Publications by authors named "Achim A Jungbluth"

186 Publications

Expression Analysis of GD2 by Immunohistochemistry in Invasive Breast Carcinoma: Clinical and Pathologic Correlation.

Appl Immunohistochem Mol Morphol 2021 Sep 21. Epub 2021 Sep 21.

Departments of Pathology Pediatric Oncology, Memorial Sloan Kettering Cancer Center, New York, NY.

The glycosphingolipid disialoganglioside GD2 is a cell surface-associated antigen expressed on tumors of neuroectodermal origin that serves as a target of immunotherapy in select cancer types. Information about the expression of GD2 in breast cancer is limited. In the present study, we investigate the utility of GD2 as a potential biomarker for targeted treatment. The study cohort consists of 386 breast carcinomas of several histologic types. GD2 expression was assessed in both whole tumor sections and tissue microarrays with anti-GD2 3F8 monoclonal antibody immunohistochemistry and correlated with clinicopathologic features and survival outcomes. A total of 134 (35%) breast carcinomas were positive for GD2, with a median H-score of 100. 3F8 staining displayed granular and predominantly cytoplasmic or perinuclear patterns, which was confined to the neoplastic tissue in nearly all cases. GD2 positivity was significantly associated with tumor histologic type (P=0.0015), low grade (P<0.0001), estrogen receptor positivity (P<0.0001), low stage (P=0.0014), and multifocality (P=0.022). Event-free survival and overall survival of patients with GD2-positive and GD2-negative tumors were not significantly different. Our results support further assessment of GD2 using the 3F8 antibody as a predictive and prognostic biomarker in breast cancer.
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http://dx.doi.org/10.1097/PAI.0000000000000974DOI Listing
September 2021

IDH2 R172 Mutations Across Poorly Differentiated Sinonasal Tract Malignancies: Forty Molecularly Homogenous and Histologically Variable Cases With Favorable Outcome.

Am J Surg Pathol 2021 09;45(9):1190-1204

Departments of Neuropathology.

IDH2 R172 mutations occur in sinonasal undifferentiated carcinoma (SNUC), large-cell neuroendocrine carcinoma (LCNEC), sinonasal adenocarcinomas, and olfactory neuroblastoma (ONB). We performed a clinical, pathologic, and genetic/epigenetic analysis of a large IDH2-mutated sinonasal tumor cohort to explore their distinct features. A total 165 sinonasal/skull base tumors included 40 IDH2 mutants studied by light microscopy, immunohistochemistry, and genome-wide DNA methylation, and 125 IDH2 wild-type tumors used for comparison. Methylation profiles were analyzed by unsupervised hierarchical clustering, t-distributed stochastic neighbor embedding dimensionality reduction and assessed for copy number alterations (CNA). Thirty-nine histologically assessable cases included 25 (64.1%) SNUC, 8 (20.5%) LCNEC, 2 (5.1%) poorly differentiated adenocarcinomas, 1 (2.7%) ONB, and 3 (7.7%) IDH2-mutated tumors with ONB features. All cases were high-grade showing necrosis (82.4%), prominent nucleoli (88.9%), and median 21 mitoses/10 HPFs. AE1/AE3 and/or CAM 5.2 were positive in all and insulinoma-associated protein 1 (INSM1) in 80% cases. All IDH2 mutants formed one distinct group by t-distributed stochastic neighbor embedding dimensionality reduction separating from all IDH2 wild-type tumors. There was no correlation between methylation clusters and histopathologic diagnoses. Recurrent CNA included 1q gain (79.3%), 17p loss (75.9%), and 17q gain (58.6%). No CNA differences were observed between SNUC and LCNEC. IDH2 mutants showed better disease-specific survival than SMARCB1-deficient (P=0.027) and IDH2 wild-type carcinomas overall (P=0.042). IDH2-mutated sinonasal tumors are remarkably homogeneous at the molecular level and distinct from IDH2 wild-type sinonasal malignancies. Biology of IDH2-mutated sinonasal tumors might be primarily defined by their unique molecular fingerprint rather than by their respective histopathologic diagnoses.
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http://dx.doi.org/10.1097/PAS.0000000000001697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373679PMC
September 2021

PD-L1 Expression in Metaplastic Breast Carcinoma Using the PD-L1 SP142 Assay and Concordance Among PD-L1 Immunohistochemical Assays.

Am J Surg Pathol 2021 09;45(9):1274-1281

Departments of Pathology.

Immunotherapy for the treatment of programmed death-ligand 1 (PD-L1) positive locally advanced or metastatic triple negative breast cancer may benefit patients with metaplastic breast cancer (MpBC). Previous study of PD-L1 in MpBC scored tumor cells (TCs), different from Food and Drug Administration-approved scoring methods. We sought to define PD-L1 expression in MpBCs and to evaluate concordance of 3 PD-L1 assays. Primary, treatment naive MpBC treated at our Center from 1998 to 2019 were identified. PD-L1 expression was assessed using SP142, E1L3n, and 73-10. We evaluated PD-L1 expression on tumor infiltrating immune cells (IC) and also in TCs. For each assay, we scored PD-L1 expression using ≥1% IC expression according to the IMpassion130 trial criteria and using combined positive score (CPS) ≥10 according to the KEYNOTE-355 trial cutoff. A total of 42 MpBCs were identified. Most MpBC had PD-L1 positivity in ≥1% IC with all 3 assays (95%, 95%, 86%) in contrast to a maximum 71% with a CPS ≥10. PD-L1 IC expression was comparable between the SP142 and 73-10 assays and was lowest with E1L3n. PD-L1 TC expression was lowest using SP142. The overall concordance for IC scoring was 88% while 62% had concordant CPS. For each assay, the results of the 2 scoring algorithms were not interchangeable. The SP142 assay showed distinct expression patterns between IC (granular, dot-like) and TC (membranous) while 73-10 and E1L3n showed membranous and/or cytoplasmic expression in both IC and TC. Most MpBC in our cohort were positive for PD-L1 indicating eligibility for anti-PD-L1/programmed death-1 immunotherapy.
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http://dx.doi.org/10.1097/PAS.0000000000001760DOI Listing
September 2021

PRAME Immunohistochemistry as an Ancillary Test for the Assessment of Melanocytic Lesions.

Surg Pathol Clin 2021 Jun 28;14(2):165-175. Epub 2021 Apr 28.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

PRAME (PReferentially expressed Antigen in MElanoma) is a melanoma-associated antigen expressed in cutaneous and ocular melanomas and some other malignant neoplasms, while its expression in normal tissue and benign tumors is limited. Detection of PRAME protein expression by immunohistochemistry in a cohort of 400 melanocytic tumors showed diffuse nuclear immunoreactivity for PRAME in most metastatic and primary melanomas. In contrast, most nevi were negative for PRAME or showed nondiffuse immunoreactivity. The difference in the extent of immunoreactivity for PRAME in unambiguous melanocytic tumors prompted the study of PRAME as an ancillary tool for evaluating melanocytic lesions in more challenging scenarios.
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http://dx.doi.org/10.1016/j.path.2021.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8152939PMC
June 2021

Immunohistochemical Detection of Cancer-Testis Antigen PRAME.

