NTRK fusion detection across multiple assays and 33,997 cases: diagnostic implications and pitfalls.

With the FDA approval of larotrectinib, NTRK fusion assessment has recently become a standard part of management for patients with locally advanced or metastatic cancers. Unlike somatic mutation assessment, the detection of NTRK fusions is not straightforward, and various assays exist at the DNA, RNA, and protein level. Here, we investigate the performance of immunohistochemistry and DNA-based next-generation sequencing to indirectly or directly detect NTRK fusions relative to an RNA-based next-generation sequencing approach in the largest cohort of NTRK fusion positive solid tumors to date. A retrospective analysis of 38,095 samples from 33,997 patients sequenced by a targeted DNA-based next-generation sequencing panel (MSK-IMPACT), 2189 of which were also examined by an RNA-based sequencing assay (MSK-Fusion), identified 87 patients with oncogenic NTRK1-3 fusions. All available institutional NTRK fusion positive cases were assessed by pan-Trk immunohistochemistry along with a cohort of control cases negative for NTRK fusions by next-generation sequencing. DNA-based sequencing showed an overall sensitivity and specificity of 81.1% and 99.9%, respectively, for the detection of NTRK fusions when compared to RNA-based sequencing. False negatives occurred when fusions involved breakpoints not covered by the assay. Immunohistochemistry showed overall sensitivity of 87.9% and specificity of 81.1%, with high sensitivity for NTRK1 (96%) and NTRK2 (100%) fusions and lower sensitivity for NTRK3 fusions (79%). Specificity was 100% for carcinomas of the colon, lung, thyroid, pancreas, and biliary tract. Decreased specificity was seen in breast and salivary gland carcinomas (82% and 52%, respectively), and positive staining was often seen in tumors with neural differentiation. Both sensitivity and specificity were poor in sarcomas. Selection of the appropriate assay for NTRK fusion detection therefore depends on tumor type and genes involved, as well as consideration of other factors such as available material, accessibility of various clinical assays, and whether comprehensive genomic testing is needed concurrently.

Loss of Activin Receptor Type 1B Accelerates Development of Intraductal Papillary Mucinous Neoplasms in Mice With Activated KRAS.

BACKGROUND & AIMS: Activin, a member of the transforming growth factor-ß (TGFB) family, might be involved in pancreatic tumorigenesis, similar to other members of the TGFB family. Human pancreatic ductal adenocarcinomas contain somatic mutations in the activin A receptor type IB (ACVR1B) gene, indicating that ACVR1B could be a suppressor of pancreatic tumorigenesis. METHODS: We disrupted Acvr1b specifically in pancreata of mice (Acvr1b(flox/flox);Pdx1-Cre mice) and crossed them with LSL-KRAS(G12D) mice, which express an activated form of KRAS and develop spontaneous pancreatic tumors. The resulting Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice were monitored; pancreatic tissues were collected and analyzed by histology and immunohistochemical analyses. We also analyzed p16(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice and Cre-negative littermates (controls). Genomic DNA, total RNA, and protein were isolated from mouse tissues and primary pancreatic tumor cell lines and analyzed by reverse-transcription polymerase chain reaction, sequencing, and immunoblot analyses. Human intraductal papillary mucinous neoplasm (IPMN) specimens were analyzed by immunohistochemistry. RESULTS: Loss of ACVR1B from pancreata of mice increased the proliferation of pancreatic epithelial cells, led to formation of acinar to ductal metaplasia, and induced focal inflammatory changes compared with control mice. Disruption of Acvr1b in LSL-KRAS(G12D);Pdx1-Cre mice accelerated the growth of pancreatic IPMNs compared with LSL-KRAS(G12D);Pdx1-Cre mice, but did not alter growth of pancreatic intraepithelial neoplasias. We associated perinuclear localization of the activated NOTCH4 intracellular domain to the apical cytoplasm of neoplastic cells with the expansion of IPMN lesions in Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice. Loss of the gene that encodes p16 (Cdkn2a) was required for progression of IPMNs to pancreatic ductal adenocarcinomas in Acvr1b(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice. We also observed progressive loss of p16 in human IPMNs of increasing grades. CONCLUSIONS: Loss of ACVR1B accelerates growth of mutant KRAS-induced pancreatic IPMNs in mice; this process appears to involve NOTCH4 and loss of p16. ACVR1B suppresses early stages of pancreatic tumorigenesis; the activin signaling pathway therefore might be a therapeutic target for pancreatic cancer.

