A Model System for Rapid Identification and Functional Testing of Genes Involved in Early Breast Cancer Progression

Early breast cancer progression involves advancement through specific morphologic stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not always in a linear fashion. Histological studies have examined differences in breast tissues representing these stages of progression, but model systems which allow for experimental testing of factors influencing transition through these stages are scarce. The purpose of these studies was to develop a 3D in vitro model of early breast cancer progression that reflected the in vivo sequence of events, and to use this system to identify and functionally test genes important in controlling the processes. The 21T series cell lines, originally derived from the same patient with metastatic breast cancer, were shown to mimic specific stages of human breast cancer progression (21PT, ADH; 21NT, DCIS; 21MT-1, IMC) when grown in the mammary fat pad of nude mice. When grown in 3D Matrigel, the cell lines showed characteristic morphologies in which aspects of the stage-specific in vivo behaviours were recapitulated. Gene expression profiling of the 21T cells revealed characteristic patterns for each, with differential expression of certain key genes in common with clinical specimens. Subsequent studies focused on functionally characterizing the roles of VANGL1, S100A2 and TBX3 in breast cancer progression. Genes were up- or down-regulated to determine if alterations could affect transitioning between stages of progression. VANGL1 was differentially expressed in the ADH (21PT) to DCIS (21NT) transition (higher in DCIS) and was found functionally to promote the transition from ADH to a malignant phenotype (DCIS and even IMC). S100A2 and TBX3 were both differentially expressed in the DCIS to IMC (21MT-1) transition (S100A2 lower in IMC, TBX3 higher in IMC). S100A2 was found to functionally inhibit the transition from DCIS to IMC, while TBX3 promoted progression to invasion. These studies demonstrate that the 21T series cell lines provide a model of early breast cancer progression when grown in 3D. In addition, the model provides a means of testing the functional effects of genes on transitions between stages of pre-malignant to malignant growth, which may elucidate potential therapeutic targets

Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy

CONTEXT.—: The US Food and Drug Administration (FDA) approved immune checkpoint inhibitor therapy for patients with advanced solid tumors that have DNA mismatch repair defects or high levels of microsatellite instability; however, the FDA provided no guidance on which specific clinical assays should be used to determine mismatch repair status. OBJECTIVE.—: To develop an evidence-based guideline to identify the optimal clinical laboratory test to identify defects in DNA mismatch repair in patients with solid tumor malignancies who are being considered for immune checkpoint inhibitor therapy. DESIGN.—: The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. Using the National Academy of Medicine-endorsed Grading of Recommendations Assessment, Development and Evaluation approach, the recommendations were derived from available evidence, strength of that evidence, open comment feedback, and expert panel consensus. Mismatch repair immunohistochemistry, microsatellite instability derived from both polymerase chain reaction and next-generation sequencing, and tumor mutation burden derived from large panel next-generation sequencing were within scope. RESULTS.—: Six recommendations and 3 good practice statements were developed. More evidence and evidence of higher quality were identified for colorectal cancer and other cancers of the gastrointestinal (GI) tract than for cancers arising outside the GI tract. CONCLUSIONS.—: An optimal assay depends on cancer type. For most cancer types outside of the GI tract and the endometrium, there was insufficient published evidence to recommend a specific clinical assay. Absent published evidence, immunohistochemistry is an acceptable approach readily available in most clinical laboratories

Molecular Biomarker Testing for the Diagnosis of Diffuse Gliomas:Guideline from the College of American Pathologists in Collaboration with the American Association of Neuropathologists, Association for Molecular Pathology, and Society for Neuro-Oncology