Int J Surg Pathol 2021 Apr 23:10668969211012085. Epub 2021 Apr 23.

5803Memorial Sloan-Kettering Cancer Center, New York, USA.

Cancer-testis (CT) antigens were identified by their ability to elicit T- or B-cell immune responses in the autologous host. They are typically expressed in a wide variety of neoplasms and in normal adult tissues are restricted to testicular germ cells. PReferentially expressed Antigen of Melanoma (PRAME) is a member of the family of nonclassical CT antigens being expressed in a few other normal tissues besides testis. Interestingly, knowledge about the protein expression of many CT antigens is still incomplete due to the limited availability of reagents for their immunohistochemical detection. Here, we tested several commercially available serological reagents and identified a monoclonal antibody suitable for the immunohistochemical detection of PRAME in formalin-fixed paraffin-embedded specimens. We also tested a wide array of normal and neoplastic tissues. PRAME protein expression in normal tissues is congruent with original molecular data being present in the testis, and at low levels in the endometrium, adrenal cortex, and adult as well as fetal ovary. In tumors, there is diffuse PRAME immunoreactivity in most metastatic melanomas, myxoid liposarcomas, and synovial sarcomas. Other neoplasms such as seminomas and carcinomas of various origins including endometrial, serous ovarian, mammary ductal, lung, and renal showed an intermediate proportion of cases and variable extent of tumor cells positive for PRAME protein expression. As seen with other CT antigens, hepatocellular and colorectal carcinoma, Leydig cell tumors, mesothelioma, and leiomyosarcoma are poor expressers of PRAME.
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http://dx.doi.org/10.1177/10668969211012085DOI Listing
April 2021

Genomic characterization of small cell carcinomas of the uterine cervix.

Mol Oncol 2021 Apr 8. Epub 2021 Apr 8.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Small cell carcinoma (SCC) of the uterine cervix is a rare and aggressive form of neuroendocrine carcinoma, which resembles small cell lung cancer (SCLC) in its histology and poor survival rate. Here, we sought to define the genetic underpinning of SCCs of the uterine cervix and compare their mutational profiles with those of human papillomavirus (HPV)-positive head and neck squamous cell carcinomas, HPV-positive cervical carcinomas, and SCLCs using publicly available data. Using a combination of whole-exome and targeted massively parallel sequencing, we found that the nine uterine cervix SCCs, which were HPV18-positive (n = 8) or HPV16-positive (n = 1), harbored a low mutation burden, few copy number alterations, and other than TP53 in two cases no recurrently mutated genes. The majority of mutations were likely passenger missense mutations, and only few affected previously described cancer-related genes. Using RNA-sequencing, we identified putative viral integration sites on 18q12.3 and on 8p22 in two SCCs of the uterine cervix. The overall nonsilent mutation rate of uterine cervix SCCs was significantly lower than that of SCLCs, HPV-driven cervical adeno- and squamous cell carcinomas, or HPV-positive head and neck squamous cell carcinomas. Unlike SCLCs, which are reported to harbor almost universal TP53 and RB1 mutations and a dominant tobacco smoke-related signature 4, uterine cervix SCCs rarely harbored mutations affecting these genes (2/9, 22% TP53; 0% RB1) and displayed a dominant aging (67%) or APOBEC mutational signature (17%), akin to HPV-driven cancers, including cervical adeno- and squamous cell carcinomas and head and neck squamous cell carcinomas. Taken together, in contrast to SCLCs, which are characterized by highly recurrent TP53 and RB1 alterations, uterine cervix SCCs were positive for HPV leading to inactivation of the suppressors p53 and RB, suggesting that these SCCs are convergent phenotypes.
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http://dx.doi.org/10.1002/1878-0261.12962DOI Listing
April 2021

A phase II trial of durvalumab and tremelimumab in metastatic, non-urothelial carcinoma of the urinary tract.

Cancer Med 2021 02 31;10(3):1074-1083. Epub 2020 Dec 31.

Genitourinary Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Background: Immune checkpoint blockade has made a significant impact on the clinical outcomes of patients with metastatic urothelial carcinoma (UC). However, evidence for this approach in patients with non-UC of the urinary tract is limited.

Methods: This was a phase II open-label study of durvalumab 1500 mg and tremelimumab 75 mg every 4 weeks for four cycles followed by durvalumab 1500 mg every 4 weeks. Eligible patients had metastatic non-UC with ECOG PS 0-1 regardless of prior therapy (except small cell carcinoma who were pretreated). The primary endpoint was overall response rate per RECIST v1.1. A Simon's minimax two-stage design was employed, with 13 patients planned for stage one. Pre-treatment tumors underwent PD-L1 staining and next-generation sequencing.

Results: Thirteen patients were treated, including seven small cell carcinoma, three squamous cell carcinoma, and three adenocarcinoma. Eleven patients had visceral metastases. No responses were observed; 11 patients had PD and 2 patients had SD. Median PFS was 1.8 months (95% CI, 1.25-not reached [NR]) with a median follow-up of 7.38 months (range, 5.23-21.99 months). Median OS was 6.97 months (95% CI, 4.34-NR). One patient's tumor was PD-L1 positive and all sequenced tumors (n = 8) were microsatellite stable. Grades 3-4 treatment-related adverse events occurred in 38.4% of patients.

Conclusions: In a poor prognosis cohort of patients with non-UC, durvalumab and tremelimumab lacked clinical activity while demonstrating a manageable safety profile.
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http://dx.doi.org/10.1002/cam4.3699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897955PMC
February 2021

Clonal relationship and directionality of progression of synchronous endometrial and ovarian carcinomas in patients with DNA mismatch repair-deficiency associated syndromes.

Mod Pathol 2021 05 16;34(5):994-1007. Epub 2020 Dec 16.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Sporadic synchronous endometrial (ECs) and ovarian cancers (OCs), although clinically considered to be independent primaries, have been shown to be clonally related and likely constitute metastases from each other. We sought to define whether synchronous ECs/OCs in patients with DNA mismatch repair (MMR)-deficiency syndromes would be clonally related. We subjected synchronous ECs/OCs from four patients (LS3-LS6) with clinically confirmed Lynch syndrome (LS) and one patient with constitutional mismatch repair-deficiency syndrome (CMMRD) to massively parallel sequencing targeting 468 cancer-related genes. Somatic mutations, copy number alterations (CNAs), clonal relatedness and clonal decomposition analyses were performed using previously described bioinformatics methods. All synchronous ECs/OCs analyzed were considered independent primaries based on clinicopathologic criteria. Sequencing analysis revealed that the ECs/OCs of three cases (LS2-CMMRD, L3, L4) harbored similar repertoires of somatic mutations and CNAs and were clonally related. In these three cases, a subset of subclonal mutations in the EC became clonal in the OC, suggesting that the EC was likely the substrate from which the OC developed. LS5's EC/OC had distinct mutational profiles but shared specific CNAs. In contrast, LS6's EC/OC harbored distinct somatic mutations and lacked CNAs, consistent with each tumor constituting an independent primary lesion. In LS5 and LS6, PTEN mutations and PTEN loss of protein expression were found to be restricted to the EC. Finally, re-analysis of sequencing data of sporadic synchronous ECs/OCs supported the observations made in the current study that the directionality of progression is likely from the endometrium to the ovary. In conclusion, contrary to sporadic synchronous ECs/OCs, which are almost invariably clonally related, ECs/OCs simultaneously involving the uterus and ovary in LS patients may represent distinct primary tumors. A subset of MMR-deficiency syndrome-related synchronous ECs/OCs, however, may originate from a single primary tumor at variance with their clinical diagnosis, with the endometrium being the likeliest site of origin.
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http://dx.doi.org/10.1038/s41379-020-00721-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076061PMC
May 2021

Cancer Testis Antigens and Immunotherapy: Expression of PRAME Is Associated with Prognosis in Soft Tissue Sarcoma.

