APOBEC3 mutagenesis drives therapy resistance in breast cancer.

Acquired genetic alterations commonly drive resistance to endocrine and targeted therapies in metastatic breast cancer 1-7 , however the underlying processes engendering these diverse alterations are largely uncharacterized. To identify the mutational processes operant in breast cancer and their impact on clinical outcomes, we utilized a well-annotated cohort of 3,880 patient samples with paired tumor-normal sequencing data. The mutational signatures associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) enzymes were highly prevalent and enriched in post-treatment compared to treatment-naïve hormone receptor-positive (HR+) cancers. APOBEC3 mutational signatures were independently associated with shorter progression-free survival on antiestrogen plus CDK4/6 inhibitor combination therapy in patients with HR+ metastatic breast cancer. Whole genome sequencing (WGS) of breast cancer models and selected paired primary-metastatic samples demonstrated that active APOBEC3 mutagenesis promoted resistance to both endocrine and targeted therapies through characteristic alterations such as RB1 loss-of-function mutations. Evidence of APOBEC3 activity in pre-treatment samples illustrated a pervasive role for this mutational process in breast cancer evolution. The study reveals APOBEC3 mutagenesis to be a frequent mediator of therapy resistance in breast cancer and highlights its potential as a biomarker and target for overcoming resistance.

Somatic mutations in FAS pathway increase hemophagocytic lymphohistiocytosis risk in patients with T- and/or NK-cell lymphoma.

ABSTRACT: Although significant progress has been made in understanding the genetic basis of primary hemophagocytic lymphohistiocytosis (HLH), the pathogenesis of secondary HLH, the more prevalent form, remains unclear. Among the various conditions giving rise to secondary HLH, HLH in patients with lymphoma (HLH-L) accounts for a substantial proportion. In this study, we investigated the role of somatic mutations in the pathogenesis of HLH-L in a cohort of patients with T- and/or natural killer-cell lymphoma. We identified a 3-time higher frequency of mutations in FAS pathway in patients with HLH-L. Patients harboring these mutations had a 5-time increased HLH-L risk. These mutations were independently associated with inferior outcome. Hence, our study demonstrates the association between somatic mutations in FAS pathway and HLH-L. Further studies are warranted on the mechanistic role of these mutations in HLH-L.

Comparison of Immunohistochemistry, Next-generation Sequencing and Fluorescence In Situ Hybridization for Detection of MTAP Loss in Pleural Mesothelioma.

9p21 deletions involving MTAP/CDKN2A genes are detected in diffuse pleural mesotheliomas (DPM) but are absent in benign mesothelial proliferations. Loss of MTAP expression by immunohistochemistry (IHC) is well accepted as a surrogate for 9p21 deletion to support a diagnosis of DPM. Accurate interpretation can be critical in the diagnosis of DPM, but variations in antibody performance may impact interpretation. The objectives of this study were to compare the performance of MTAP monoclonal antibodies (mAbs) EPR6893 and 1813 and to compare MTAP expression by IHC with 9p21 copy number status in DPM. Cytoplasmic expression of MTAP IHC with mAbs EPR6893 (ab126770; Abcam) and 1813 (NBP2-75730, Novus Biologicals) was evaluated in 56 DPM (47 epithelioid, 7 biphasic, and 2 sarcomatoid) profiled by targeted next-generation sequencing. 9p21 Copy number status was assessed by Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) analysis and also by CDKN2A fluorescence in situ hybridization in discrepant cases when material was available. MTAP mAb 1813 showed stronger immunoreactivity, more specific staining, and no equivocal interpretations compared to mAb EPR6893 which showed equivocal staining in 19 (34%) of cases due to weak or heterogenous immunoreactivity, lack of definitive internal positive control, and/or nonspecific background staining. MTAP expression with mAb 1813 showed near perfect agreement with 9p21 copy number by combined FACETS/fluorescence in situ hybridization calls (κ = 0.85; 95% CI, 0.71-0.99; P < .001). MTAP IHC with mAb 1813 was 96% sensitive, 86% specific, and 93% accurate for 9p21 homozygous deletion. The findings of this study suggest that interpretation of MTAP IHC is improved with mAb 1813 because mAb EPR6893 was often limited by equivocal interpretations. We show that MTAP IHC and molecular assays are complementary in detecting 9p21 homozygous deletion. MTAP IHC may be particularly useful for low tumor purity samples and in low-resource settings.

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

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.

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

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.

Immunohistochemical Detection of Cancer-Testis Antigen PRAME.

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.

mTOR Pathway Activation Assessed by Immunohistochemistry in Cervical Biopsies of HPV-associated Endocervical Adenocarcinomas (HPVA): Correlation With Silva Invasion Patterns.

The Silva pattern of invasion, recently introduced to stratify patients at risk for lymph node metastases in human papillomavirus-associated endocervical adenocarcinomas (HPVAs), can only be assessed in cone and loop electrosurgical excision procedure excisions with negative margins or in a hysterectomy specimen. Previous studies found associations between destructive stromal invasion patterns (Silva patterns B and C) and mutations in genes involved in the MEK/PI3K pathways that activate the mammalian target of rapamycin (mTOR) pathway. The primary aim of this study was to use cervical biopsies to determine whether markers of mTOR pathway activation associate with aggressive invasion patterns in matched excision specimens. The status of the markers in small biopsy specimens should allow us to predict the final and biologically relevant pattern of invasion in a resection specimen. Being able to predict the final pattern of invasion is important, since prediction as Silva A, for example, might encourage conservative clinical management. If the pattern in the resection specimen is B with lymphovascular invasion or C, further surgery can be performed 34 HPVA biopsies were evaluated for expression of pS6, pERK, and HIF1α. Immunohistochemical stains were scored semiquantitatively, ranging from 0 to 4+ with scores 2 to 4+ considered positive, and Silva pattern was determined in follow-up excisional specimens. Silva patterns recognized in excisional specimens were distributed as follows: pattern A (n=8), pattern B (n=4), and pattern C (n=22). Statistically significant associations were found comparing pS6 and pERK immunohistochemistry with Silva pattern (P=0.034 and 0.05, respectively). Of the 3 markers tested, pERK was the most powerful for distinguishing between pattern A and patterns B and C (P=0.026; odds ratio: 6.75, 95% confidence interval: 1.111-41.001). Although the negative predictive values were disappointing, the positive predictive values were encouraging: 90% for pERK, 88% for pS6 and 100% for HIF1α. mTOR pathway activation assessed by immunohistochemistry in cervical biopsies of HPVA correlate with Silva invasion patterns.

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

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.

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

PURPOSE: Selpercatinib and pralsetinib induce deep and durable responses in patients with advanced RET fusion-positive lung and thyroid cancer. RET 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 RET structural variant of unknown significance (SVUS) were present. Canonical DNA-level RET fusions and RNA-confirmed RET 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 RET 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 RET fusions. The most common RET fusion-positive tumor types were lung (65.6%) and thyroid (23.2%). The most common partners were KIF5B (45%), CCDC6 (29.1%), and NCOA4 (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 NCOA4-RET (66.7%, 8/12). A total of 87.5% (7/8) of RET SVUS negative for RNA-level fusions demonstrated rearrangement by FISH. The sensitivity of IHC varied by fusion partner: KIF5B sensitivity was highest (100%, 31/31), followed by CCDC6 (88.9%, 16/18) and NCOA4 (50%, 6/12). Specificity of RET IHC was 82% (73/89). CONCLUSIONS: Although DNA sequencing has high sensitivity and specificity, RNA sequencing of RET SVUS is necessary. Both FISH and IHC demonstrated lower sensitivity for NCOA4-RET fusions.

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

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.

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

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.

Chromosome 3p loss of heterozygosity and reduced expression of H3K36me3 correlate with longer relapse-free survival in sacral conventional chordoma.

