Programmed Death Ligand-1 and Tumor Mutation Burden Testing of Patients With Lung Cancer for Selection of Immune Checkpoint Inhibitor Therapies: Guideline From the College of American Pathologists, Association for Molecular Pathology, International Association for the Study of Lung Cancer, Pulmonary Pathology Society, and LUNGevity Foundation.

CONTEXT.­: Rapid advancements in the understanding and manipulation of tumor-immune interactions have led to the approval of immune therapies for patients with non-small cell lung cancer. Certain immune checkpoint inhibitor therapies require the use of companion diagnostics, but methodologic variability has led to uncertainty around test selection and implementation in practice. OBJECTIVE.­: To develop evidence-based guideline recommendations for the testing of immunotherapy/immunomodulatory biomarkers, including programmed death ligand-1 (PD-L1) and tumor mutation burden (TMB), in patients with lung cancer. DESIGN.­: The College of American Pathologists convened a panel of experts in non-small cell lung cancer and biomarker testing to develop evidence-based recommendations in accordance with the standards for trustworthy clinical practice guidelines established by the National Academy of Medicine. A systematic literature review was conducted to address 8 key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach, recommendations were created from the available evidence, certainty of that evidence, and key judgments as defined in the GRADE Evidence to Decision framework. RESULTS.­: Six recommendation statements were developed. CONCLUSIONS.­: This guideline summarizes the current understanding and hurdles associated with the use of PD-L1 expression and TMB testing for immune checkpoint inhibitor therapy selection in patients with advanced non-small cell lung cancer and presents evidence-based recommendations for PD-L1 and TMB testing in the clinical setting.

Novel MED15::ATF1 fusion in a pediatric melanoma with spitzoid features and aggressive presentation.

Childhood melanoma is a rare and biologically heterogeneous pediatric malignancy. The differential diagnosis of pediatric melanoma is usually broad, including a wide variety of spindle cell or epithelioid neoplasms. Different molecular alterations affecting the MAPK and PI3K/AKT/mTOR pathways, tumor suppressor genes, and telomerase reactivation have been implicated in melanoma tumorigenesis and progression. Here, we report a novel MED15::ATF1 fusion in a pediatric melanoma with spitzoid features and an aggressive clinical course.

Constitutional balanced translocations involving SMARCB1: A rare cause of rhabdoid tumor predisposition syndrome.

Rhabdoid Tumor Predisposition Syndrome 1 (RTPS1) confers an increased risk of developing rhabdoid tumors and is caused by germline mutations in SMARCB1. RTPS1 should be evaluated in all individuals with rhabdoid tumor and is more likely in those with a young age at presentation (occasionally congenital presentation), multiple primary tumors, or a family history of rhabdoid tumor or RTPS1. Proband genetic testing is the standard method for diagnosing RTPS1. Most known RTPS1-related SMARCB1 gene mutations are copy number variants (CNVs) or single nucleotide variants/indels, but structural variant analysis (SVA) is not usually included in the molecular evaluation. Here, we report two children with RTPS1 presenting with atypical teratoid/rhabdoid tumor (ATRT) who had constitutional testing showing balanced chromosome translocations involving SMARCB1. Patient 1 is a 23-year-old female diagnosed with pineal region ATRT at 7 months who was found to have a de novo, constitutional t(16;22)(p13.3;q11.2). Patient 2 is a 24-month-old male diagnosed with a posterior fossa ATRT at 14 months, with subsequent testing showing a constitutional t(5;22)(q14.1;q11.23). These structural rearrangements have not been previously reported in RTPS1. While rare, these cases suggest that structural variants should be considered in the evaluation of children with rhabdoid tumors to provide more accurate genetic counseling on the risks of developing tumors, the need for surveillance, and the risks of passing the disorder on to future children. Further research is needed to understand the prevalence, clinical features, and tumor risks associated with RTPS1-related constitutional balanced translocations.

CBFA2T3::GLIS2-positive acute leukemia with RAM and mixed T/megakaryocytic phenotype.

Herein, we present a rare case of acute myeloid leukemia (AML) with CBFA2T3-rearrangement and the expression of megakaryocytic and lymphoid markers, highlighting the need for a high suspicion index in differential diagnosis and applying adequate workup to avoid misdiagnosing this entity. CBFA2T3::GLIS2-positive AML is primarily found in infants with non-down syndrome acute megakaryoblastic leukemia (non-DSAMKL). Flow cytometry immunophenotyping plays an important role in recognizing the unique immunophenotype of bright CD56 expression with dim/negative expression of HLA-DR, CD38, and CD45 termed the RAM immunophenotype in this entity. Still, CBFA2T3::GLIS2-positive acute leukemia with T/megakaryocytic markers could be misdiagnosed as T-lymphoblastic leukemia/lymphoma, early T-cell precursor acute lymphoblastic leukemia/lymphoma, NK lymphoblastic leukemia, AML with minimal differentiation, or AML with myelodysplasia-related changes.