Transfusion of human volunteers with older, stored red blood cells produces extravascular hemolysis and circulating non-transferrin-bound iron.

Transfusions of RBCs stored for longer durations are associated with adverse effects in hospitalized patients. We prospectively studied 14 healthy human volunteers who donated standard leuko-reduced, double RBC units. One unit was autologously transfused "fresh" (3-7 days of storage), and the other "older" unit was transfused after 40 to 42 days of storage. Of the routine laboratory parameters measured at defined times surrounding transfusion, significant differences between fresh and older transfusions were only observed in iron parameters and markers of extravascular hemolysis. Compared with fresh RBCs, mean serum total bilirubin increased by 0.55 mg/dL at 4 hours after transfusion of older RBCs (P = .0003), without significant changes in haptoglobin or lactate dehydrogenase. In addition, only after the older transfusion, transferrin saturation increased progressively over 4 hours to a mean of 64%, and non-transferrin-bound iron appeared, reaching a mean of 3.2µM. The increased concentrations of non-transferrin-bound iron correlated with enhanced proliferation in vitro of a pathogenic strain of Escherichia coli (r = 0.94, P = .002). Therefore, circulating non-transferrin-bound iron derived from rapid clearance of transfused, older stored RBCs may enhance transfusion-related complications, such as infection.

Costs of biomarker testing among patients with metastatic lung or thyroid cancer in the USA: a real-world commercial claims database study.

OBJECTIVE: This real-world retrospective database study quantified the costs of biomarker testing in a US population of patients with lung or thyroid cancers. MATERIALS AND METHODS: The commercial claims IBM Marketscan database, a de-identified real-world dataset, was used to identify patients diagnosed with lung or thyroid cancer between 1/2015 and 12/2019. Eligible patients were 18 years or older with two or more lung or thyroid diagnosis codes. Patients were excluded who had evidence of prior cancer diagnoses. Subgroup analyses evaluated eligible patients with metastatic disease. Descriptive statistics were used to evaluate commercial insurance plan payer and patient out-of-pocket costs for diagnostic testing overall as well as by test procedure code and payer type. Costs were adjusted to 2020 US dollars. RESULTS: A total of 23,633 patients with lung cancer were eligible, 13,320 of whom had metastatic disease. There were 36,867 patients with thyroid cancer, 2,241 of whom had metastatic disease. Biomarker codes were observed among 68.4/75.8% (lung/metastatic lung) and 18.2/42.3% (thyroid/metastatic thyroid). Few patients had codes for comprehensive biomarker tests (5.2/6.7% lung/metastatic lung, 0.3/2.2% thyroid/metastatic thyroid) Among those with biomarker tests, the median per-patient total payer lifetime costs of all biomarker testing were $394/$462 (lung/metastatic lung) and $148/$232 (thyroid/metastatic thyroid). Total lifetime biomarker costs for payers ranged from a median of $128 (consumer-driven health plans) to $477 (preferred provider organizations). Median lifetime patient out-of-pocket costs were $0.00 for both tumor types and all payer types except for consumer-driven health plans ($12 for thyroid and $10 for metastatic lung). CONCLUSIONS: While comprehensive testing adds to the cost of biomarker testing, these data suggest the relatively low lifetime cost of biomarker testing for both payers and patients. Costs for biomarker testing should not be a limitation to access among these populations with commercial insurance plans in the US.

This real-world retrospective database study found that there is a relatively low lifetime total cost of biomarker testing for the care of patients with lung or thyroid cancers. While comprehensive testing adds to the cost of biomarker testing, these data suggest the relatively low lifetime cost of biomarker testing for both payers and patients. Payer costs for biomarker testing do not appear to be limitation to access among populations with commercial insurance plans in this study.

Biomarker testing in patients diagnosed with advanced/metastatic medullary thyroid cancer in the USA.

