Molecular Oncology Testing Research Articles

General Applicability of Existing College of American Pathologists Accreditation Requirements to Clinical Implementation of Machine Learning-Based Methods in Molecular Oncology Testing.

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. 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. 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. 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. 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.

SPOT/Dx Pilot Reanalysis and College of American Pathologists Proficiency Testing for KRAS and NRAS Demonstrate Excellent Laboratory Performance.

The Sustainable Predictive Oncology Therapeutics and Diagnostics quality assurance pilot study (SPOT/Dx pilot) on molecular oncology next-generation sequencing (NGS) reportedly demonstrated performance limitations of NGS laboratory-developed tests, including discrepancies with a US Food and Drug Administration-approved companion diagnostic. The SPOT/Dx pilot methods differ from those used in proficiency testing (PT) programs. To reanalyze SPOT/Dx pilot data using PT program methods and compare to PT program data.Also see p. 136. The College of American Pathologists (CAP) Molecular Oncology Committee reanalyzed SPOT/Dx pilot data applying PT program methods, adjusting for confounding conditions, and compared them to CAP NGS PT program performance (2019-2022). Overall detection rates of KRAS and NRAS single-nucleotide variants (SNVs) and multinucleotide variants (MNVs) by SPOT/Dx pilot laboratories were 96.8% (716 of 740) and 81.1% (129 of 159), respectively. In CAP PT programs, the overall detection rates for the same SNVs and MNVs were 97.2% (2671 of 2748) and 91.8% (1853 of 2019), respectively. In 2022, the overall detection rate for 5 KRAS and NRAS MNVs in CAP PT programs was 97.3% (1161 of 1193). CAP PT program data demonstrate that laboratories consistently have high detection rates for KRAS and NRAS variants. The SPOT/Dx pilot has multiple design and analytic differences with established PT programs. Reanalyzed pilot data that adjust for confounding conditions demonstrate that laboratories proficiently detect SNVs and less successfully detect rare to never-observed MNVs. The SPOT/Dx pilot results are not generalizable to all molecular oncology testing and should not be used to market products or change policy affecting all molecular oncology testing.

Neoplastic Cellularity Assessment in Molecular Testing.

Neoplastic cellularity assessment has become an essential component of molecular oncology testing; however, there are currently no best practice recommendations or guidelines for this potentially variable step in the testing process. To describe the domestic and international practices of neoplastic cellularity assessment and to determine how variations in laboratory practices affect neoplastic cellularity assessment accuracy. Data were derived from 57 US and international laboratories that participated in the 2019 College of American Pathologists Neoplastic Cellularity Proficiency Testing Survey (NEO-B 2019). NEO-B 2019 included 29 laboratory practice questions and 5 images exhibiting challenging histologic features. Participants assessed the neoplastic cellularity of hematoxylin-eosin-stained digital images, and results were compared to a criterion standard derived from a manual cell count. The survey responses showed variations in the laboratory practices for the assessment of neoplastic cellularity, including the definition of neoplastic cellularity, assessment methodology, counting practices, and quality assurance practices. In some instances, variation in laboratory practice affected neoplastic cellularity assessment performance. The results highlight the need for a consensus definition and improved standardization of the assessment of neoplastic cellularity. We put forth an initial set of best practice recommendations to begin the process of standardizing neoplastic cellularity assessment.

Donor Engraftment of a HSC-Transplanted CLL Patient Experiencing Richter’ Transformation to DLBCL

Abstract A patient diagnosed with Chronic Lymphocytic Leukemia (CLL) received HSC transplantation, and displayed stable donor engraftment. Three years after transplantation, the patient experienced a relapse in the form of Diffuse Large B cell Lymphoma (DLBCL). Total blood, bone marrow, lymph node (LN) and lineage specific Chimerism measurements were performed using AmpFlSTR+ (Applied Biosystems) kit. The patient showed full myeloid engraftment and mixed T and B Chimerism immediately after transplant. Within 6–12 weeks all lineages studied (T, B, NK, myeloid and erythroid) were fully donor (99–100%), which remained stable for over 3 years. A miniscule drop in the B cell lineage Chimerism was noted (97.3%) 42 months after transplantation. Concurrent clinical status (lymphadenopathy, abdominal pain) suggested relapse. Histopathology and flow cytometry studies showed that the patient was experiencing Richter’s transformation (RT) to DLBCL. Molecular oncology testing demonstrated that the DLBCL IgH rearrangement was identical to the original CLL disease. Leftover LN tissue which had been submitted for flow cytometry studies and demonstrated the presence of immunophenotypically aberrant monotypic B cells (>50% of cellularity) was processed for Chimerism and showed 21% donor cells. RT is defined as transformation from CLL into a more aggressive lymphoma, typically DLBCL, with a reported incidence ranging from 1 to 9%. The median time to RT is 1.9 years after diagnosis. Importantly, RT changes the nature of the hematological neoplasm from leukemia, residing mostly in the blood and bone marrow, to lymphoma, a disease which mainly affects secondary lymphoid organs. Therefore, this patient displayed complete or near complete donor engraftment in the peripheral blood (including in lymphoid lineages) by routine Chimerism testing. The reemergence of neoplastic cells of recipient origin was clearly evident when the test was applied to an affected lymph node. In conclusion RT from leukemia to lymphoma may represent a significant challenge to monitoring disease relapse by Chimerism.

