Recommendations for Cell-Free DNA Assay Validations: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists.

Diagnosing, selecting therapy for, and monitoring cancer in patients using a minimally invasive blood test represents a significant advance in precision medicine. Wide variability exists in how circulating tumor DNA (ctDNA) assays are developed, validated, and reported in the literature, which hinders clinical adoption and may negatively impact patient care. Standardization is needed for factors affecting ctDNA assay performance and reporting, including pre-analytical variables, analytical considerations, and elements of laboratory assay reporting. The Association for Molecular Pathology Clinical Practice Committee's Liquid Biopsy Working Group (LBxWG), including organizational representation from the American Society of Clinical Oncology and the College of American Pathologists, has undertaken a full-text data extraction of 1228 ctDNA publications that describe assays performed in patients with lymphoma and solid tumor malignancies. With an emphasis on clinical assay validation, the LBxWG has developed a set of 13 best practice consensus recommendations for validating, reporting, and publishing clinical ctDNA assays. Recommendations include reporting key pre-analytical considerations and assay performance metrics; this analysis demonstrates these elements are inconsistently included in publications. The LBxWG recommendations are intended to assist clinical laboratories with validating and reporting ctDNA assays and to ensure high-quality data are included in publications. It is expected that these recommendations will need to be updated as the body of literature continues to mature.

The Spectrum of Clinical Utilities in Molecular Pathology Testing Procedures for Inherited Conditions and Cancer: A Report of the Association for Molecular Pathology.

Clinical utility describes the benefits of each laboratory test for that patient. Many stakeholders have adopted narrow definitions for the clinical utility of molecular testing as applied to targeted pharmacotherapy in oncology, regardless of the population tested or the purpose of the testing. This definition does not address all of the important applications of molecular diagnostic testing. Definitions consistent with a patient-centered approach emphasize and recognize that a clinical test result's utility depends on the context in which it is used and are particularly relevant to molecular diagnostic testing because of the nature of the information they provide. Debates surrounding levels and types of evidence needed to properly evaluate the clinical value of molecular diagnostics are increasingly important because the growing body of knowledge, stemming from the increase of genomic medicine, provides many new opportunities for molecular testing to improve health care. We address the challenges in defining the clinical utility of molecular diagnostics for inherited diseases or cancer and provide assessment recommendations. Starting with a modified analytic validity, clinical validity, clinical utility, and ethical, legal, and social implications model for addressing clinical utility of molecular diagnostics with a variety of testing purposes, we recommend promotion of patient-centered definitions of clinical utility that appropriately recognize the valuable contribution of molecular diagnostic testing to improve patient care.

Do circulating tumor cells, exosomes, and circulating tumor nucleic acids have clinical utility? A report of the association for molecular pathology.

Diagnosing and screening for tumors through noninvasive means represent an important paradigm shift in precision medicine. In contrast to tissue biopsy, detection of circulating tumor cells (CTCs) and circulating tumor nucleic acids provides a minimally invasive method for predictive and prognostic marker detection. This allows early and serial assessment of metastatic disease, including follow-up during remission, characterization of treatment effects, and clonal evolution. Isolation and characterization of CTCs and circulating tumor DNA (ctDNA) are likely to improve cancer diagnosis, treatment, and minimal residual disease monitoring. However, more trials are required to validate the clinical utility of precise molecular markers for a variety of tumor types. This review focuses on the clinical utility of CTCs and ctDNA testing in patients with solid tumors, including somatic and epigenetic alterations that can be detected. A comparison of methods used to isolate and detect CTCs and some of the intricacies of the characterization of the ctDNA are also provided.

The role of MGMT testing in clinical practice: a report of the association for molecular pathology.

Recent advances in modern molecular technologies allow for the examination and measurement of cancer-related genomic changes. The number of molecular tests for evaluation of diagnostic, prognostic, or predictive markers is expected to increase. In recent years, O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been firmly established as a biomarker in patients diagnosed with gliomas, for both clinical trials and routine clinical management. Similarly, molecular markers, such as loss of heterozygosity (LOH) for 1p/19q have already demonstrated clinical utility in treatment of oligodendroglial tumors, and others might soon show clinical utility. Furthermore, nonrandom associations are being discovered among MGMT, 1p/19q LOH, isocitrate dehydrogenase (IDH) mutations, and other tumor-specific modifications that could possibly enhance our ability to predict outcome and response to therapy. While pathologists are facing new and more complicated requests for clinical genomic testing, clinicians are challenged with increasing numbers of molecular data coming from molecular pathology and genomic medicine. Both pathologists and oncologists need to understand the clinical utility of molecular tests and test results, including issues of turnaround time, and their impact on the application of targeted treatment regimens. This review summarizes the existing data that support the rationale for MGMT promoter methylation testing and possibly other molecular testing in clinically defined glioma subtypes. Various molecular testing platforms for evaluation of MGMT methylation status are also discussed.