Aim: To describe real-world testing patterns for RET in US patients with advanced/metastatic medullary thyroid cancer and determine consistency of real-world testing practices with national guidelines. Materials & methods: The authors performed a retrospective medical record analysis of patients with advanced/metastatic medullary thyroid cancer who initiated systemic therapy between 2013 and 2018. Seventy-five US-based oncologists collected the data using a customized electronic data collection form. Results: A total of 59.6% (121 of 203) of patients underwent testing for RET, and 37.2% (45 of 121) had a RET mutation, of which 55.6% were identified as RET mutation-positive before initial diagnosis. Overall, 90 (44.3%) patients were tested for biomarkers on or after initial diagnosis, with RET being the most tested (95.6%) biomarker. Conclusion: The authors' findings suggest an opportunity to improve testing rates in accordance with treatment guidelines.

Mutations in the RET gene are common in patients with medullary thyroid cancer (MTC). As RET mutations are involved in the development of MTC, several treatment guidelines recommend counseling patients and testing for mutations in the RET gene in all patients with MTC. However, limited data are available on RET testing patterns in the USA in this patient population. In this study, the authors determined testing patterns for RET in patients with advanced or metastatic MTC in the USA using real-world data and found that only 60% of patients were tested for RET (i.e., testing for presence of RET mutations was observed in less than two-thirds of all patients included in the study). These results demonstrate the need for improved testing for RET mutations in patients with MTC in alignment with the treatment guidelines in routine clinical practice in the USA.

Real-World Biomarker Testing Patterns in Patients With Metastatic Non-Squamous Non-Small Cell Lung Cancer (NSCLC) in a US Community-Based Oncology Practice Setting.

INTRODUCTION/BACKGROUND: This study was designed to describe real-world changes in biomarker testing among patients with non-squamous, metastatic non-small cell lung cancer (mNSCLC) in a community oncology setting from 2015 to 2020. PATIENTS AND METHODS: This retrospective study randomly selected 500 adult patients diagnosed with nonsquamous mNSCLC to undergo chart review and data extraction. Data were extracted and validated by 2 independent abstractors. Biomarker testing rates were described before and after national guideline updates and FDA approval of targeted agents. RESULTS: At least 1 biomarker test was received by 89.4% of patients with mNSCLC. Of all patients, 46.6%, 34.6%, and 8.2% received both single-gene and next generation sequencing (NGS)-based testing, single-gene testing only, and NGS-based testing only, respectively. However, there were changes in testing rates at the time of drug approvals for targeted agents. Biomarker testing increased for ALK (45.0% before to 78.3% after ALK-targeted drug approval), BRAF (from 20.0% to 67.8%), EGFR (from 20.0% to 78.2%), NTRK (from 34.6% to 55.7%), and ROS1 (increased from 29.6% before approval to 74.2% after). Biomarker testing increased after changes were made to national guidelines for BRAF (from 18.8% before to 68.1% after inclusion in guidelines), NTRK (from 37.2% to 56.5%), and ROS1 (increased from 40.8% to 74.5% after guideline updates). Targeted therapy was received by 62.4% of patients with a positive biomarker. CONCLUSION: Increases in biomarker testing rates were observed relative to targeted agent approvals and national guideline updates. However, many patients with non-squamous mNSCLC did not receive full genotyping in accordance with national guidelines and represent an opportunity to identify reasons and solutions for barriers to care.

Biomarker Testing for Patients With Advanced/Metastatic Nonsquamous NSCLC in the United States of America, 2015 to 2021.

Introduction: NSCLC is a solid tumor with a growing number of actionable biomarkers that may inform treatment. Current guidelines recommend a broad, panel-based approach be taken to identify actionable markers. This retrospective study used a deidentified electronic health records database in the United States to evaluate utilization of various testing modalities. Methods: Data from all adult patients diagnosed with having advanced/metastatic nonsquamous NSCLC between January 2015 and March 2021 were eligible if there was evidence of systemic therapy within 90 days of diagnosis. Results: Records from a total of 17,513 patients (91.6% from community-based practices) were eligible with 83,064 genomic biomarker tests recorded from 2015 to 2021. The proportion of patients who received biomarker testing by next-generation sequencing (NGS)-based methods ranged from 28.3% in 2015 to 68.1% in 2020. The proportion of biomarker testing methods with inconclusive or unsuccessful results ranged from 3.4% for NGS to 9.7% for fluorescence in situ hybridization. The median time to receive results ranged from 4.0 days for polymerase chain reaction-based tests to 10.0 days for immunohistochemistry- and NGS-based tests. Median time to receive results was 8 days for academic and 9 days for community practices. Conclusions: These real-world data suggest increased adoption of NGS-based testing, yet nearly one-third of all patients with advanced/metastatic nonsquamous NSCLC still did not receive broad-based genomic testing by 2020.