Rapid BRAF Mutation Testing in Pigmented Melanomas.

BRAF mutations are present in ∼40%-60% of melanomas, and targeted therapy in advanced-stage melanoma is associated with improvement in overall and progression-free survival. Accordingly, BRAF mutation determination is standard-of-care in metastatic melanoma, and a rapid, accurate assay is desirable. Melanin can present unique challenges due to inhibition of the polymerase chain reaction. The novel cartridge-based Idylla platform offers rapid molecular oncology testing; however, a formal evaluation of the impact of melanin on testing heretofore has not been explored. In this study, we evaluated the performance of Idylla BRAF mutation detection in 23 melanomas including resections, small biopsies, and cytology cell blocks. Pathologists assigned each case a pigment score from 0 to 2 based on extent of melanin content. Samples with a pigment score of 2 were successfully resulted, thus demonstrating that high melanin content did not inhibit the assay. Sensitivity and specificity of BRAF mutation detection were 100% compared with reference laboratory testing. Tissue input requirements were low, with the Idylla successfully detecting a BRAF mutation in cell block material containing only ∼400 tumor cells. The assay was simple and quick to perform, with total hands-on time of 5-10 minutes and instrument time ∼90 minutes. In summary, the Idylla BRAF mutation assay provides rapid, robust testing for melanomas with high melanin content, including samples with limited material. The assay requires minimal technical expertise, making mutation status determination accessible in a range of clinical laboratory settings. The total assay time of <2 hours facilitates prompt results to guide patient care decisions.

A Window Into Clinical Next-Generation Sequencing–Based Oncology Testing Practices

- Detection of acquired variants in cancer is a paradigm of precision medicine, yet little has been reported about clinical laboratory practices across a broad range of laboratories. - To use College of American Pathologists proficiency testing survey results to report on the results from surveys on next-generation sequencing-based oncology testing practices. - College of American Pathologists proficiency testing survey results from more than 250 laboratories currently performing molecular oncology testing were used to determine laboratory trends in next-generation sequencing-based oncology testing. - These presented data provide key information about the number of laboratories that currently offer or are planning to offer next-generation sequencing-based oncology testing. Furthermore, we present data from 60 laboratories performing next-generation sequencing-based oncology testing regarding specimen requirements and assay characteristics. The findings indicate that most laboratories are performing tumor-only targeted sequencing to detect single-nucleotide variants and small insertions and deletions, using desktop sequencers and predesigned commercial kits. Despite these trends, a diversity of approaches to testing exists. - This information should be useful to further inform a variety of topics, including national discussions involving clinical laboratory quality systems, regulation and oversight of next-generation sequencing-based oncology testing, and precision oncology efforts in a data-driven manner.

Identification of major factors associated with failed clinical molecular oncology testing performed by next generation sequencing (NGS)

PurposeDNA analysis by NGS has become important to direct the clinical care of cancer patients. However, NGS is not successful in all cases, and the factors responsible for test failures have not been systematically evaluated. Materials and methodsA series of 1528 solid and hematolymphoid tumor specimens was tested by an NGS comprehensive cancer panel during 2012–2014. DNA was extracted and 2×101 bp paired-end sequence reads were generated on cancer-related genes utilizing Illumina HiSeq and MiSeq platforms. ResultsTesting was unsuccessful in 343 (22.5%) specimens. The failure was due to insufficient tissue (INST) in 223/343 (65%) cases, insufficient DNA (INS-DNA) in 99/343 (28.9%) cases, and failed library (FL) in 21/343 (6.1%) cases. 87/99 (88%) of the INS-DNA cases had below 10 ng DNA available for testing. Factors associated with INST and INS-DNA failures were site of biopsy (SOB) and type of biopsy (TOB) (both p < 0.0001), and clinical setting of biopsy (CSB, initial diagnosis or recurrence) (p < 0.0001). Factors common to INST and FL were age of specimen (p ≤ 0.006) and tumor viability (p ≤ 0.05). Factors common to INS-DNA and FL were DNA purity and DNA degradation (all p ≤ 0.005). In multivariate analysis, common predictors for INST and INS-DNA included CSB (p = 0.048 and p < 0.0001) and TOB (both p ≤ 0.003), respectively. SOB (p = 0.004) and number of cores (p = 0.001) were specific for INS-DNA, whereas TOB and DNA degradation were associated with FL (p = 0.04 and 0.02, respectively). ConclusionsPre-analytical causes (INST and INS-DNA) accounted for about 90% of all failed cases; independent of test design. Clinical setting; site and type of biopsy; and number of cores used for testing all correlated with failure. Accounting for these factors at the time of tissue biopsy acquisition could improve the analytic success rate.