Cancers (Basel) 2020 Dec 3;12(12). Epub 2020 Dec 3.

Institute of Pathology, Ludwig-Maximilians-Universität (LMU) Munich, Thalkirchner Str. 36, 80337 Munich, Germany.

(1) Background: PRAME, NY-ESO-1, and SSX2 are cancer testis antigens (CTAs), which are expressed in testicular germ cells with re-expression in numerous cancer types. Their ability to elicit humoral and cellular immune responses have rendered them promising targets for cancer immunotherapy, but they have never been studied in a large and well-characterised cohort of soft tissue sarcomas (STS). (2) Methods: On a protein level, we examined PRAME, NY-ESO-1, and SSX2 expression in tumour tissues of 249 high-risk STS using immunohistochemistry. We correlated expression levels with clinicopathological parameters including tumour-infiltrating lymphocyte (TIL) counts, grading, and long-term survival. (3) Results: Expression of PRAME, NY-ESO-1, and SSX2 was observed in 25 (10%), 19 (8%), and 11 (4%) of 249 specimens with distinct patterns for histo-subtypes. Expression of PRAME was associated with shorter patient survival ( = 0.005) and higher grade (G2 vs. G3, = 0.001), while NY-ESO-1 expression was correlated with more favourable survival ( = 0.037) and lower grade (G2 vs. G3, = 0.029). Both PRAME and NY-ESO-1 expression were more frequent in STS with low TIL counts. In multivariate analysis, high PRAME and low SSX2 expression levels as well as metastatic disease and non-radical resections were independent predictors of shorter overall survival. (4) Conclusions: CTAs PRAME, NY-ESO-1, and SSX2 show distinct expression patterns in different STS subtypes. These results demonstrate their prognostic relevance and may guide future immunotherapeutic approaches in STS.
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http://dx.doi.org/10.3390/cancers12123612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761656PMC
December 2020

A Pan-Cancer Study of Somatic TERT Promoter Mutations and Amplification in 30,773 Tumors Profiled by Clinical Genomic Sequencing.

J Mol Diagn 2021 02 5;23(2):253-263. Epub 2020 Dec 5.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York. Electronic address:

TERT gene promoter mutations are known in multiple cancer types. Other TERT alterations remain poorly characterized. Sequencing data from 30,773 tumors analyzed by a hybridization capture next-generation sequencing assay (Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets) were analyzed for the presence of TERT alterations. Promoter rearrangements (500 bases upstream of the transcriptional start site), hypermethylation (n = 57), and gene expression (n = 155) were evaluated for a subset of cases. Mutually exclusive and recurrent promoter mutations were identified at three hot spots upstream of the transcriptional start site in 11.3% of cases (-124: 74%; -146: 24%; and -138: <2%). Mutually exclusive amplification events were identified in another 2.3% of cases, whereas mutually exclusive rearrangements proximal to the TERT gene were seen in 24 cases. The highest incidence of TERT promoter mutations was seen in cutaneous melanoma (82%), whereas amplification events significantly outnumbered promoter mutations in well-differentiated/dedifferentiated liposarcoma (14.1% versus 2.4%) and adrenocortical carcinoma (13.6% versus 4.5%). Gene expression analysis suggests that the highest levels of gene expression are seen in cases with amplifications and rearrangements. Hypermethylation events upstream of the TERT coding sequence were not mutually exclusive with known pathogenic alterations. Studies aimed at defining the prevalence and prognostic impact of TERT alterations should incorporate other pathogenic TERT alterations as these may impact telomerase function.
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http://dx.doi.org/10.1016/j.jmoldx.2020.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874333PMC
February 2021

A Performance Comparison of Commonly Used Assays to Detect RET Fusions.

Clin Cancer Res 2021 Mar 3;27(5):1316-1328. Epub 2020 Dec 3.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York.

Purpose: Selpercatinib and pralsetinib induce deep and durable responses in patients with advanced fusion-positive lung and thyroid cancer. fusion testing strategies with rapid and reliable results are critical given recent FDA approval. Here, we assess various clinical assays in a large pan-cancer cohort.

Experimental Design: Tumors underwent DNA-based next-generation sequencing (NGS) with reflex to RNA-based NGS if no mitogenic driver or if a structural variant of unknown significance (SVUS) were present. Canonical DNA-level fusions and RNA-confirmed fusions were considered true fusions. Break-apart FISH and IHC performance were assessed in subgroups.

Results: A total of 171 of 41,869 patients with DNA NGS harbored structural variants, including 139 canonical fusions and 32 SVUS. Twelve of 32 (37.5%) SVUS were transcribed into RNA-level fusions, resulting in 151 oncogenic fusions. The most common fusion-positive tumor types were lung (65.6%) and thyroid (23.2%). The most common partners were (45%), (29.1%), and (13.3%). DNA NGS showed 100% (46/46) sensitivity and 99.6% (4,459/4,479) specificity. FISH showed 91.7% (44/48) sensitivity, with lower sensitivity for - (66.7%, 8/12). A total of 87.5% (7/8) of SVUS negative for RNA-level fusions demonstrated rearrangement by FISH. The sensitivity of IHC varied by fusion partner: sensitivity was highest (100%, 31/31), followed by (88.9%, 16/18) and (50%, 6/12). Specificity of RET IHC was 82% (73/89).

Conclusions: Although DNA sequencing has high sensitivity and specificity, RNA sequencing of SVUS is necessary. Both FISH and IHC demonstrated lower sensitivity for - fusions.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285056PMC
March 2021

Identification of Immunohistochemical Reagents for In Situ Protein Expression Analysis of Coronavirus-associated Changes in Human Tissues.

Appl Immunohistochem Mol Morphol 2021 01;29(1):5-12

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York.