Analytical Accuracy of RET Fusion Detection by Break-Apart Fluorescence In Situ Hybridization.

CONTEXT.­: RET gene fusions are oncogenic drivers in nonsmall cell lung cancer and nonmedullary thyroid cancer. Selpercatinib (RETEVMO), a targeted inhibitor of RET, was approved by the US Food and Drug Administration for the treatment of RET fusion-positive nonsmall cell lung cancer and nonmedullary thyroid cancer emphasizing the need for rapid and accurate diagnosis of RET fusions. Fluorescence in situ hybridization (FISH) has been used to detect gene rearrangements, but its performance detecting RET rearrangements is understudied. OBJECTIVE.­: To validate and describe the performance of Abbott Molecular RET break-apart FISH probes for detecting RET rearrangements. DESIGN.­: A training set with RET fusion-positive (13) and RET fusion-negative nonsmall cell lung cancer and nonmedullary thyroid cancer samples (12) was used to establish criteria for FISH scoring. The scoring criteria was then applied to a larger validation set of samples (96). RESULTS.­: A cutoff of 19% or more positive nuclei by FISH was established in the training set and determined by the mean ±3 SD. The validation set was tested using Abbott Molecular RET break-apart FISH compared with sequencing. With this cutoff, a sensitivity of 86% (12 of 14) and specificity of 99% (81 of 82) was achieved. Bootstrapping showed sensitivity could be optimized by using a greater than 13% cutoff with indeterminate samples of 13% to 18% abnormal nuclei requiring confirmation by an orthogonal method. Using this 3-tier scoring system sensitivity increased to 100% (14 of 14) and specificity was 96% (79 of 82). CONCLUSIONS.­: Abbott Molecular break-apart FISH probes can be used to detect RET fusions. Laboratories can optimize cutoffs and/or testing algorithms to maximize sensitivity and specificity to ensure appropriate patients receive effective, timely therapy.

Aberrant T-cell antigen expression in B lymphoblastic leukaemia.

B lymphoblastic leukaemia (B-ALL) cells are characterized by the expression of various B-cell antigens. Expression of T/Natural Killer-cell antigens, however, has rarely been reported in B-ALL (TAg+ B-ALL), and the significance of this aberrant antigen expression is unclear. We thus analysed the frequency of TAg+ B-ALL at our institution and investigated its significance in the context of immunophenotypes, cytogenetic/molecular findings, and prognosis. We reviewed 134 consecutive cases of B-ALL and found 18 cases (13·4%) of TAg+ B-ALL. The most common aberrant T-cell antigens expressed were CD2, CD5, and CD7 at equivalent rates (each in six cases), CD4 (two cases), and CD56 (three cases). Adverse cytogenetic abnormalities were seen in a significantly larger proportion of the TAg+ cases (72·2%) than the TAg- cases (32·2%; P = 0·003). Multivariate Cox-regression analysis showed that the risk of relapse over time was higher in the TAg+ cases, independent of high risk status (based on age and white blood cell count) and the presence of adverse cytogenetic abnormalities (hazard ratio = 2·256, P = 0·065). These findings suggest that T-cell antigen expression in B-ALL may be an independent predictor of poor prognosis, and a useful marker to identify patients at increased risk for relapse and for harbouring adverse cytogenetic abnormalities.

Efficacy of immune checkpoint inhibitor therapy in patients with RET fusion-positive non-small-cell lung cancer.