Manual microdissection technique in a case of subcutaneous panniculitis-like T-cell lymphoma: a case report and review.

Demonstration of T-cell receptor gene monoclonality often plays an important role in the diagnosis of T-cell lymphoma. When a test to detect monoclonality is performed on whole tissue sections, the presence of a reactive lymphocyte population may reduce sensitivity. This may be especially true for early or borderline cases of lymphoma. Microdissection techniques may be utilized to more readily identify a clonal population of lymphocytes. Subcutaneous panniculitis-like T-cell lymphoma represents a cutaneous lymphoid neoplasm whose clinical course may vary from an indolent, waxing and waning course to an aggressive course resulting in death. We report the first case of a microdissection technique used to facilitate diagnosing a case of subcutaneous panniculitis-like T-cell lympoma.

Clinical performance of JAK2 V617F mutation detection assays in a molecular diagnostics laboratory: evaluation of screening and quantitation methods.

The presence of the JAK2 V617F mutation is now part of clinical diagnostic algorithms, and JAK2 status is routinely assessed when BCR/ABL- chronic myeloproliferative neoplasms (MPNs) are suspected. The aim of this study was to evaluate performance of 3 screening and 1 quantitative method for JAK2 V617F detection. For the study, 43 samples (27 bone marrow aspirates and 16 peripheral blood samples) were selected. The screening assays were the JAK2 Activating Mutation Assay (InVivoScribe, San Diego, CA), JAK2 MutaScreen kit (Ipsogen, Luminy Biotech, Marseille, France), and a home-brew melting curve analysis method. Ipsogen's JAK2 MutaQuant assay was used for quantification of mutant and wild-type alleles. The limit of detection was 1% for the kit-based screening methods and 10% for the melting curve method. The JAK2 MutaQuant assay demonstrated analytic sensitivity of 0.01%. All 4 methods detected cases of BCR/ABL- MPNs and gave negative results with BCR/ABL+ chronic myelogenous leukemia, multiple myeloma, myelodysplastic syndrome, and normal cases.

The Henry Ford production system: LEAN process redesign improves service in the molecular diagnostic laboratory: a paper from the 2008 William Beaumont hospital symposium on molecular pathology.

Accurate and timely molecular test results play an important role in patient management; consequently, there is a customer expectation of short testing turnaround times. Baseline data analysis revealed that the greatest challenge to timely result generation occurred in the preanalytic phase of specimen collection and transport. Here, we describe our efforts to improve molecular testing turnaround times by focusing primarily on redesign of preanalytic processes using the principles of LEAN production. Our goal was to complete greater than 90% of the molecular tests in less than 3 days. The project required cooperation from different laboratory disciplines as well as individuals outside of the laboratory. The redesigned processes involved defining and standardizing the protocols and approaching blood and tissue specimens as analytes for molecular testing. The LEAN process resulted in fewer steps, approaching the ideal of a one-piece flow for specimens through collection/retrieval, transport, and different aspects of the testing process. The outcome of introducing the LEAN process has been a 44% reduction in molecular test turnaround time for tissue specimens, from an average of 2.7 to 1.5 days. In addition, extending LEAN work principles to the clinician suppliers has resulted in a markedly increased number of properly collected and shipped blood specimens (from 50 to 87%). These continuous quality improvements were accomplished by empowered workers in a blame-free environment and are now being sustained with minimal management involvement.

A simplified laboratory validated assay for MGMT promoter hypermethylation analysis of glioma specimens from formalin-fixed paraffin-embedded tissue.

Glioma, and in particular high-grade astrocytoma termed glioblastoma multiforme (GBM), is the most common primary tumor of the brain. Epigenetic silencing of the MGMT (O(6)-methylguanine-DNA Methyl transferase) DNA repair gene by promoter methylation compromises DNA repair and has been associated with longer survival in patients with GBM who receive alkylating agents. The methylation status of the MGMT promoter is determined by methylation-specific polymerase chain reaction analysis (MSP). This protocol is often challenging with GBM specimens, because of extensive necrosis and scarcity of malignant cells. The objective of this study was to develop a reliable, clinically validated assay for detection of epigenetic silencing of the MGMT gene using formalin-fixed, paraffin-embedded brain tumor resections and methylation-specific PCR.