Clinical and genomic characterization of an ATRA-insensitive acute promyelocytic leukemia variant with a FNDC3B::RARB fusion.

The promyelocytic leukemia-retinoic acid receptor-α (PML::RARA) fusion is the hallmark of acute promyelocytic leukemia (APL) and is observed in over 95% of APL cases. RARA and homologous receptors RARB and RARG are occasionally fused to other gene partners, which differentially affect sensitivity to targeted therapies. Most APLs without RARA fusions have rearrangements involving RARG or RARB, both of which frequently show resistance to all-trans-retinoic acid (ATRA) and/or multiagent chemotherapy for acute myeloid leukemia (AML). We present a 13-year-old male diagnosed with variant APL with a novel FNDC3B::RARB in-frame fusion that showed no response to ATRA but responded well to conventional AML therapy. While FNDC3B has been identified as a rare RARA translocation partner in ATRA-sensitive variant APL, it has never been reported as a fusion partner with RARB and it is only the second known fusion partner with RARB in variant APL. We also show that this novel fusion confers an RNA expression signature that is similar to APL, despite clinical resistance to ATRA monotherapy.

Novel HNRNPM::LEUTX fusion resulting from chromothripsis of chromosome 19 in a pediatric undifferentiated small round cell neoplasm.

Small round cell neoplasms comprise a diverse group of tumors characterized by a primitive/undifferentiated appearance. Although several entities are associated with recurrent gene fusions, many of these neoplasms have not been fully characterized, and novel molecular alterations are being discovered. Here, we report an undifferentiated small round cell neoplasm arising in the anterior mediastinum of a 17-month-old female. The tumor harbored a novel HNRNPM::LEUTX fusion resulting from chromothripsis of chromosome 19, which was identified by whole transcriptome sequencing, but not by targeted sequencing. The structural variations caused by the chromothripsis event also challenged the interpretation of the targeted sequencing findings. This report expands the spectrum of gene partners involved in LEUTX fusions and underscores the value of whole transcriptome sequencing in the diagnostic workup of undifferentiated small round cell tumors. It also highlights the interpretive challenges associated with complex genomic alterations. A careful evidence-based analysis of sequencing data along with histopathologic correlation is essential to ensure correct categorization of fusions.

Progressive metastatic infantile fibrosarcoma with multiple acquired mutations.

Infantile fibrosarcoma is the most common soft-tissue sarcoma in children under the age of 1 yr and is defined molecularly by NTRK fusion proteins. This tumor is known to be locally invasive; however, although rare, metastases can occur. The NTRK fusion acts as a driver for tumor formation, which can be targeted by first- and second-generation TRK inhibitors. Although NTRK gatekeeper mutations have been well-described as mechanisms of resistance to these agents, alternative pathway mutations are rare. Here, we report the case of a patient with infantile fibrosarcoma treated with chemotherapy and TRK inhibition that developed metastatic, progressive disease with multiple acquired mutations, including TP53, SUFU, and an NTRK F617L gatekeeper mutation. Alterations in pathways of SUFU and TP53 have been widely described in the literature in other tumors; however, not yet in infantile fibrosarcoma. Although most patients have a sustained response to TRK inhibitors, a subset will go on to develop mechanisms of resistance that have implications for clinical management, such as in our patient. We hypothesize this constellation of mutations contributed to the patient's aggressive clinical course. Taken together, we report the first case of infantile fibrosarcoma with ETV6::NTRK3 and acquired SUFU, TP53, and NTRK F617L gatekeeper mutation along with detailed clinical course and management. Our report highlights the importance of genomic profiling in recurrent infantile fibrosarcoma to reveal actionable mutations, such as gatekeeper mutations, that can improve patient outcomes.

Pathogenic Variants in Adult-Onset Cancer Predisposition Genes in Pediatric Cancer: Prevalence and Impact on Tumor Molecular Features and Clinical Management.