We studied the suitability of commercially available monoclonal antibodies (mAbs) for the immunohistochemical (IHC) detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) in standard archival specimens. Antibodies were screened on HEK293 cells transfected with viral nucleoprotein, S1 subunit and S2 subunit of spike protein and on untransfected cells, as well as a panel of normal tissue. Lung tissue with presence of SARS-CoV2 confirmed by in situ hybridization (ISH) was also used. A total of 7 mAbs were tested: (1) mAb 001 (Sino Biological, 40143-R001), (2) mAb 007 (Sino Biological, 40150-R007), (3) mAb 019 (Sino Biological, 40143-R019), (4) mAb 1A9 (GeneTex, GTX632604), (5) mAb ABM19C9 (Abeomics, 10-10007), (6) FIPV3-70 (Santa Cruz, SC-65653), and (7) mAb 6F10 (BioVision, A2060). Only 2 mAbs, clone 001 to the nucleoprotein and clone 1A9 to the S2 subunit spike protein displayed specific immunoreactivity. Both clones showed strong staining in the acute phase of COVID-19 pneumonia, mostly in areas of acute diffuse alveolar damage, but were not completely congruent. Viral protein was also found in kidney tubules, endothelia of multiple organs and a nasal swab of a patient with persistent SARS-CoV2 infection. The other tested reagents were either poorly reactive or demonstrated nonspecific staining in tissues and lesions not infected by SARS-CoV2. Our study demonstrates that rigid specificity testing is mandatory for the evaluation of mAbs to SARS-CoV2 and that clones 001 to nucleoprotein and 1A9 to S2 subunit spike protein are useful for the in situ detection of SARS-CoV2.
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http://dx.doi.org/10.1097/PAI.0000000000000878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713639PMC
January 2021

Molecular epidemiology of IDH2 hotspot mutations in cancer and immunohistochemical detection of R172K, R172G, and R172M variants.

Hum Pathol 2020 12 2;106:45-53. Epub 2020 Oct 2.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

IDH1/2 hotspot mutations occur in glioma, cholangiocarcinoma, chondrosarcoma, sinonasal carcinoma, and T-cell lymphoma and have diagnostic, prognostic, and/or therapeutic value. Availability of immunohistochemistry (IHC) protocols for specific IDH2 mutation detection is limited. A targeted exome sequencing assay MSK-IMPACT cohort comprising >38,000 cancer cases was explored for the presence of IDH1/2 mutations in solid malignancies and select T-cell lymphomas. Seventy-four formalin-fixed paraffin-embedded IDH1/2-mutated (n = 62) and wild-type (n = 12) samples were used for testing and optimization of anti-IDH2 monoclonal antibodies (mAbs) 14H7, 3C11, and MMab1 targeting R172K, R172G, and R172M mutant proteins, respectively. IDH1/2 mutations were common in glioma (26.8% and 1.6%), intrahepatic cholangiocarcinoma (23.1% and 5.7%), chondrosarcoma (19.4% and 10.7%), sinonasal undifferentiated/large-cell neuroendocrine carcinoma (0% and 84.2%), angioimmunoblastic T-cell lymphoma (0% and 22%), and peripheral T-cell lymphoma (0 and 5.1%). In other cancers, IDH2 mutations were rare. IDH2 R172 variants included R172K (39%), R172S (29%), R172W (12%), R172G (10%), R172M (5%), and R172T (4%). 14H7, 3C11, and MMab1 detected all IDH2 R172K, R172G, and R172M, respectively, and produced a crisp, granular cytoplasmic staining pattern. 3C11 was also positive in 5 of 6 IDH1 R132G mutants showing a homogeneous, smooth cytoplasmic staining. All 3 mAbs were negative in other IDH1/2 mutant or wild-type cases. IHC using mAbs 14H7, 3C11, and MMab1 can facilitate molecular diagnosis as a reliable, fast, and inexpensive alternative for specific IDH2 variant detection. Given the distinct distribution of IDH2 R172 mutations in cancers, these mAbs could also serve as useful pathologic diagnostic markers.
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http://dx.doi.org/10.1016/j.humpath.2020.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721993PMC
December 2020

SCLC Subtypes Defined by ASCL1, NEUROD1, POU2F3, and YAP1: A Comprehensive Immunohistochemical and Histopathologic Characterization.

J Thorac Oncol 2020 12 1;15(12):1823-1835. Epub 2020 Oct 1.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York. Electronic address:

Introduction: Recent studies have identified subtypes of small cell lung carcinoma (SCLC) defined by the RNA expression of ASCL1, NEUROD1, POU2F3, and YAP1 transcriptional regulators. There are only limited data on the distribution of these markers at the protein level and associated pathologic characteristics in clinical SCLC samples.

Methods: The expression of ASCL1, NEUROD1, POU2F3, and YAP1 was analyzed by immunohistochemistry in 174 patient samples with SCLC. Subtypes defined by these markers were correlated with histologic characteristics, expression of classic neuroendocrine markers (synaptophysin, chromogranin A, CD56, INSM1), and other SCLC markers, including the neuroendocrine phenotype-associated markers TTF-1 and DLL3.

Results: ASCL1 and NEUROD1 expression had the following distribution: (1) 41% ASCL1+/NEUROD1-; (2) 37% ASCL1+/NEUROD1+; (3) 8% ASCL1-/NEUROD1+; and (4) 14% ASCL1-/NEUROD1-. On the basis of their relative expression, 69% of cases were ASCL1-dominant and 17% were NEUROD1-dominant. POU2F3 was expressed in 7% of SCLC and was mutually exclusive of ASCL1 and NEUROD1. YAP1 was expressed at low levels, primarily in combined SCLC, and was not exclusive of other subtypes. Both ASCL1-dominant and NEUROD1-dominant subtypes were associated with neuroendocrine marker/TTF-1/DLL3 profile, whereas POU2F3 and other ASCL1/NEUROD1 double-negative tumors were neuroendocrine marker/TTF-1/DLL3.

Conclusions: This is the first comprehensive immunohistochemical and histopathologic analysis of novel SCLC subtypes in patient samples. We confirm that ASCL1/NEUROD1 double-negative tumors represent a distinct neuroendocrine-low subtype of SCLC, which is either uniquely associated with POU2F3 or lacks a known dominant regulator. The expression profiles of these markers appear more heterogeneous in native samples than in experimental models, particularly with regard to the high prevalence of ASCL1/NEUROD1 coexpression. These findings may have prognostic and therapeutic implications and warrant further clinical investigation.
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http://dx.doi.org/10.1016/j.jtho.2020.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8362797PMC
December 2020

Genomic Profiling Aids Classification of Diagnostically Challenging Uterine Mesenchymal Tumors With Myomelanocytic Differentiation.

Am J Surg Pathol 2021 01;45(1):77-92

Departments of Pathology.