Angioimmunoblastic T-cell lymphoma with supervening Epstein-Barr virus-associated large B-cell lymphoma.

Patients with angioimmunoblastic T-cell lymphoma can have profound immune dysfunction and immunodeficiency. Epstein-Barr virus-driven B-cell lymphoid proliferation can occur in angioimmunoblastic T-cell lymphoma, as in other immunodeficiency states. However, few cases of Epstein-Barr virus-positive B-cell lymphoma arising in patients with preexisting angioimmunoblastic T-cell lymphoma have been reported. We report a case of angioimmunoblastic T-cell lymphoma in which diffuse large B-cell lymphoma developed 56 months after the diagnosis of angioimmunoblastic T-cell lymphoma. The patient survived for 9 years after the initial diagnosis of angioimmunoblastic T-cell lymphoma, and molecular studies performed on multiple biopsy specimens during this period revealed the dynamic nature of clonal lymphoid expansion. Epstein-Barr virus latent membrane protein 1 and Epstein-Barr virus-encoded RNA were detected in the diffuse large B-cell lymphoma, suggesting that Epstein-Barr virus may have played a role in the pathogenesis of the diffuse large B-cell lymphoma.

Failure to detect human herpes simplex virus, cytomegalovirus, and Epstein-Barr virus viral genomes in giant cell arteritis biopsy specimens by real-time quantitative polymerase chain reaction.

A study provided evidence of human herpes simplex virus (HSV) DNA in giant cell arteritis (GCA) biopsy specimens. This prompted us to study our own GCA biopsy specimens using real-time quantitative polymerase chain reaction for the detection of HSV1, cytomegalovirus, and Epstein-Barr virus DNAs. Our study failed to confirm an association between HSV1 and GCA, revealing no viral genome in 35 biopsy specimens of histologically positive temporal arteries.

Detection of non-maternal components of gestational choriocarcinoma by PCR-based microsatellite DNA assay.

OBJECTIVE: Gestational and non-gestational choriocarcinomas have distinctly different tissues of origin, parental genotypes, natural histories, and responses to therapy. Our objective was to develop a convenient, fast, and reliable assay that would, using only patient tissue, allow separation of gestational from non-gestational choriocarcinomas. METHOD: Benign and malignant tissues, preserved in paraffin blocks and separated by microdissection, were examined using a commercial PCR-based tissue identity assay (ABI AmpFlSTR Profiler Plus Kit and ABI 377 DNA sequencer) to detect genetic profiles of 9 microsatellite markers, along with X and Y chromosome markers. Cases included 6 choriocarcinomas. Controls included eight non-germ cell reproductive tract tumors and two hydatidiform moles. RESULTS: The microsatellite markers identified the five choriocarcinomas diagnosed on clinical and histological grounds as gestational, to be of genetically non-maternal (androgenic) origin. The neoplasm previously classified as a non-gestational choriocarcinoma was demonstrated to be of maternal origin, as were the non-germ cell reproductive tract tumors. Samples from hydatidiform moles contained either androgenic markers only or a mix of maternal and androgenic markers, as previously seen in complete and partial moles, respectively. CONCLUSION: A commercially available microsatellite DNA diagnostic assay is a quick and convenient way to discriminate between gestational and non-gestational choriocarcinoma.

Use of microsatellite analysis in detection of tumor lineage as a cause of death in a liver transplant patient.

Malignant tumors are a significant cause of long-term morbidity and mortality in allograft recipients. Most solid tumors in transplant recipients are assumed to arise de novo in the setting of chronic immunosuppressive therapy; however, there have been instances in which malignant tumors have been transplanted in donated tissue from apparently healthy donors. We report a case of a 49-year-old liver transplant patient who presented with metastatic melanoma 9 months after transplantation for hepatocellular carcinoma and who later succumbed to the disease. To investigate the possibility that melanoma was derived from the donor liver, we used a commercially available polymerase chain reaction-based microsatellite marker assay to perform tissue identity testing. The genetic profiles of the patient's original hepatocellular carcinoma and the melanoma from the autopsy specimen were compared with the profile of the normal donor liver tissue, which was still available for testing. The pattern of microsatellite allelic expression strongly suggested that the melanoma detected at autopsy originated from the transplanted liver.