PURPOSE: Clinical genomic sequencing of pediatric tumors is increasingly uncovering pathogenic variants in adult-onset cancer predisposition genes (aoCPG). Nevertheless, it remains poorly understood how often aoCPG variants are of germline origin and whether they influence tumor molecular profiles and/or clinical care. In this study, we examined the prevalence, spectrum, and impacts of aoCPG variants on tumor genomic features and patient management at our institution. EXPERIMENTAL DESIGN: This is a retrospective study of 1,018 children with cancer who underwent clinical genomic sequencing of their tumors. Tumor genomic data were queried for pathogenic variants affecting 24 preselected aoCPGs. Available tumor whole-genome sequencing (WGS) data were evaluated for second hit mutations, loss of heterozygosity (LOH), DNA mutational signatures, and homologous recombination deficiency (HRD). Patients whose tumors harbored one or more pathogenic aoCPG variants underwent subsequent germline testing based on hereditary cancer evaluation and family or provider preference. RESULTS: Thirty-three patients (3%) had tumors harboring pathogenic variants affecting one or more aoCPGs. Among 21 tumors with sufficient WGS sequencing data, six (29%) harbored a second hit or LOH affecting the remaining aoCPG allele with four of these six tumors (67%) also exhibiting a DNA mutational signature consistent with the altered aoCPG. Two additional tumors demonstrated HRD, of uncertain relation to the identified aoCPG variant. Twenty-one of 26 patients (81%) completing germline testing were positive for the aoCPG variant in the germline. All germline-positive patients were counseled regarding future cancer risks, surveillance, and risk-reducing measures. No patients had immediate cancer therapy changed due to aoCPG data. CONCLUSIONS: AoCPG variants are rare in pediatric tumors; however, many originate in the germline. Almost one third of tumor aoCPG variants examined exhibited a second hit and/or conferred an abnormal DNA mutational profile suggesting a role in tumor formation. aoCPG information aids in cancer risk prediction but is not commonly used to alter the treatment of pediatric cancers.

Four-Year Laboratory Performance of the First College of American Pathologists In Silico Next-Generation Sequencing Bioinformatics Proficiency Testing Surveys.

CONTEXT.­: In 2016, the College of American Pathologists (CAP) launched the first next-generation sequencing (NGS) in silico bioinformatics proficiency testing survey to evaluate the performance of clinical laboratory bioinformatics pipelines for the detection of oncology-associated variants at varying allele fractions. This survey focused on 2 commonly used oncology panels, the Illumina TruSeq Amplicon Cancer Panel and the Thermo Fisher Ion AmpliSeq Cancer Hotspot v2 Panel. OBJECTIVE.­: To review the analytical performance of laboratories participating in the CAP NGS bioinformatics (NGSB) surveys, comprising NGSB1 for Illumina users and NGSB2 for Thermo Fisher Ion Torrent users, between 2016 and 2019. DESIGN.­: Responses from 78 laboratories were analyzed for accuracy and associated performance characteristics. RESULTS.­: The analytical sensitivity was 90.0% (1901 of 2112) for laboratories using the Illumina platform and 94.8% (2153 of 2272) for Thermo Fisher Ion Torrent users. Variant type and variant allele fraction were significantly associated with performance. False-negative results were seen mostly for multi-nucleotide variants and variants engineered at variant allele fractions of less than 25%. Analytical specificity for all participating laboratories was 99.8% (9303 of 9320). There was no statistically significant association between deletion-insertion length and detection rate. CONCLUSIONS.­: These results demonstrated high analytical sensitivity and specificity, supporting the feasibility and utility of using in silico mutagenized NGS data sets as a supplemental challenge to CAP surveys for oncology-associated variants based on physical samples. This program demonstrates the opportunity and challenges that can guide future surveys inclusive of customized in silico programs.

Standardized evidence-based approach for assessment of oncogenic and clinical significance of NTRK fusions.

Gene fusions involving the neurotrophic receptor tyrosine kinase genes NTRK1, NTRK2, and NTRK3, are well established oncogenic drivers in a broad range of pediatric and adult tumors. These fusions are also important actionable markers, predicting often dramatic response to FDA approved kinase inhibitors. Accurate interpretation of the clinical significance of NTRK fusions is a high priority for diagnostic laboratories, but remains challenging and time consuming given the rapid pace of new data accumulation, the diversity of fusion partners and tumor types, and heterogeneous and incomplete information in variant databases and knowledgebases. The ClinGen NTRK Fusions Somatic Cancer Variant Curation Expert Panel (SC-VCEP) was formed to systematically address these challenges and create an expert-curated resource to support clinicians, researchers, patients and their families in making accurate interpretations and informed treatment decisions for NTRK fusion-driven tumors. We describe a system for NTRK fusion interpretation (including compilation of key elements and annotations) developed by the NTRK fusions SC-VCEP. We illustrate this stepwise process on examples of LMNA::NTRK1 and KANK1::NTRK2 fusions. Finally, we provide detailed analysis of current representation of NTRK fusions in public fusion databases and the CIViC knowledgebase, performed by the NTRK fusions SC-VCEP to determine existing gaps and prioritize future curation activities.

