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Tumor mutational burden (TMB) has been recognized as a predictive biomarker for immunotherapy response in several tumor types. Several laboratories offer TMB testing, but there is significant variation in how TMB is calculated, reported, and interpreted among laboratories. TMB standardization efforts are underway, but no published guidance for TMB validation and reporting is currently available. Recognizing the current challenges of clinical TMB testing, the Association for Molecular Pathology convened a multidisciplinary collaborative working group with representation from the American Society of Clinical Oncology, the College of American Pathologists, and the Society for the Immunotherapy of Cancer to review the laboratory practices surrounding TMB and develop recommendations for the analytical validation and reporting of TMB testing based on survey data, literature review, and expert consensus. These recommendations encompass pre-analytical, analytical, and postanalytical factors of TMB analysis, and they emphasize the relevance of comprehensive methodological descriptions to allow comparability between assays.
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Biomarcadores Tumorais , Mutação , Neoplasias , Humanos , Neoplasias/genética , Neoplasias/terapia , Neoplasias/imunologia , Biomarcadores Tumorais/genética , Imunoterapia/métodos , Patologia Molecular/métodos , Consenso , Sociedades Médicas , Estados Unidos , Patologistas , Reprodutibilidade dos Testes , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/normasRESUMO
CONTEXT.: The College of American Pathologists (CAP) accreditation requirements for clinical laboratory testing help ensure laboratories implement and maintain systems and processes that are associated with quality. Machine learning (ML)-based models share some features of conventional laboratory testing methods. Accreditation requirements that specifically address clinical laboratories' use of ML remain in the early stages of development. OBJECTIVE.: To identify relevant CAP accreditation requirements that may be applied to the clinical adoption of ML-based molecular oncology assays, and to provide examples of current and emerging ML applications in molecular oncology testing. DESIGN.: CAP accreditation checklists related to molecular pathology and general laboratory practices (Molecular Pathology, All Common and Laboratory General) were reviewed. Examples of checklist requirements that are generally applicable to validation, revalidation, quality management, infrastructure, and analytical procedures of ML-based molecular oncology assays were summarized. Instances of ML use in molecular oncology testing were assessed from literature review. RESULTS.: Components of the general CAP accreditation framework that exist for traditional molecular oncology assay validation and maintenance are also relevant for implementing ML-based tests in a clinical laboratory. Current and emerging applications of ML in molecular oncology testing include DNA methylation profiling for central nervous system tumor classification, variant calling, microsatellite instability testing, mutational signature analysis, and variant prediction from histopathology images. CONCLUSIONS.: Currently, much of the ML activity in molecular oncology is within early clinical implementation. Despite specific considerations that apply to the adoption of ML-based methods, existing CAP requirements can serve as general guidelines for the clinical implementation of ML-based assays in molecular oncology testing.
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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.
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Antígeno B7-H1 , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas , Inibidores de Checkpoint Imunológico , Neoplasias Pulmonares , Mutação , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/diagnóstico , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Inibidores de Checkpoint Imunológico/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , ImunoterapiaRESUMO
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.
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Glicina , Melanoma , Nevo de Células Epitelioides e Fusiformes , Proteínas de Fusão Oncogênica , Pirróis , Neoplasias Cutâneas , Criança , Humanos , Diagnóstico Diferencial , Glicina/análogos & derivados , Complexo Mediador , Melanoma/diagnóstico , Melanoma/genética , Melanoma/patologia , Nevo de Células Epitelioides e Fusiformes/diagnóstico , Fosfatidilinositol 3-Quinases , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Proteínas de Fusão Oncogênica/genéticaAssuntos
Neoplasias , Coativador 3 de Receptor Nuclear , beta Catenina , Criança , Humanos , beta Catenina/imunologia , beta Catenina/metabolismo , Biomarcadores Tumorais/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Coativador 3 de Receptor Nuclear/genética , Fibroblastos/patologiaRESUMO
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.