Although diagnosis of high-grade uterine mesenchymal tumors (UMTs) exhibiting classic morphologic features is straightforward, diagnosis is more challenging in tumors in which prototypical features are poorly developed, focal, and/or coexist with features seen in other neoplasms. Here, we sought to define the repertoire of somatic genetic alterations in diagnostically challenging UMTs with myomelanocytic differentiation, including some reported as perivascular epithelioid cell tumors (PEComas). In 17 samples from 15 women, the tumors were histologically heterogenous. Immunohistochemical expression of at least 1 melanocytic marker (HMB45, Melan-A, or MiTF) was identified in all tumors, and of myogenic markers (desmin or smooth muscle actin) in most tumors. Targeted massively parallel sequencing revealed several genetic alterations, most commonly in TP53 (41% mutation, 12% deletion), TSC2 (29% mutation, 6% deletion), RB1 (18% deletion), ATRX (24% mutation), MED12 (12% mutation), BRCA2 (12% deletion), CDKN2A (6% deletion) as well as FGFR3, NTRK1, and ERBB3 amplification (each 6%). Gene rearrangements (JAZF1-SUZ12; DNAJB6-PLAG1; and SFPQ-TFE3) were identified in 3 tumors. Integrating histopathologic, immunohistochemical, and genetic findings, tumors from 4 patients were consistent with malignant PEComa (1 TFE3-rearranged); 6 were classified as leiomyosarcomas; 3 showed overlapping features of PEComa and other sarcoma types (leiomyosarcoma or low-grade endometrial stromal sarcoma); and 2 were classified as sarcoma, not otherwise specified. Our findings suggest that diagnostically challenging UMTs with myomelanocytic differentiation represent a heterogenous group of neoplasms which harbor a diverse repertoire of somatic genetic alterations; these genetic alterations can aid classification.
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http://dx.doi.org/10.1097/PAS.0000000000001572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276853PMC
January 2021

Proteomic analysis shows that the main constituent of subepidermal localised cutaneous amyloidosis is not galectin-7.

Amyloid 2021 Mar 1;28(1):35-41. Epub 2020 Sep 1.

Hematopathology Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Lichen or macular localised cutaneous amyloidoses have long been described as keratinic amyloidoses and believed to be due to the deposition of cytokeratin peptides originating from epidermis in the dermal papillae. However, recently it was suggested that galectin-7 is the causative protein for this type of amyloidosis. This was based on the detection of galectin-7 in a biopsy from a patient diagnosed with Bowen's disease and localised cutaneous amyloidosis. In this study we report mass spectrometry-based proteomic analysis of the protein composition of localised cutaneous amyloid deposits from seven patients using laser microdissection and show that basal keratins are the main constituents of the amyloid deposits. Galectin-7 was not present in the dermal amyloid deposits and was only present in the overlying Congo red negative epidermis.
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http://dx.doi.org/10.1080/13506129.2020.1811962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962860PMC
March 2021

Insights into pathogenesis of fatal COVID-19 pneumonia from histopathology with immunohistochemical and viral RNA studies.

Histopathology 2020 Dec 16;77(6):915-925. Epub 2020 Oct 16.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Introduction: We describe post-mortem pulmonary histopathologic findings of COVID-19 pneumonia in patients with a spectrum of disease course, from rapid demise to prolonged hospitalisation.

Methods And Results: Histopathologic findings in post-mortem lung tissue from eight patients who died from COVID-19 pneumonia were reviewed. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were performed to detect virus. Diffuse alveolar damage (DAD) was seen in all cases with a spectrum of acute phase and/or organising phase. IHC with monoclonal antibodies against SARS-CoV-2 viral nucleoprotein and spike protein detected virus in areas of acute but not organising DAD, with intracellular viral antigen and RNA expression seen predominantly in patients with duration of illness less than 10 days. Major vascular findings included thrombi in medium- and large-calibre vessels, platelet microthrombi detected by CD61 IHC and fibrin microthrombi.

Conclusions: Presence of SARS-CoV-2 viral RNA by NGS early in the disease course and expression of viral antigen by IHC exclusively in the acute, but not in the organising phase of DAD, suggests that the virus may play a major role in initiating the acute lung injury of DAD, but when DAD progresses to the organising phase the virus may have been cleared from the lung by the patient's immune response. These findings suggest the possibility of a major change during the disease course of COVID-19 pneumonia that may have therapeutic implications. Frequent thrombi and microthrombi may also present potential targets for therapeutic intervention.
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http://dx.doi.org/10.1111/his.14201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361244PMC
December 2020

A Molecular Reappraisal of Glomus Tumors and Related Pericytic Neoplasms With Emphasis on NOTCH-gene Fusions.

Am J Surg Pathol 2020 11;44(11):1556-1562

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY.

Glomus tumors (GTs), together with myofibroma (MF), myopericytoma (MP), and angioleiomyoma (AL) are classified as members of the perivascular myoid family of tumors. The reported genetic abnormalities across these neoplasms is dissimilar, arguing against a pathogenetically unified family; half of the GT showing NOTCH-gene fusions and a smaller subset BRAF V600E mutations, while PDGFRB mutations are noted in a subset of MF and MP. This study aimed to investigate the prevalence and specificity of NOTCH-gene fusions in a large group of GT and correlate with clinical features. BRAF-VE1 and PDGFRB immunoexpression was also investigated in this cohort. A total of 93 GT and 43 other pericytic lesions (11 MP, 13 MF, and 19 AL) were selected. All cases were tested by fluorescence in situ hybridization for NOTCH1-4 and MIR143 gene abnormalities and 6 cases were investigated by targeted RNA-sequencing. Fluorescence in situ hybridization revealed NOTCH-gene rearrangements in 50 (54%) GT, 2 MP (18%), and 2 AL (11%). NOTCH-rearrangements were present in 34 (68%) benign and 16 (32%) malignant GT. Fusion-positive benign GT were overwhelmingly seen in males with a predilection for extremities, while the malignant GT occurred mostly in viscera. Among the fusion-negative GT, 88% were benign, 9% uncertain malignant potential, and 2% malignant. Half of the fusion-negative GTs occurred in the finger/subungual region. In summary, rearrangements of NOTCH genes are seen in over half of GT, with NOTCH2-MIR143 being the most common fusion (73%), while only a small subset of AL and MP share these abnormalities. The common subungual GT subset lack NOTCH-gene fusions suggesting an alternative pathogenesis. BRAF-VE1 was negative in all 37 cases studied, while strong PDGFRB staining was seen in 14 (21%) cases. Additional studies are needed to investigate the genetic alterations in the fusion-negative cases.
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http://dx.doi.org/10.1097/PAS.0000000000001531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554116PMC
November 2020

Comparison of Immunohistochemistry for PRAME With Cytogenetic Test Results in the Evaluation of Challenging Melanocytic Tumors.

Am J Surg Pathol 2020 07;44(7):893-900

Department of Pathology, Memorial Sloan Kettering Cancer, New York, NY.

PRAME (PReferentially expressed Antigen in MElanoma) is a melanoma-associated antigen. Although diffuse immunoreactivity for PRAME is found in most primary cutaneous melanomas, melanocytic nevi express PRAME usually only in a subpopulation of tumor cells or not at all. Hence, testing for PRAME expression has the potential to provide useful information for the assessment for diagnostically ambiguous melanocytic neoplasms. Many of the latter tumors are currently studied by cytogenetic methods for ancillary evidence in support of or against a diagnosis of melanoma. In this study we analyzed 110 diagnostically problematic melanocytic tumors comparing results for PRAME immunohistochemistry (IHC) with those from fluorescence in situ hybridization and/or single nucleotide polymorphism-array, and each with the final diagnostic interpretation. In 90% of cases there was concordance between PRAME IHC and cytogenetic tests results, and in 92.7% concordance between PRAME IHC and the final diagnosis. The high concordance between PRAME IHC and cytogenetic test results as well as the final diagnosis supports the use of PRAME IHC as an ancillary test in the evaluation of ambiguous primary cutaneous melanocytic neoplasms, especially given its practical advantage of lower cost and faster turnaround over cytogenetic or gene expression studies. However, our results indicate that PRAME IHC and cytogenetic tests for melanocytic tumors are not entirely interchangeable and on occasion each type of test may yield false-negative or false-positive results.
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http://dx.doi.org/10.1097/PAS.0000000000001492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289661PMC
July 2020

Genomic Landscape of Uterine Sarcomas Defined Through Prospective Clinical Sequencing.