Aim: To describe outcomes of patients with rearraned during transfection (RET) fusion-positive non-small-cell lung cancer (NSCLC) who received immune checkpoint inhibitor (ICI)-based treatments in the US. Patients & methods: Using de-identified Flatiron Health-Foundation Medicine NSCLC Clinico-Genomic and Guardant Health databases, treatment patterns and outcomes of 69 patients with advanced/metastatic RET fusion-positive NSCLC who received ICI-based treatment were described. Results: Median real-world progression-free survival and overall survival months were 4.2 (95% CI: 1.4-8.4) and 19.1 (6.9-not reached), respectively, among patients in Clinico-Genomic database (n = 17) receiving first-line ICI-based therapy. In the Guardant Health database, progression-free survival was unavailable, and the median overall survival was not reached (n = 29). Conclusion: Outcomes associated with ICI-based treatments in the first-line setting among patients with RET fusion-positive NSCLC are consistent with unselected populations reported in literature.

Lay abstract Treatment options are rapidly expanding for patients who are diagnosed with advanced/metastatic non-small-cell lung cancer. The current standard of care for patients who do not have an actionable genomic alteration includes immune checkpoint inhibitor (ICI)-based therapy. However, the role of ICI-based therapy is not well understood in patients with rearranged during transfection (RET) fusions. In this study, the survival outcomes observed in patients with RET fusion-positive non-small-cell lung cancer who received first-line ICI-based therapy were comparable with published data from patients without genomic alterations. This study is limited due to the small sample size.

Clinical Utilization, Utility, and Reimbursement for Expanded Genomic Panel Testing in Adult Oncology.

PURPOSE: The routine use of large next-generation sequencing (NGS) pan-cancer panels is required to identify the increasing number of, but often uncommon, actionable alterations to guide therapy. Inconsistent coverage and variable payment is hindering NGS adoption into clinical practice. A review of test utilization, clinical utility, coverage, and reimbursement was conducted in a cohort of patients diagnosed with high-risk cancer who received pan-cancer panel testing as part of their clinical care. MATERIALS AND METHODS: The Columbia Combined Cancer Panel (CCCP), a 467-gene panel designed to detect DNA variations in solid and liquid tumors, was performed in the Laboratory of Personalized Genomic Medicine at Columbia University Irving Medical Center. Utilization was characterized at test order. Results were reviewed by a molecular pathologist, followed by a multidisciplinary molecular tumor board where clinical utility was classified by consensus. Reimbursement was reviewed after payers provided final coverage decisions. RESULTS: NGS was performed on 359 high-risk tumors from 349 patients. Reimbursement data were available for 246 cases. The most common reason providers ordered CCCP testing was for patients diagnosed with a treatment-resistant or recurrent tumor (n = 214; 61%). Findings were clinically impactful for 229 cases (64%). Molecular alterations that may inform future therapy in the event of progression or relapse were found in 42% of cases, and a targeted therapy was initiated in 23 cases (6.6%). The majority of tests were denied coverage by payers (n = 190; 77%). On average, insurers reimbursed 10.75% of the total NGS service charge. CONCLUSION: CCCP testing identified clinically impactful alterations in 64% of cases. Limited coverage and low reimbursement remain barriers, and broader reimbursement policies are needed to adopt pan-cancer NGS testing that benefits patients into clinical practice.

Molecular Pathology Economics 101: An Overview of Molecular Diagnostics Coding, Coverage, and Reimbursement: A Report of the Association for Molecular Pathology.

Widespread indications for use of molecular diagnostics in various aspects of clinical medicine have driven proliferation of testing. The rapid adoption and continuous technological evolution of molecular diagnostics have often strained the development and maintenance of a functional underlying framework of coding, coverage, and reimbursement policies, thereby presenting challenges to various stakeholders, including molecular professionals, payers, and patients. A multidisciplinary working group convened by the Association for Molecular Pathology Economic Affairs Committee was tasked to describe the complex landscape of molecular pathology economics and highlight opportunities for member engagement. In this article, on the basis of review and synthesis of government regulations and procedures, published payer policy documents, peer-reviewed literature, and expert consensus, the Working Group navigates the ecosystem of molecular pathology economics in terms of stakeholders, coding systems and processes, coverage policy determination, and pricing mechanisms. The composition and interrelatedness of various working groups and committees are emphasized to highlight the functional underpinnings of the system. Molecular professionals must be conversant in the language and complex inner workings of molecular pathology economics to lead successful, viable laboratories and advocate effectively for policy development on their behalf. This overview is provided to be a resource to molecular professionals as they navigate the reimbursement landscape.

Detection of Tumor NTRK Gene Fusions to Identify Patients Who May Benefit from Tyrosine Kinase (TRK) Inhibitor Therapy.