A CTNNB1-altered medulloblastoma shows the immunophenotypic, DNA methylation and transcriptomic profiles of SHH-activated, and not WNT-activated, medulloblastoma.

Recent advancements in molecular characterisation have identified four principal molecular groups of medulloblastoma: WNT, SHH, group 3 and group 4. Each has its characteristic clinical features, signature genetic alterations and distinct DNA methylome profiles. Thus far, CTNNB1 mutations have been considered pathognomonic of WNT-activated medulloblastoma. Furthermore, it has been shown that CTNNB1 mutations dominantly drive the WNT-activated phenotype in medulloblastoma, even in the presence of alterations in the SHH pathway. We herein report an illustrative case that challenges this belief-a medulloblastoma with a pathogenic CTNNB1 mutation that otherwise showed the histopathology, immunophenotype and methylation and transcriptomic profiles of an SHH-activated medulloblastoma. Detailed molecular analyses, including whole exome sequencing, transcriptome analysis and DNA methylation profiling with DKFZ brain tumour classifier and St. Jude MLPnet neural network classifier analyses, have been performed on the tumour. Our example emphasises the diagnostic value of the immunohistochemistry panel with YAP1, GAB1 and ß-catenin and DNA methylation profiling, combined with exome sequencing, in the characterisation of medulloblastoma. CTNNB1 mutations are not specific for WNT-activated medulloblastoma, and different CTNNB1 mutations have diverse oncogenic potential.

Histologic and Molecular Characterization of Non-Small Cell Lung Carcinoma With Discordant ROS1 Immunohistochemistry and Fluorescence In Situ Hybridization.

INTRODUCTION: ROS1 immunohistochemical (IHC) positivity requires follow-up with confirmatory testing such as fluorescence in situ hybridization (FISH). Identifying predictive characteristics of false positive ROS1 IHC cases could aid in optimizing testing algorithms, decrease testing costs and preserve tissue. MATERIALS AND METHODS: Retrospective results were retrieved for 2054 patients with non-small cell lung carcinoma submitted to our laboratory for molecular testing. Reflex ROS1 FISH was done on all ROS1 immunoreactive cases using ROS1 D4D6 antibody. Staining intensity and histo-score was recorded for all ROS1 immunoreactive cases. Results of any additional molecular testing (KRAS, BRAF, EGFR, ALK FISH, RET FISH, MET FISH) were also tabulated. RESULTS: ROS1 immunoreactivity was seen in 305/2054 (14.8%) cases. Immunoreactivity was weak in majority of the cases with only 4.6% cases having an histo-score >100 and 5.9% of cases had moderate staining intensity. FISH was negative in 99% (302/305) cases with any degree of IHC expression (discordant cases) while 3 cases were positive by FISH. Diffuse strong IHC staining in greater than 90% of the tumor was noted in 6 cases, 3 (0.98%) of which were confirmed to have ROS1 rearrangement by FISH. The discordant cases had significantly higher rates of EGFR mutations (P<0.0005) in comparison to ROS1 IHC negative cases, were seen more often in adenocarcinoma and adenosquamous cell carcinoma (P<0.0005) with lepidic and acinar patterns, and more likely to occur in primary lung carcinomas (P<0.0005). CONCLUSIONS: False positive ROS1 immunoreactivity was very frequent, occurred more commonly in primary NSCLC cases with acinar and/or lepidic histologies and was more likely in EGFR mutated cases. Using higher positivity thresholds for ROS1 IHC and incorporating the histologic and molecular correlates into algorithmic strategies could result in increased specificity and clinical utility of ROS1 IHC assay.

PD-L1 Tumor Cell Expression in Upper Tract Urothelial Carcinomas is Associated With Higher Pathologic Stage.

BACKGROUND: Upper tract urothelial carcinomas (UTUCs) are a rare and unique subset of urothelial carcinoma (UC). Patients with UTUC may qualify for treatment with immune checkpoint inhibitors if their tumor cells express programmed death ligand-1 (PD-L1). While several large studies have looked at PD-L1 expression in UC, most have not investigated UTUC as a separate group, and most have not used Food and Drug Administration approved PD-L1 stains and scoring systems. Moreover, comparison between studies of PD-L1 expression is challenging as a wide variety of different PD-L1 antibody clones, testing platforms, and cutoff values have been used in the literature. METHODS: This is a retrospective study of 37 cases of resected UTUC. Representative tissue from each case was compiled into tissue microarrays and immunohistochemical stains for PD-L1 (Dako antibody clones 22C3 and 28-8) were performed. PD-L1 staining was evaluated using several established Food and Drug Administration approved scoring systems: tumor proportion score (TPS), combined positive score, and immune cell score. Associations between PD-L1 expression and clinicopathologic features were investigated. RESULTS: Overall expression of PD-L1 in UTUC was 29.7% when using a TPS cutoff of ≥1%. Total of, 55.6% of cases with higher pathologic stage (pT3 or pT4) were positive for PD-L1, compared with only 5.3% of cases with lower pathologic stage (pTis, pT1, or pT2; P=0.0011). When using a combined positive score cutoff of ≥10, there was no significant association between tumor stage and PD-L1 expression. There was no association between PD-L1 positivity and tumor grade, tumor location, sex, or age. There was 100% concordance between 22C3 and 28-8 in terms of positivity rate. CONCLUSIONS: Our study using approved testing methods shows that PD-L1 expression in UTUC is more often associated with high pathologic stage, which may reflect an immune response evasion mechanism that UC cells acquire later in disease progression. In addition we show that 29.7% of UTUCs are positive for PD-L1 TPS expression, comparable to the 20% to 30% reported in UC literature. Finally, PD-L1 22C3 and 28-8 clones show similar overall patterns of staining in this setting.

Metastatic HPV-Associated Oropharyngeal Versus Primary Pulmonary Squamous Cell Carcinoma: is p16 Immunostain Useful?

The lungs are a common site of metastasis of head and neck (H&N) squamous cell carcinomas (SCC). This study attempts to define p16 immunoexpression and presence of HPV in primary SCC of the lung and determine their usefulness in discriminating between primary lung SCC and metastasis from HPV-associated oropharyngeal primary. Pathology archives were searched for patients with SCC of the lung without SCC elsewhere. Tissue microarray was constructed and immunohistochemistry performed using anti-p40 and anti-p16 antibodies. All cases were tested for HPV viral proteins E6/E7 by RNA in situ hybridization (ISH) and available positive cases for HPV DNA by polymerase chain reaction (PCR). Eight of 25 (32%) showed cytoplasmic and nuclear expression of p16: 2 (8%) strong and 2 (8%) moderate in > 70% of tumor cells; 1 (4%) strong, 1 (4%) moderate, and 1 (4%) weak in 50-70% of tumor cells; 1 (4%) weak in < 50% of tumor cells. E6/E7 mRNA ISH was negative in all cases. Seven of 8 (87.5%) p16-expressing cases were available for testing by HPV PCR; all were negative for HPV DNA. A retrospective control group of 12 patients with possible SCC metastatic to lung was also identified; high-risk HPV DNA was present in 3, confirming metastasis. p16 expression in lung SCC is not uncommon and may not discriminate between primary pulmonary SCC and metastasis from HPV-associated oropharyngeal primary. Confirmatory HPV testing (high risk HPV DNA or E6/E7 mRNA) is recommended to differentiate metastasis from oropharyngeal primary from two separate primaries.

Successful lung cancer EGFR sequencing from DNA extracted from TTF-1 immunohistochemistry slides: a new means to extend insufficient tissue.

Lung cancer biopsy material is limited and is used for morphologic diagnosis and immunohistochemical and molecular testing. This can lead to tissue exhaustion, resulting in repeat biopsies (when clinically possible), delayed testing, and increased risks. Consequently, there is a need to optimize preanalytical specimen use for molecular testing. Although hematoxylin/eosin can be used for as a DNA source for molecular testing, little is known regarding the potential use of immunohistochemistry (IHC) slides, as these are subject to harsh conditions that can lead to DNA degradation. Our aim was to evaluate whether DNA extracted from TTF-1 IHC slides, a common stain for lung adenocarcinoma, can be tested for EGFR mutations. Twenty-two lung adenocarcinoma samples (11 EGFR wild type and 11 mutated) were selected. Slides were stained for TTF-1 IHC. Following TTF-1 staining, tissue underwent DNA extraction. Pyrosequencing for mutations in exons 18, 19, 20, and 21 of EGFR was performed, and results were compared to clinical EGFR testing data. All 22 TTF-1 samples produced successful results, and 21 were concordant. Of the 11 originally EGFR-mutated cases, 10 TTF-1 samples showed identical mutations in all exons of interest. One case with an L858R mutation on original testing was negative on sequencing of the TTF-1 sample, possibly due to lower tumor burden on the TTF-1 stained slide. All 11 originally EGFR wild-type cases showed identical results on the TTF-1 samples. TTF-1 IHC slides can be a viable DNA source for molecular testing, especially important in lung biopsies with insufficient material following diagnostic evaluation.