Treatment recommendations to cancer patients in the context of FDA guidance for next generation sequencing.

BACKGROUND: Regulatory approval of next generation sequencing (NGS) by the FDA is advancing the use of genomic-based precision medicine for the therapeutic management of cancer as standard care. Recent FDA guidance for the classification of genomic variants based on clinical evidence to aid clinicians in understanding the actionability of identified variants provided by comprehensive NGS panels has also been set forth. In this retrospective analysis, we interpreted and applied the FDA variant classification guidance to comprehensive NGS testing performed for advanced cancer patients and assessed oncologist agreement with NGS test treatment recommendations. METHODS: NGS comprehensive genomic profiling was performed in a CLIA certified lab (657 completed tests for 646 patients treated at Roswell Park Comprehensive Cancer Center) between June 2016 and June 2017. Physician treatment recommendations made within 120 days post-test were gathered from tested patients' medical records and classified as targeted therapy, precision medicine clinical trial, immunotherapy, hormonal therapy, chemotherapy/radiation, surgery, transplant, or non-therapeutic (hospice, surveillance, or palliative care). Agreement between NGS test report targeted therapy recommendations based on the FDA variant classification and physician targeted therapy treatment recommendations were evaluated. RESULTS: Excluding variants contraindicating targeted therapy (i.e., KRAS or NRAS mutations), at least one variant with FDA level 1 companion diagnostic supporting evidence as the most actionable was identified in 14% of tests, with physicians most frequently recommending targeted therapy (48%) for patients with these results. This stands in contrast to physicians recommending targeted therapy based on test results with FDA level 2 (practice guideline) or FDA level 3 (clinical trial or off label) evidence as the most actionable result (11 and 4%, respectively). CONCLUSIONS: We found an appropriate "dose-response" relationship between the strength of clinical evidence supporting biomarker-directed targeted therapy based on application of FDA guidance for NGS test variant classification, and subsequent treatment recommendations made by treating physicians. In view of recent changes at FDA, it is paramount to define regulatory grounds and medical policy coverage for NGS testing based on this guidance.

Deletion and deletion/insertion mutations in the juxtamembrane domain of the FLT3 gene in adult acute myeloid leukemia.

In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF) deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del) deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations.

Assessment of DNA damage at the dimer level: measurement of the formamide lesion.

UVC-radiation-induced DNA damage was measured in mouse fibroblast cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with isotopically labeled internal standards. The thymine glycol and formamide lesions were assayed in the form of modified dinucleoside monophosphates. The 8-oxo-7,8-dihydroguanine lesion was measured as the modified nucleoside. DNA damage in cells treated with tirapazamine was also measured. Tirapazamine is a chemotherapeutic agent that acts via a free radical mechanism. The two agents, UVC radiation and tirapazamine, produce markedly different profiles of DNA damage, reflecting their respective mechanisms of action. Both agents produce significant amounts of thymine glycol and formamide damage, but only the former produced a measurable amount of the 8-oxo-7,8-dihydroguanine lesion. The merits of measuring DNA damage at the dimer level are discussed.

A novel approach to DNA damage assessments: measurement of the thymine glycol lesion.

A different approach to the measurement of DNA damage has been developed based on the fact that many lesions can be excised from DNA in the form of modified dinucleoside monophosphates. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used in conjunction with isotopically labeled internal standards to quantify the lesion. The method has several advantages, including high sensitivity for the detection of dinucleoside monophosphates. The method was applied to the measurement of the 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol) lesion in the DNA of mouse fibroblast cells exposed in culture to various treatments including ionizing radiation, UVC light and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. The application of the method to the measurement of other DNA lesions is discussed.

Singlet oxygen-induced DNA damage.

Singlet oxygen, hydrogen peroxide, hydroxyl radical and hydrogen peroxide are the reactive oxygen species (ROS) considered most responsible for producing oxidative stress in cells and organisms. Singlet oxygen interacts preferentially with guanine to produce 8-oxo-7,8-dihydroguanine and spiroiminodihydantoin. DNA damage due to the latter lesion has not been detected directly in the DNA of cells exposed to singlet oxygen. In this study, the singlet oxygen-induced lesion was isolated from a short synthetic oligomer after exposure to UVA radiation in the presence of methylene blue. The lesion could be enzymatically excised from the oligomer in the form of a modified dinucleoside monophosphate. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), the singlet oxygen lesion was detected in the form of modified dinucleoside monophosphates in double-stranded DNA and in the DNA of HeLa cells exposed to singlet oxygen. Pentamer containing the singlet oxygen-induced lesion and an isotopic label was synthesized as an internal standard for quantifying the lesion and served as well as for correcting for losses of product during sample preparation.