Chromosomal rearrangements involving the NTRK1, NTRK2, and NTRK3 genes (NTRK genes), which encode the high-affinity nerve growth factor receptor (TRKA), brain-derived neurotrophic factor/neurotrophin-3 (BDNF/NT-3) growth factor receptor (TRKB), and neurotrophin-3 (NT-3) growth factor receptor (TRKC) tyrosine kinases (TRK proteins), act as oncogenic drivers in a broad range of pediatric and adult tumor types. NTRK gene fusions have been shown to be actionable genomic events that are predictive of response to TRK kinase inhibitors, making their routine detection an evolving clinical priority. In certain exceedingly rare tumor types, NTRK gene fusions may be seen in the overwhelming majority of cases, whereas in a range of common cancers, reported incidences are in the range of 0.1% to 2%. Herein, we review the structure of the three NTRK genes and the nature and incidence of NTRK gene fusions in different solid tumor types, and we summarize the clinical data showing the importance of identifying tumors harboring such genomic events. We also outline the laboratory techniques that can be used to diagnose NTRK gene fusions in clinical samples. Finally, we propose a diagnostic algorithm for solid tumors to facilitate the identification of patients with TRK fusion cancer. This algorithm accounts for the widely varying frequencies by tumor histology and the underlying prevalence of TRK expression in the absence of NTRK gene fusions and is based on a combination of fluorescence in situ hybridization, next-generation sequencing, and immunohistochemistry assays.

Plasma-thrombin cell blocks: Potential source of DNA contamination.

BACKGROUND: Cell blocks are being used more frequently in cytology for ancillary testing, including molecular diagnostics. There are several different methods of processing cell blocks, with plasma-thrombin being one of the most common. Plasma is a blood-derived product and may be a source of DNA. The aim of this study was to determine whether the plasma used for the plasma-thrombin cell block method has amplifiable DNA that may potentially interfere with molecular testing results. METHODS: Expired bags of fresh frozen plasma were collected from a blood bank. From each sample, DNA was extracted from a 1-mL aliquot with the QIAsymphony MIDI kit (Qiagen). The concentration of DNA was measured on a NanoDrop instrument. The amplifiable DNA quality was assessed by polymerase chain reaction (PCR) with primers to generate amplicons of various sizes. Characterization was performed with the AmpFLSTR Identifiler Plus PCR kit with capillary electrophoresis. RESULTS: Twenty samples from 20 bags were collected. All samples showed amplifiable DNA despite low DNA concentrations in a few cases. PCR amplification revealed the presence of high-quality amplifiable DNA (up to 600 base pairs). DNA was amplified at the 16 loci interrogated in all samples tested with the AmpFLSTR Identifiler Plus PCR kit. CONCLUSIONS: The presence of genomic DNA in plasma may theoretically interfere with results of molecular testing. Particularly in clinical samples with low cellularity, the DNA in plasma may potentially either mask the presence of minute amounts of tumor-derived DNA or lead to a false-positive result.

Fat-predominant mixed epithelial stromal tumor (MEST): report of a unique case mimicking angiomyolipoma.

A unique case of a mixed epithelial stromal tumor (MEST) that was predominantly composed of adipose tissue is reported here. Radiographically and grossly, the lesion was thought to be an angiomyolipoma, based upon its fatty appearance. Microscopically, the lesion was predominantly composed of mature adipose tissue but also contained clusters of bland tubules surrounded by smooth muscle bundles and collagen. By immunohistochemistry, the stroma labeled diffusely for estrogen and progesterone receptors, while the muscle bundles labeled for desmin. Melanocytic markers HMB45 and Melan A, typically positive in angiomyolipoma, were nonreactive. This case expands the morphologic spectrum of MEST to include mimics of angiomyolipoma.

The History and Impact of Molecular Coding Changes on Coverage and Reimbursement of Molecular Diagnostic Tests: Transition from Stacking Codes to the Current Molecular Code Set Including Genomic Sequencing Procedures.