Multiplatform comparison of molecular oncology tests performed on cytology specimens and formalin‐fixed, paraffin‐embedded tissue

Molecular oncology testing is important for patient management, and requests for the molecular analysis of cytology specimens are increasingly being made. Formalin-fixed, paraffin-embedded (FFPE) cell blocks of such specimens have been routinely used for molecular diagnosis. However, the inability to assess cellularity before cell block preparation is a pitfall of their use. In this study, various cytologic preparations were tested with several molecular test platforms, and the results were compared with paired FFPE tissue. Seventy-seven cytology cases, including fine-needle aspiration smears, touch preparations, and SurePath thin-layer preparations, were selected from the archives. Areas of interest were removed from the slide with a matrix capture solution. DNA extracted from the cells was evaluated by mutation analysis for BRAF, epidermal growth factor receptor (EGFR), RAS, and a 50-gene panel with various testing platforms (single-nucleotide primer extension assay, Sanger sequencing, and next-generation sequencing). Thirty-eight tumors with available FFPE tissue were tested in parallel. The average DNA concentration was 299 ng/µL for the cytology specimens and 171 ng/µg for the paired FFPE tissue. Point mutations and large deletions were detected in BRAF, KRAS, NRAS, HRAS, and EGFR genes. In comparison with FFPE tissue, 5- to 8-fold less input DNA was needed when cytology preparations were used. The concordance between cytology specimens and FFPE tissue was 100%. Cytologic preparations were found to be a reliable source for molecular oncology testing. DNA derived from cytology specimens performed well on multiple platforms, and 100% concordance was observed between cytology specimens and FFPE tissue.

A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing.

BackgroundExtraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column.MethodsThree DNA extraction methods were compared: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student’s t-test.ResultsThe combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods.ConclusionsUsing H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.

Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing

The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement-negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel-based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.

Molecular Oncology Testing in Resource-Limited Settings

Molecular Oncology Testing in Resource-Limited Settings

Personalized medicine and pharmacogenetic biomarkers: progress in molecular oncology testing

In the field of oncology, clinical molecular diagnostics and biomarker discoveries are constantly advancing as the intricate molecular mechanisms that transform a normal cell into an aberrant state in concert with the dysregulation of alternative complementary pathways are increasingly understood. Progress in biomarker technology, coupled with the companion clinical diagnostic laboratory tests, continue to advance this field, where individualized and customized treatment appropriate for each individual patient define the standard of care. Here, we discuss the current commonly used predictive pharmacogenetic biomarkers in clinical oncology molecular testing: BRAF V600E for vemurafenib in melanoma; EML4–ALK for crizotinib and EGFR for erlotinib and gefitinib in non-small-cell lung cancer; KRAS against the use of cetuximab and panitumumab in colorectal cancer; ERBB2 (HER2/neu) for trastuzumab in breast cancer; BCR–ABL for tyrosine kinase inhibitors in chronic myeloid leukemia; and PML/RARα for all-trans-retinoic acid and arsenic trioxide treatment for acute promyelocytic leukemia.

Postal survey of physicians and laboratories: Practices and perceptions of molecular oncology testing

BackgroundMolecular oncology testing (MOT) to detect genomic alterations underlying cancer holds promise for improved cancer care. Yet knowledge limitations regarding the delivery of testing services may constrain the translation of scientific advancements into effective health care.MethodsWe conducted a cross-sectional, self-administered, postal survey of active cancer physicians in Ontario, Canada (N = 611) likely to order MOT, and cancer laboratories (N = 99) likely to refer (i.e., referring laboratories) or conduct (i.e., testing laboratories) MOT in 2006, to assess respondents' perceptions of the importance and accessibility of MOT and their preparedness to provide it.Results54% of physicians, 63% of testing laboratories and 60% of referring laboratories responded. Most perceived MOT to be important for treatment, diagnosis or prognosis now, and in 5 years (61% – 100%). Yet only 45% of physicians, 59% of testing labs and 53% of referring labs agreed that patients in their region were receiving MOT that is indicated as a standard of care. Physicians and laboratories perceived various barriers to providing MOT, including, among 70% of physicians, a lack of clear guidelines regarding clinical indications, and among laboratories, a lack of funding (73% – 100%). Testing laboratories were confident of their ability to determine whether and which MOT was indicated (77% and 82% respectively), and perceived that key elements of formal and continuing education were helpful (75% – 100%). By contrast, minorities of physicians were confident of their ability to assess whether and which MOT was indicated (46% and 34% respectively), and while majorities considered various continuing educational resources helpful (68% – 75%), only minorities considered key elements of formal education helpful in preparing for MOT (17% – 43%).ConclusionPhysicians and laboratory professionals were enthusiastic about the value of MOT for cancer care but most did not believe patients were gaining adequate access to clinically necessary testing. Further, our results suggest that many were ill equipped as individual stakeholders, or as a coordinated system of referral and interpretation, to provide MOT. These challenges should inspire educational, training and other interventions to ensure that developments in molecular oncology can result in optimal cancer care.