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Neoplasias Encefálicas , Transtornos Cromossômicos , Tumor Rabdoide , Teratoma , Criança , Feminino , Masculino , Humanos , Adulto Jovem , Adulto , Lactente , Tumor Rabdoide/genética , Tumor Rabdoide/patologia , Proteína SMARCB1/genética , Neoplasias Encefálicas/genética , Mutação em Linhagem Germinativa , Translocação Genética , Teratoma/genética , Teratoma/patologiaRESUMO
CONTEXT.: Machine learning applications in the pathology clinical domain are emerging rapidly. As decision support systems continue to mature, laboratories will increasingly need guidance to evaluate their performance in clinical practice. Currently there are no formal guidelines to assist pathology laboratories in verification and/or validation of such systems. These recommendations are being proposed for the evaluation of machine learning systems in the clinical practice of pathology. OBJECTIVE.: To propose recommendations for performance evaluation of in vitro diagnostic tests on patient samples that incorporate machine learning as part of the preanalytical, analytical, or postanalytical phases of the laboratory workflow. Topics described include considerations for machine learning model evaluation including risk assessment, predeployment requirements, data sourcing and curation, verification and validation, change control management, human-computer interaction, practitioner training, and competency evaluation. DATA SOURCES.: An expert panel performed a review of the literature, Clinical and Laboratory Standards Institute guidance, and laboratory and government regulatory frameworks. CONCLUSIONS.: Review of the literature and existing documents enabled the development of proposed recommendations. This white paper pertains to performance evaluation of machine learning systems intended to be implemented for clinical patient testing. Further studies with real-world clinical data are encouraged to support these proposed recommendations. Performance evaluation of machine learning models is critical to verification and/or validation of in vitro diagnostic tests using machine learning intended for clinical practice.
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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.
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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.
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Cromotripsia , Sarcoma , Humanos , Criança , Feminino , Lactente , Cromossomos Humanos Par 19 , Sarcoma/genética , Fusão Gênica , Biomarcadores Tumorais/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Proteínas de Homeodomínio/genéticaRESUMO
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.
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Leucemia Mieloide Aguda , Leucemia Promielocítica Aguda , Masculino , Humanos , Adolescente , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Translocação Genética , Tretinoína/uso terapêutico , Leucemia Mieloide Aguda/genética , Receptor alfa de Ácido Retinoico/genética , Genômica , Proteínas de Fusão Oncogênica/genética , Fibronectinas/genéticaRESUMO
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.
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Fibrossarcoma , Segunda Neoplasia Primária , Sarcoma , Criança , Humanos , Fibrossarcoma/genética , Mutação , Inibidores de Proteínas Quinases , Progressão da DoençaRESUMO
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.
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Predisposição Genética para Doença , Neoplasias , Criança , Adulto , Humanos , Estudos Retrospectivos , Prevalência , Neoplasias/epidemiologia , Neoplasias/genética , Sequenciamento Completo do Genoma , Mutação em Linhagem GerminativaRESUMO
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.
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Laboratórios , Neoplasias , Humanos , Patologistas , Neoplasias/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ensaio de Proficiência Laboratorial/métodos , Biologia ComputacionalAssuntos
Neoplasias do Sistema Nervoso Central , Neoplasias Meníngeas , Neoplasias Neuroepiteliomatosas , Humanos , Neoplasias do Sistema Nervoso Central/genética , Cromossomos , Cromossomos Humanos Par 1/genética , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/patologia , Metilação , Neoplasias Neuroepiteliomatosas/genética , Neoplasias Neuroepiteliomatosas/patologia , PrognósticoRESUMO
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.
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Neoplasias , Receptor trkA , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/uso terapêutico , Adulto , Biomarcadores Tumorais/genética , Carcinogênese , Criança , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/uso terapêutico , Fusão Gênica , Humanos , Neoplasias/diagnóstico , Proteínas de Fusão Oncogênica/genética , Receptor trkA/genética , Receptor trkA/uso terapêuticoRESUMO
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.