Clin Cancer Res 2020 07 16;26(14):3881-3888. Epub 2020 Apr 16.

Gynecologic Medical Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.

Purpose: We examined whether prospective molecular characterization of advanced metastatic disease can reveal grade and/or histology-specific differences to inform diagnosis and facilitate enrollment onto clinical trials.

Experimental Design: Patients with uterine sarcoma consented to a prospective study of next-generation sequencing (NGS). Clinical annotations were extracted from their medical record. Tumor and matched normal DNA were subjected to NGS, and the genomic landscape was explored for survival correlations and therapeutic targetability.

Results: Tumors from 107 women were sequenced and included leiomyosarcoma ( = 80), high-grade non-leiomyosarcoma ( = 22), low-grade endometrial stromal sarcoma (LG-ESS, = 4), and smooth muscle tumor of uncertain malignant potential (STUMP, = 2). Genomic profiling influenced histologic diagnosis in three cases. Common uterine leiomyosarcoma alterations were loss-of-function mutations in (56%), (51%), and (31%). Homozygous deletions of were present in 5% of these patients. alteration frequency was higher in the metastases samples as compared with the primary samples. Genomes of low-grade tumors were largely silent, while 50.5% of high-grade tumors had whole-genome duplication. Two metastatic uterine leiomyosarcoma cases were hypermutated. Both had prolonged disease-free survival. Potentially actionable mutations were identified in 48 patients (45%), 8 (17%) of whom received matched therapy with 2 achieving clinical responses. Among patients with uterine leiomyosarcoma with somatic alterations, sustained partial responses were observed with PARP inhibitor-containing therapy.

Discussion: Prospective genomic profiling can contribute to diagnostic precision and inform treatment selection in patients with uterine sarcomas. There was evidence of clinical benefit in patients with uterine leiomyosarcoma with somatic alterations treated with PARP inhibitors.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367750PMC
July 2020

Immunohistochemical analysis of IDH2 R172 hotspot mutations in breast papillary neoplasms: applications in the diagnosis of tall cell carcinoma with reverse polarity.

Mod Pathol 2020 06 2;33(6):1056-1064. Epub 2020 Jan 2.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Tall cell carcinoma with reverse polarity is a rare subtype of breast carcinoma with solid and papillary growth and nuclear features reminiscent of those of the tall cell variant of papillary thyroid carcinoma. These tumors harbor recurrent IDH2 R172 hotspot mutations or TET2 mutations, co-occurring with mutations in PI3K pathway genes. Diagnosis of tall cell carcinomas with reverse polarity is challenging in view of their rarity and the range of differential diagnosis. We sought to determine the sensitivity and specificity of IDH2 R172 immunohistochemistry for the detection of IDH2 R172 hotspot mutations in this entity. We evaluated 14 tall cell carcinomas with reverse polarity (ten excision and five core needle biopsy specimens), 13 intraductal papillomas, 16 solid papillary carcinomas, and 5 encapsulated papillary carcinomas by Sanger sequencing of the IDH2 R172 hotspot locus and of exons 9 and 20 of PIK3CA, and by immunohistochemistry using monoclonal antibodies (11C8B1) to the IDH2 R172S mutation. The 14 tall cell carcinomas with reverse polarity studied harbored IDH2 R172 hotspot mutations, which co-occurred with PIK3CA hotspot mutations in 50% of cases. None of the other papillary neoplasms analyzed displayed IDH2 R172 mutations, however PIK3CA hotspot mutations were detected in 54% of intraductal papillomas, 6% of solid papillary carcinomas, and 20% of encapsulated papillary carcinomas tested. Immunohistochemical analysis with anti-IDH2 R172S antibodies (11C8B1) detected IDH2 R172 mutated protein in 93% (14/15) of tall cell carcinomas with reverse polarity samples including excision (n = 9/10) and core needle biopsy specimens (n = 5), whereas the remaining papillary neoplasms (n = 34) were negative. Our findings demonstrate that immunohistochemical analysis of IDH2 R172 is highly sensitive and specific for the detection of IDH2 R172 hotspot mutations, and likely suitable as a diagnostic tool in the evaluation of excision and core needle biopsy material of tall cell carcinomas with reverse polarity.
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http://dx.doi.org/10.1038/s41379-019-0442-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286791PMC
June 2020

Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary.

Nat Commun 2020 01 2;11(1):44. Epub 2020 Jan 2.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

Sclerosing stromal tumor (SST) of the ovary is a rare type of sex cord-stromal tumor (SCST), whose genetic underpinning is currently unknown. Here, using whole-exome, targeted capture and RNA-sequencing, we report recurrent FHL2-GLI2 fusion genes in 65% (17/26) of SSTs and other GLI2 rearrangements in additional 15% (4/26) SSTs, none of which are detected in other types of SCSTs (n = 48) or common cancer types (n = 9,950). The FHL2-GLI2 fusions result in transcriptomic activation of the Sonic Hedgehog (SHH) pathway in SSTs. Expression of the FHL2-GLI2 fusion in vitro leads to the acquisition of phenotypic characteristics of SSTs, increased proliferation, migration and colony formation, and SHH pathway activation. Targeted inhibition of the SHH pathway results in reversal of these oncogenic properties, indicating its role in the pathogenesis of SSTs. Our results demonstrate that the FHL2-GLI2 fusion is likely the oncogenic driver of SSTs, defining a genotypic-phenotypic correlation in ovarian neoplasms.
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http://dx.doi.org/10.1038/s41467-019-13806-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940380PMC
January 2020

Immunohistochemical assessment of HRAS Q61R mutations in breast adenomyoepitheliomas.

Histopathology 2020 May;76(6):865-874

Department of Histopathology, Nottingham University Hospitals NHS Trust, Nottingham, UK.

Aims: Breast adenomyoepitheliomas (AMEs) are uncommon tumours. Most oestrogen receptor (ER)-positive AMEs have mutations in phosphoinositide 3-kinase (PI3K) pathway genes, whereas ER-negative AMEs usually harbour concurrent mutations affecting the HRAS Q61 hotspot and PI3K pathway genes. Here, we sought to determine the sensitivity and specificity of RAS Q61R immunohistochemical (IHC) analysis for detection of HRAS Q61R mutations in AMEs.

Methods And Results: Twenty-six AMEs (14 ER-positive; 12 ER-negative) previously subjected to massively parallel sequencing (n = 21) or Sanger sequencing (n = 5) of the HRAS Q61 hotspot locus were included in this study. All AMEs were subjected to IHC analysis with a monoclonal (SP174) RAS Q61R-specific antibody, in addition to detailed histopathological analysis. Nine ER-negative AMEs harboured HRAS mutations, including Q61R (n = 7) and Q61K (n = 2) mutations. Five of seven (71%) AMEs with HRAS Q61R mutations were immunohistochemically positive, whereas none of the AMEs lacking HRAS Q61R mutations (n = 17) were immunoreactive. RAS Q61R immunoreactivity was restricted to the myoepithelium in 80% (4/5) of cases, whereas one case showed immunoreactivity in both the epithelial component and the myoepithelial component. RAS Q61R immunohistochemically positive AMEs were associated with infiltrative borders (P < 0.001), necrosis (P < 0.01) and mitotic index in the epithelial (P < 0.05) and myoepithelial (P < 0.01) components. RAS Q61R IHC assessment did not reveal Q61K mutations (0/2).

Conclusions: IHC analysis of RAS Q61R shows high specificity (100%) and moderate sensitivity (71%) for detection of HRAS Q61R mutations in breast AMEs, and appears not to detect HRAS Q61K mutations. IHC analysis of RAS Q61R may constitute a useful technique in the diagnostic workup of ER-negative AMEs.
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http://dx.doi.org/10.1111/his.14057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225035PMC
May 2020

Histone H3K36I mutation in a metastatic histiocytic tumor of the skull and response to sarcoma chemotherapy.

Cold Spring Harb Mol Case Stud 2019 10 23;5(5). Epub 2019 Oct 23.

Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

Recurrent somatic missense mutations in histone H3 genes have been identified in subsets of pediatric cancers. H3K36 histone mutations have recently been recognized as oncogenic drivers in rare subsets of malignant soft tissue sarcomas but have not been reported in histiocytic neoplasms. Currently, the histological and molecular spectrum, as well as the clinical behavior of H3K36-mutant soft tissue malignancies, is largely unknown. We describe a pediatric patient with a K36I-mutant histiocytic tumor arising in the skull. After the failure of upfront therapy for histiocytosis and development of widely disseminated metastatic disease, the patient had an exceptional response to empiric chemotherapy and remains in complete disease remission for more than 5 years. Our report expands the histological spectrum of H3K36M/I-mutant soft tissue malignancies to histiocytic neoplasms and indicates that multiagent sarcoma-like chemotherapy can be highly effective even in the setting of widely disseminated metastatic disease.
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http://dx.doi.org/10.1101/mcs.a004606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824254PMC
October 2019

Immunohistochemistry for PRAME in the Distinction of Nodal Nevi From Metastatic Melanoma.

Am J Surg Pathol 2020 04;44(4):503-508

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY.

The distinction of metastatic melanoma from melanocytic nevi in lymph nodes can on occasion be difficult. As diffuse immunohistochemical (IHC) PRAME (PReferentially expressed Antigen in MElanoma) expression is detected in the majority of primary and metastatic melanomas, but rarely in nevi, we reasoned that PRAME could be a useful adjunct marker for the diagnosis of melanocytes in lymph nodes. In this study, we examined 45 nodal melanocytic deposits comprising 30 nodal nevi and 15 melanoma metastases. The latter were diagnostically not straightforward because they either coexisted with nodal nevi or were present in perinodal fibrous tissue. All nodal nevi (30/30) were negative for PRAME, whereas all melanoma metastases (15/15) were diffusely positive for PRAME IHC. We additionally report the novel use of a PRAME/Melan A dual-label immunostain. Our results show that PRAME IHC may be useful in the assessment of diagnostically challenging nodal melanocytic deposits, such as intraparenchymal nodal nevi, metastases confined to the capsular fibrous tissue, or in the setting of small metastases coexisting with a nodal nevus in the same lymph node.
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http://dx.doi.org/10.1097/PAS.0000000000001393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071962PMC
April 2020

TFEB Expression Profiling in Renal Cell Carcinomas: Clinicopathologic Correlations.

Am J Surg Pathol 2019 11;43(11):1445-1461

Departments of Pathology.

TFEB is overexpressed in TFEB-rearranged renal cell carcinomas as well as in renal tumors with amplifications of TFEB at 6p21.1. As recent literature suggests that renal tumors with 6p21.1 amplification behave more aggressively than those with rearrangements of TFEB, we compared relative TFEB gene expression in these tumors. This study included 37 TFEB-altered tumors: 15 6p21.1-amplified and 22 TFEB-rearranged (including 5 cases from The Cancer Genome Atlas data set). TFEB status was verified using a combination of fluorescent in situ hybridization (n=27) or comprehensive molecular profiling (n=13) and digital droplet polymerase chain reaction was used to quantify TFEB mRNA expression in 6p21.1-amplified (n=9) and TFEB-rearranged renal tumors (n=19). These results were correlated with TFEB immunohistochemistry. TFEB-altered tumors had higher TFEB expression when normalized to B2M (mean: 168.9%, n=28), compared with non-TFEB-altered controls (mean: 7%, n=18, P=0.005). Interestingly, TFEB expression in tumors with rearrangements (mean: 224.7%, n=19) was higher compared with 6p21.1-amplified tumors (mean: 51.2%, n=9; P=0.06). Of note, classic biphasic morphology was only seen in TFEB-rearranged tumors and when present correlated with 6.8-fold higher TFEB expression (P=0.00004). Our results suggest that 6p21.1 amplified renal tumors show increased TFEB gene expression but not as much as t(6;11) renal tumors. These findings correlate with the less consistent/diffuse expression of downstream markers of TFEB activation (cathepsin K, melan A, HMB45) seen in the amplified neoplasms. This suggests that the aggressive biological behavior of 6p21.1 amplified renal tumors might be secondary to other genes at the 6p21.1 locus that are co-amplified, such as VEGFA and CCND3, or other genetic alterations.
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http://dx.doi.org/10.1097/PAS.0000000000001307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6788764PMC
November 2019

Evaluation of cancer testis antigen (CT10, PRAME) and MHC I expression in high-grade urothelial carcinoma of the bladder.

Virchows Arch 2020 Apr 2;476(4):535-542. Epub 2019 Sep 2.

Division of Anatomic Pathology, Department of Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Sciences Centre, 2075 Bayview Avenue, Toronto, ON, M4N 3M5, Canada.

Immunotherapeutic strategies are increasingly used in the treatment of a number of malignancies including high-grade urothelial carcinoma (HGUC) of the bladder. Because of this, detailed and accurate assessment of the tumor immune microenvironment is paramount. In this study, we aimed to correlate the composition of the tumor immune microenvironment with oncologic outcome and the expression of two cancer testis antigens (CTAs), CT10 and PRAME, potential cancer vaccine targets, as well as major histocompatibility complex I (MHC I), a molecule associated with tumor immune escape and resistance to immunotherapy. Triplicate tissue microarrays (TMAs) were constructed using 207 cases of HGUC of the bladder. Oncologic outcome data was gathered for each case. Consecutive sections from the TMA blocks were stained with CD3, CD4, CD8, FOXP3, PD1, PD-L1, CT10, PRAME, and MHC I. Twenty-one percent and 15% of cases expressed CT10 and PRAME, respectively. Eighty-eight percent of cases showed absent or decreased MHC I expression. CT10-expressing tumors showed a significantly worse disease specific survival (p = 0.007, hazard ratio 2.245, confidence interval 1.223-4.122). CT10, PRAME, and MHC I expression significantly correlated with other some immune parameters. CT10 and PRAME are expressed in a subset of HGUC and CTA and MHC I expression correlate with a number of important immune parameters. Together, these findings highlight the potential for exploring novel immune therapeutic strategies in HGUC. Additional studies evaluating the clinical relevance of these findings are warranted.
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http://dx.doi.org/10.1007/s00428-019-02661-2DOI Listing
April 2020

Pan-Trk immunohistochemistry is a sensitive and specific ancillary tool for diagnosing secretory carcinoma of the salivary gland and detecting ETV6-NTRK3 fusion.