Conventional chordoma is a rare slow-growing malignant tumor of notochordal origin primarily arising at the base of the skull and sacrococcygeal bones. Chordoma may arise from its benign counterpart, benign notochordal cell tumors, and can also undergo dedifferentiation progressing into dedifferentiated chordoma. No study has directly compared the genomic alterations among these tumors comprising a morphologic continuum. Our prior study identified frequent chromosome 3p loss of heterozygosity and minimal deleted regions on chromosome 3 encompassing SETD2, encoding a histone methyltransferase involved in histone H3 lysine 36 trimethylation (H3K36me3). In the present study, we expanded our study to include 65 sacral conventional chordoma cases, 3 benign notochordal cell tumor cases, and 2 dedifferentiated chordoma cases using single nucleotide polymorphism (SNP) array, targeted next-generation sequencing analysis, and immunohistochemistry. We performed immunohistochemical analysis of histone, H3K36me3, and investigated whether there is any association between the clinical behavior and recurrent chromosome or aneuploidy or H3K36me3 protein expression. We found that there is increased genomic instability from benign notochordal cell tumor to conventional chordoma to dedifferentiated chordoma. The highly recurrent genomic aberration, chromosome 3p loss of heterozygosity (occurred in 70% of conventional chordomas), is correlated with longer relapse-free survival, but not with overall survival or metastasis-free survival in sacral chordoma. Chordomas demonstrate variable patterns and levels of H3K36me3 expression, and reduced expression of H3K36me3 showed marginally significant correlation with longer relapse-free survival. Copy number alterations in the genes encoding the H3K36me3 methylation transferase complex and demethylase may account for the altered H3K36me3 expression levels.

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

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.

V-domain Ig-containing suppressor of T-cell activation (VISTA), a potentially targetable immune checkpoint molecule, is highly expressed in epithelioid malignant pleural mesothelioma.

V-domain Ig-containing suppressor of T-cell activation (VISTA) is an immune checkpoint gene that inhibits anti-tumor immune responses. Since most malignant pleural mesotheliomas do not respond to anti-programmed cell death(-ligand)1 (PD-(L)1)/cytotoxic T-lymphocyte-associated protein 4 (CTLA4) therapy and given the recent finding of The Cancer Genome Atlas Study that pleural mesothelioma displays the highest expression of VISTA among all cancers studied, we examined VISTA expression in a large pleural mesothelioma cohort. VISTA and PD-L1 immunohistochemistry were performed on tissue microarray of immunotherapy-naive pleural mesotheliomas (254 epithelioid, 24 biphasic and 41 sarcomatoid) and ten whole-tissue sections of benign pleura (VISTA only). Percentages of tumor and inflammatory cells with positive staining were assessed. Optimal prognostic cutoff percentages were determined using maximally selected rank statistics. Overall survival was evaluated using Kaplan-Meier methods and Cox proportional hazard analysis. All benign mesothelium expressed VISTA. Eighty-five percent of 319 and 38% of 304 mesotheliomas expressed VISTA and PD-L1 (88% and 33% of epithelioid, 90% and 43% of biphasic, and 42% and 75% of sarcomatoid), respectively. Median VISTA score was significantly higher in epithelioid (50%) (vs. biphasic [20%] and sarcomatoid [0]) (p < 0.001), while median PD-L1 score was significantly higher in sarcomatoid tumors (20%) (vs. biphasic and epithelioid [both 0%]) (p < 0.001). VISTA and PD-L1 were expressed in inflammatory cells in 94% (n = 317) and 24% (n = 303) of mesothelioma, respectively. Optimal prognostic cutoffs for VISTA and PD-L1 were 40% and 30%, respectively. On multivariable analysis, VISTA and PD-L1 expression in mesothelioma were associated with better and worse overall survival (p = 0.001 and p = 0.002), respectively, independent of histology. In a large cohort of mesothelioma, we report frequent expression of VISTA and infrequent expression of PD-L1 with favorable and unfavorable survival correlations, respectively. These findings may explain poor responses to anti-PD-(L)1 immunotherapy and suggest VISTA as a potential novel target in pleural mesothelioma.

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

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.

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

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.

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

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.

The immune microenvironment and expression of PD-L1, PD-1, PRAME and MHC I in salivary duct carcinoma.