Changes in coding and coverage generate an uncertain reimbursement environment for molecular pathology laboratories. We analyzed our experience with two representative molecular oncology tests: a T-cell receptor (TCR) ß rearrangement test and a large (467-gene) cancer next-generation sequencing panel, the Columbia Combined Cancer Panel (CCCP). Before 2013, the TCR ß test was coded using stacked current procedural terminology codes and subsequently transitioned to a tier 1 code. CCCP was coded using a combination of tier 1 and 2 codes until 2015, when a new Genomic Sequencing Procedure code was adopted. A decrease in reimbursement of 61% was observed for the TCR ß test on moving from stacking to tier 1 codes. No initial increase in total rejection rate was observed, but a subsequent increase in rejection rates in 2015 and 2016 was noted. The CCCP test showed a similar decrease (48%) in reimbursement after adoption of the new Genomic Sequencing Procedure code and was accompanied by a sharp increase in rejection rates both on implementation of the new code and over time. Changes in coding can result in substantial decreases in reimbursement. This may be a barrier to patient access because of the high cost of molecular diagnostics. Revisions to the molecular code set will continue. These findings help laboratories and manufacturers prepare for the financial impact and advocate appropriately.

Clinical Genomic Profiling of a Diverse Array of Oncology Specimens at a Large Academic Cancer Center: Identification of Targetable Variants and Experience with Reimbursement.

Large cancer panels are being increasingly used in the practice of precision medicine to generate genomic profiles of tumors with the goal of identifying targetable variants and guiding eligibility for clinical trials. To facilitate identification of mutations in a broad range of solid and hematological malignancies, a 467-gene oncology panel (Columbia Combined Cancer Panel) was developed in collaboration with pathologists and oncologists and is currently available and in use for clinical diagnostics. Herein, we share our experience with this testing in an academic medical center. Of 255 submitted specimens, which encompassed a diverse range of tumor types, we were able to successfully sequence 92%. The Columbia Combined Cancer Panel assay led to the detection of a targetable variant in 48.7% of cases. However, although we show good clinical performance and diagnostic yield, third-party reimbursement has been poor. Reimbursement from government and third-party payers using the 81455 Current Procedural Terminology code was at 19.4% of billed costs, and 55% of cases were rejected on first submission. Likely contributing factors to this low level of reimbursement are the delays in valuation of the 81455 Current Procedural Terminology code and in establishing national or local coverage determinations. In the absence of additional demonstrations of clinical utility and improved patient outcomes, we expect the reimbursement environment will continue to limit the availability of this testing more broadly.

Precision Medicine in Children and Young Adults with Hematologic Malignancies and Blood Disorders: The Columbia University Experience.

BACKGROUND: The advent of comprehensive genomic profiling has markedly advanced the understanding of the biology of pediatric hematological malignancies, however, its application to clinical care is still unclear. We present our experience integrating genomic data into the clinical management of children with high-risk hematologic malignancies and blood disorders and describe the broad impact that genomic profiling has in multiple aspects of patient care. METHODS: The Precision in Pediatric Sequencing Program at Columbia University Medical Center instituted prospective clinical next-generation sequencing (NGS) for high-risk malignancies and blood disorders. Testing included cancer whole exome sequencing (WES) of matched tumor-normal samples or targeted sequencing of 467 cancer-associated genes, when sample adequacy was a concern, and tumor transcriptome (RNA-seq). A multidisciplinary molecular tumor board conducted interpretation of results and final tiered reports were transmitted to the electronic medical record according to patient preferences. RESULTS: Sixty-nine samples from 56 patients with high-risk hematologic malignancies and blood disorders were sequenced. Patients carried diagnoses of myeloid malignancy (n = 25), lymphoid malignancy (n = 25), or histiocytic disorder (n = 6). Six patients had only constitutional WES, performed for a suspicion of an inherited predisposition for their disease. For the remaining 50 patients, tumor was sequenced with matched normal tissue when available. The mean number of somatic variants per sample was low across the different disease categories (2.85 variants/sample). Interestingly, a gene fusion was identified by RNA-seq in 58% of samples who had adequate RNA available for testing. Molecular profiling of tumor tissue led to clinically impactful findings in 90% of patients. Forty patients (80%) had at least one targetable gene variant or fusion identified in their tumor tissue; however, only seven received targeted therapy. Importantly, NGS findings contributed to the refinement of diagnosis and prognosis for 34% of patients. Known or likely pathogenic germline alterations were discovered in 24% of patients involving cancer predisposition genes in 12% of cases. CONCLUSION: Incorporating whole exome and transcriptome profiling of tumor and normal tissue into clinical practice is feasible, and the value that comprehensive testing provides extends beyond the ability to target-specific mutations.

