, 46.2%) had β(2) microglobulin level over 3 mg/L at diagnosis, and 107 patients (107/163, 65.6%) had≥2 extranodal involvement. Sixty-five patients (65/166, 39.2%) had B symptoms, 131 patients (131/165, 79.4%) had stage Ⅲ and Ⅳ disease at initial diagnosis, 41 patients (41/161, 25.5%) had an International Prognostic Index (IPI) score of 0-2 at initial diagnosis, and 38 patients (38/161, 23.6%) had an IPI score of 3 at initial diagnosis. Eighty-two patients (82/161, 50.9%) had an IPI score of 4-5 at initial diagnosis. Nine (9/56, 16.1%) patients with DEL had MYD88 and CD79B mutations. Univariate analysis showed that age over 60 years (P=0.004) , increased β(2) microglobulin level (P=0.002) , and high IPI score (P=0.003) were associated with poor overall survival (OS) . Increased β(2) microglobulin level (P=0.031) , LDH level (P=0.017) , stage Ⅲ-Ⅳ (P=0.001) , high IPI score (P=0.013) , immunohistochemical p53 mutation (P=0.049) , and PIM1 mutation (P=0.039) were associated with poor progression-free survival (PFS) . Multivariate analysis showed that IPI score of 4-5 was an independent risk factor for the prognosis of DEL (HR=2.622, 95% CI 1.398-4.917, P=0.003) . Survival analysis showed that there was a significant difference in the PFS between patients with DEL and those without DEL (65.6% vs 75.1%, P=0.002) . However, there was no significant difference in the OS (81.8% vs 83.6%, P=0.226) . In patients with DEL, the overall response rate of R-EPOCH regimen was higher than that of RCHOP or RCHOP-like regimen (81.5% vs 63.4%, P=0.004) . Conclusion: DEL is a group of aggressive lymphomas with relatively poor PFS. The R-EPOCH regimen may improve the overall prognosis of patients.