AIMS: Salivary duct carcinoma (SDC) is an aggressive salivary malignancy that results in high mortality rates and is often resistant to chemotherapy. Anti-programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) checkpoint inhibitors have led to dramatic improvements in patients with various cancers. Other immunotherapeutic approaches, e.g. cancer vaccines, have shown promising results. Cancer testis antigens, e.g. preferentially expressed antigen in melanoma (PRAME), are regarded as promising vaccine targets because of their tumour-specific expression pattern. METHODS AND RESULTS: We analysed the immunoexpression of PD-L1, PD-1, major histocompatibility complex class I (MHC I) and PRAME in 53 SDCs. The immunoexpression levels of PD-L1 in tumour cells (TCs) and immune cells (ICs), PD-1 in ICs, PRAME in TCs and MHC I in TCs were analysed, and were correlated with outcome. PRAME expression was seen in 83% of SDCs. No PRAME staining was present in normal salivary gland tissue. With the three established diagnostic algorithms proposed for head and neck squamous cell carcinoma, the criteria being a combined positive score of ≥1, TC% ≥1%, and TC% ≥25%, 35 (66%), 17 (32%) and three cases (6%), respectively, were deemed to be positive for PD-L1. PD-1-positive ICs were seen in 35 (66%) cases. MHC I down-regulation was seen in 82% of SDCs. There was a significant correlation among PD-L1 expression in ICs, PD-1 expression in ICs, and PRAME expression in TCs. PD-L1 expression in TCs and lack of PD-1 expression in ICs were associated with decreased disease-specific survival in SDC patients. CONCLUSIONS: Alterations of the tumour immune microenvironment are common in SDCs, including expression of PD-1/PD-L1 and PRAME, which opens the way to potential novel immune therapies, such as cancer vaccination and PD-1/PD-L1 blockade, in these tumours.

Clinical Genomic Sequencing of Pediatric and Adult Osteosarcoma Reveals Distinct Molecular Subsets with Potentially Targetable Alterations.

PURPOSE: Although multimodal chemotherapy has improved outcomes for patients with osteosarcoma, the prognosis for patients who present with metastatic and/or recurrent disease remains poor. In this study, we sought to define how often clinical genomic sequencing of osteosarcoma samples could identify potentially actionable alterations.Experimental Design: We analyzed genomic data from 71 osteosarcoma samples from 66 pediatric and adult patients sequenced using MSK-IMPACT, a hybridization capture-based large panel next-generation sequencing assay. Potentially actionable genetic events were categorized according to the OncoKB precision oncology knowledge base, of which levels 1 to 3 were considered clinically actionable. RESULTS: We found at least one potentially actionable alteration in 14 of 66 patients (21%), including amplification of CDK4 (n = 9, 14%: level 2B) and/or MDM2 (n = 9, 14%: level 3B), and somatic truncating mutations/deletions in BRCA2 (n = 3, 5%: level 2B) and PTCH1 (n = 1, level 3B). In addition, we observed mutually exclusive patterns of alterations suggesting distinct biological subsets defined by gains at 4q12 and 6p12-21. Specifically, potentially targetable gene amplifications at 4q12 involving KIT, KDR, and PDGFRA were identified in 13 of 66 patients (20%), which showed strong PDGFRA expression by IHC. In another largely nonoverlapping subset of 14 patients (24%) with gains at 6p12-21, VEGFA amplification was identified. CONCLUSIONS: We found potentially clinically actionable alterations in approximately 21% of patients with osteosarcoma. In addition, at least 40% of patients have tumors harboring PDGFRA or VEGFA amplification, representing candidate subsets for clinical evaluation of additional therapeutic options. We propose a new genomically based algorithm for directing patients with osteosarcoma to clinical trial options.

Performance of DAXX Immunohistochemistry as a Screen for DAXX Mutations in Pancreatic Neuroendocrine Tumors.

OBJECTIVES: DAXX immunohistochemistry (IHC) is often used as a surrogate for sequencing. We aimed to elucidate the sensitivity of IHC for DAXX mutation. METHODS: All pancreatic neuroendocrine tumors (PanNETs) with DAXX mutations detected by sequencing and a subset of DAXX wild-type PanNETs were analyzed for DAXX expression by IHC. RESULTS: Of 154 PanNETs with MSK-IMPACT testing, 36 (30%) harbored DAXX mutations. DAXX mutations were associated with TSC2 mutations (46% vs 10%, P < 0.0001), tended to co-occur with MEN1 mutations (63% vs 49%, P = 0.11), and tended to be mutually exclusive with ATRX mutations (11% vs 25%, P = 0.053). Of 27 available DAXX mutant PanNETs, 23 lost DAXX expression (85.2%). All 4 DAXX mutants with retained expression harbored DAXX mutations within the SUMO-interacting motif of the last exon. Telomere-specific fluorescence in situ hybridization demonstrated alternative lengthening of telomeres in all 4 cases. Of 20 PanNETs with wild-type DAXX, 19 retained DAXX IHC expression (95%). CONCLUSIONS: The sensitivity and specificity of IHC for DAXX mutation are 85% and 95%, respectively. Last exon DAXX mutant PanNETs often show alternative lengthening of telomeres despite retained DAXX expression, likely due to escape of nonmediated decay.