Hepatocellular adenoma classification: a comparative evaluation of immunohistochemistry and targeted mutational analysis.

BACKGROUND: Four subtypes of hepatocellular adenomas (HCA) are recognized: hepatocyte-nuclear-factor-1α mutated (H-HCA), ß-catenin-mutated type with upregulation of glutamine synthetase (b-HCA), inflammatory type (IHCA) with serum-amyloid-A overexpression, and unclassified type. Subtyping may be useful since b-HCA appear to have higher risk of malignant transformation. We sought to apply subtype analysis and assess histological atypia, correlating these with next-generation sequencing analysis. METHODS: Twenty-six HCA were stained with serum amyloid A (SAA), liver fatty acid-binding protein (LFABP), glutamine synthetase (GS), and ß-catenin IHC, followed by analysis with a targeted multiplex sequencing panel. RESULTS: By IHC, 4 HCA (15.4 %) were classified as b-HCA, 11 (42.3 %) as IHCA, 9 (34.6 %) as H-HCA, and two (7.7 %) unclassifiable. Eight HCA (30.8 %) showed atypia (3 b-HCA, 4 IHCA and 1 H-HCA). Targeted sequencing confirmed HNF1A mutations in all H-HCA, confirming reliability of LFABP IHC in identifying these lesions. CTNNB1 mutations were detected in 1 of 4 (25 %) of GS/ß-catenin-positive cases, suggesting that positive GS stain does not always correlate with CTNNB1 mutations. CONCLUSIONS: Immunohistochemistry does not consistently identify b-HCA. Mutational analysis improves the diagnostic accuracy of ß-catenin-mutated HCA and is an important tool to assess risk of malignancy in HCA.

Implementation of next generation sequencing into pediatric hematology-oncology practice: moving beyond actionable alterations.

BACKGROUND: Molecular characterization has the potential to advance the management of pediatric cancer and high-risk hematologic disease. The clinical integration of genome sequencing into standard clinical practice has been limited and the potential utility of genome sequencing to identify clinically impactful information beyond targetable alterations has been underestimated. METHODS: The Precision in Pediatric Sequencing (PIPseq) Program at Columbia University Medical Center instituted prospective clinical next generation sequencing (NGS) for pediatric cancer and hematologic disorders at risk for treatment failure. We performed cancer whole exome sequencing (WES) of patient-matched tumor-normal samples and RNA sequencing (RNA-seq) of tumor to identify sequence variants, fusion transcripts, relative gene expression, and copy number variation (CNV). A directed cancer gene panel assay was used when sample adequacy was a concern. Constitutional WES of patients and parents was performed when a constitutionally encoded disease was suspected. Results were initially reviewed by a molecular pathologist and subsequently by a multi-disciplinary molecular tumor board. Clinical reports were issued to the ordering physician and posted to the patient's electronic medical record. RESULTS: NGS was performed on tumor and/or normal tissue from 101 high-risk pediatric patients. Potentially actionable alterations were identified in 38% of patients, of which only 16% subsequently received matched therapy. In an additional 38% of patients, the genomic data provided clinically relevant information of diagnostic, prognostic, or pharmacogenomic significance. RNA-seq was clinically impactful in 37/65 patients (57%) providing diagnostic and/or prognostic information for 17 patients (26%) and identified therapeutic targets in 15 patients (23%). Known or likely pathogenic germline alterations were discovered in 18/90 patients (20%) with 14% having germline alternations in cancer predisposition genes. American College of Medical Genetics (ACMG) secondary findings were identified in six patients. CONCLUSIONS: Our results demonstrate the feasibility of incorporating clinical NGS into pediatric hematology-oncology practice. Beyond the identification of actionable alterations, the ability to avoid ineffective/inappropriate therapies, make a definitive diagnosis, and identify pharmacogenomic modifiers is clinically impactful. Taking a more inclusive view of potential clinical utility, 66% of cases tested through our program had clinically impactful findings and samples interrogated with both WES and RNA-seq resulted in data that impacted clinical decisions in 75% of cases.