Histopathology 2020 Feb 11;76(3):375-382. Epub 2019 Dec 11.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Aims: Secretory carcinoma (SC) of the salivary gland typically harbours ETV6-NTRK3 fusion, which can be utilised clinically to assist with diagnosis. Pan-Trk inhibitor therapy has demonstrated drastic responses in patients with NTRK-translocated tumours, including SC. Pan-Trk immunohistochemistry (IHC) is emerging as a sensitive and specific tool for detecting NTRK1, NTRK2 and NTRK3 fusions in various cancers. We aimed to establish the specificity and sensitivity of pan-Trk IHC in diagnosing SC and detecting ETV6-NTRK3 fusion. A literature review on the utility of pan-Trk IHC was conducted.

Methods And Results: Pan-Trk IHC was performed on 83 salivary gland neoplasms (29 SCs and 54 non-SCs). ETV6-NTRK3 fusion status was established in 25 cases. With any staining (nuclear or cytoplasmic) as a positive threshold, the sensitivity and specificity of pan-Trk IHC were 90% and 70% in diagnosing SC, and 100% and 0% in detecting NTRK3 fusion. When only pan-Trk nuclear staining was considered as positive, the sensitivity and specificity were 69% and 100% in diagnosing SC, and 92% and 100% in detecting NTRK3 fusion.

Conclusions: Nuclear pan-Trk IHC is highly specific for SC diagnosis, with a specificity approaching 100%, making it a useful and precise diagnostic tool for differentiating SC from its histological mimics. On the other hand, any pan-Trk staining (nuclear or cytoplasmic) is highly sensitive for SC, and can serve as an attractive, cheap, fast and accessible screening tool for selecting patients to undergo confirmative molecular testing for clinical trials using TRK inhibitors.
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http://dx.doi.org/10.1111/his.13981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994356PMC
February 2020

CytoLyt fixation significantly inhibits MIB1 immunoreactivity whereas alternative Ki-67 clone 30-9 is not susceptible to the inhibition: Critical diagnostic implications.

Cancer Cytopathol 2019 10 9;127(10):643-649. Epub 2019 Aug 9.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York.

Background: The Ki-67 proliferation marker has multiple diagnostic and prognostic applications. Although several clones to the Ki-67 antigen are commercially available, the MIB1 clone is widely recommended in the surgical pathology literature for neuroendocrine tumors. In our cytopathology practice, we have encountered unexpectedly low MIB1 immunoreactivity in CytoLyt-fixed cell blocks (CBs). The current study evaluated the impact of fixatives, CB processing, and immunocytochemical (ICC) procedures on Ki-67 immunoreactivity.

Methods: Test CBs were prepared from freshly resected tumors, and multiple variables in the MIB1 ICC procedure were tested, including CytoLyt versus formalin collection media, MIB1 versus other Ki-67 clones including 30-9, and other variables. MIB1 versus Ki-67 30-9 clones were tested in parallel on CytoLyt-fixed CBs from clinical samples of small cell lung carcinoma (SCLC).

Results: In the test CBs (n = 10), the mean MIB1 labeling index was 10% in CytoLyt versus 47% in formalin (P = .0116), with a mean loss of reactivity in matched CBs of 37% (up to 70%). None of the procedure modifications tested in 223 individual ICC reactions recovered MIB1 reactivity in CytoLyt except for switching to the Ki-67 30-9 antibody. In CytoLyt-fixed SCLC samples (n = 14), the Ki-67 30-9 antibody demonstrated expected ranges of reactivity (mean, 83%; range, 60%-100%), whereas MIB1 demonstrated markedly inhibited labeling (mean, 60%; range, 10%-95%) (P = .0058).

Conclusions: CytoLyt fixation substantially inhibits MIB1 immunoreactivity, whereas the Ki-67 30-9 clone is not susceptible to inhibition. Markedly discrepant MIB1 reactivity may present a pitfall in the diagnosis of SCLC and may lead to the incorrect prognostic stratification of other tumor types. For laboratories using CytoLyt, we recommend using the Ki-67 30-9 antibody rather than the MIB1 antibody.
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http://dx.doi.org/10.1002/cncy.22170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8375359PMC
October 2019

NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls.

Mod Pathol 2020 01 2;33(1):38-46. Epub 2019 Aug 2.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

With the FDA approval of larotrectinib, NTRK fusion assessment has recently become a standard part of management for patients with locally advanced or metastatic cancers. Unlike somatic mutation assessment, the detection of NTRK fusions is not straightforward, and various assays exist at the DNA, RNA, and protein level. Here, we investigate the performance of immunohistochemistry and DNA-based next-generation sequencing to indirectly or directly detect NTRK fusions relative to an RNA-based next-generation sequencing approach in the largest cohort of NTRK fusion positive solid tumors to date. A retrospective analysis of 38,095 samples from 33,997 patients sequenced by a targeted DNA-based next-generation sequencing panel (MSK-IMPACT), 2189 of which were also examined by an RNA-based sequencing assay (MSK-Fusion), identified 87 patients with oncogenic NTRK1-3 fusions. All available institutional NTRK fusion positive cases were assessed by pan-Trk immunohistochemistry along with a cohort of control cases negative for NTRK fusions by next-generation sequencing. DNA-based sequencing showed an overall sensitivity and specificity of 81.1% and 99.9%, respectively, for the detection of NTRK fusions when compared to RNA-based sequencing. False negatives occurred when fusions involved breakpoints not covered by the assay. Immunohistochemistry showed overall sensitivity of 87.9% and specificity of 81.1%, with high sensitivity for NTRK1 (96%) and NTRK2 (100%) fusions and lower sensitivity for NTRK3 fusions (79%). Specificity was 100% for carcinomas of the colon, lung, thyroid, pancreas, and biliary tract. Decreased specificity was seen in breast and salivary gland carcinomas (82% and 52%, respectively), and positive staining was often seen in tumors with neural differentiation. Both sensitivity and specificity were poor in sarcomas. Selection of the appropriate assay for NTRK fusion detection therefore depends on tumor type and genes involved, as well as consideration of other factors such as available material, accessibility of various clinical assays, and whether comprehensive genomic testing is needed concurrently.
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http://dx.doi.org/10.1038/s41379-019-0324-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437403PMC
January 2020
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