Context.—The diagnosis and clinical management of patients with diffuse gliomas (DGs) have evolved rapidly over the past decade with the emergence of molecular biomarkers that are used to classify, stratify risk, and predict treatment response for optimal clinical care.  Objective.—To develop evidence-based recommendations for informing molecular biomarker testing for pediatric and adult patients with DGs and provide guidance for appropriate laboratory test and biomarker selection for optimal diagnosis, risk stratification, and prediction.  Design.—The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. A systematic review of literature was conducted to address the overarching question, ‘‘What ancillary tests are needed to classify DGs and sufficiently inform the clinical management of patients?’’ Recommendations were derived from quality of evidence, open comment feedback, and expert panel consensus.  Results.—Thirteen recommendations and 3 good practice statements were established to guide pathologists and treating physicians on the most appropriate methods and molecular biomarkers to include in laboratory testing to inform clinical management of patients with DGs.  Conclusions.—Evidence-based incorporation of laboratory results from molecular biomarker testing into integrated diagnoses of DGs provides reproducible and clinically meaningful information for patient management

Molecular Biomarker Testing for the Diagnosis of Diffuse Gliomas.

Context.—The diagnosis and clinical management of patients with diffuse gliomas (DGs) have evolved rapidly over the past decade with the emergence of molecular biomarkers that are used to classify, stratify risk, and predict treatment response for optimal clinical care.Objective.—To develop evidence-based recommendations for informing molecular biomarker testing for pediatric and adult patients with DGs and provide guidance for appropriate laboratory test and biomarker selection for optimal diagnosis, risk stratification, and prediction.Design.—The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. A systematic review of literature was conducted to address the overarching question, "What ancillary tests are needed to classify DGs and sufficiently inform the clinical management of patients?" Recommendations were derived from quality of evidence, open comment feedback, and expert panel consensus.Results.—Thirteen recommendations and 3 good practice statements were established to guide pathologists and treating physicians on the most appropriate methods and molecular biomarkers to include in laboratory testing to inform clinical management of patients with DGs.Conclusions.—Evidence-based incorporation of laboratory results from molecular biomarker testing into integrated diagnoses of DGs provides reproducible and clinically meaningful information for patient management

Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology

Context: In 2013, an evidence-based guideline was published by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology to set standards for the molecular analysis of lung cancers to guide treatment decisions with targeted inhibitors. New evidence has prompted an evaluation of additional laboratory technologies, targetable genes, patient populations, and tumor types for testing. Objective: To systematically review and update the 2013 guideline to affirm its validity; to assess the evidence of new genetic discoveries, technologies, and therapies; and to issue an evidence-based update. Design: The College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology convened an expert panel to develop an evidence-based guideline to help define the key questions and literature search terms, review abstracts and full articles, and draft recommendations. Results: Eighteen new recommendations were drafted. The panel also updated 3 recommendations from the 2013 guideline. Conclusions: The 2013 guideline was largely reaffirmed with updated recommendations to allow testing of cytology samples, require improved assay sensitivity, and recommend against the use of immunohistochemistry for EGFR testing. Key new recommendations include ROS1 testing for all adenocarcinoma patients; the inclusion of additional genes (ERBB2, MET, BRAF, KRAS, and RET) for laboratories that perform next-generation sequencing panels; immunohistochemistry as an alternative to fluorescence in situ hybridization for ALK and/or ROS1 testing; use of 5% sensitivity assays for EGFR T790M mutations in patients with secondary resistance to EGFR inhibitors; and the use of cell-free DNA to “rule in” targetable mutations when tissue is limited or hard to obtain

Human 21T Breast Epithelial Cell Lines Mimic Breast Cancer Progression in Vivo and in Vitro and Show Stage-specific Gene Expression Patterns

Early breast cancer progression involves advancement through specific morphological stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after a year. To develop a more rapid, readily manipulatable in vitro assay for examining the biological differences between these cell lines, we have used a 3D Matrigel system. When the three cell lines were grown in 3D Matrigel, they showed characteristic morphologies, in which quantifiable aspects of stage-specific in vivo behaviors (ie, differences in acinar structure formation, cell polarization, colony morphology, cell proliferation, cell invasion) were recapitulated in a reproducible fashion. Gene expression profiling revealed a characteristic pattern for each of the three cell lines. Interestingly, Wnt pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasion phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth