Variant classification changes over time in the clinical molecular diagnostic laboratory setting.

BACKGROUND: Variant classification in the setting of germline genetic testing is necessary for patients and their families to receive proper care. Variants are classified as pathogenic (P), likely pathogenic (LP), uncertain significance (VUS), likely benign (LB) and benign (B) using the standards and guidelines recommended by the American College of Medical Genetics and the Association for Molecular Pathology, with modifications for specific genes. As the literature continues to rapidly expand, and evidence continues to accumulate, prior classifications can be updated accordingly. In this study, we aim to characterise variant reclassifications in Ontario. METHODS: DNA samples from patients seen at hereditary cancer clinics in Ontario from January 2012 to April 2022 were submitted for testing. Patients met provincial eligibility criteria for testing for hereditary cancer syndromes or polycystic kidney disease. Reclassification events were determined to be within their broader category of significance (B to LB or vice versa, or P to LP or vice versa) or outside of their broader category as significance (ie, significant reclassifications from B/LB or VUS or P/LP, from P/LP to VUS or B/LB, or from VUS to any other category). RESULTS: Of the 8075 unique variants included in this study, 23.7% (1912) of variants were reassessed, and 7.2% (578) of variants were reclassified. Of these, 351 (60.7%) variants were reclassified outside of their broader category of significance. Overall, the final classification was significantly different for 336 (58.1%) variants. Importantly, most reclassified variants were downgraded to a more benign classification (n=245; 72.9%). Of note, most reclassified VUS was downgraded to B/LB (n=233; 84.7%). CONCLUSIONS: The likelihood for reclassification of variants on reassessment is high. Most reclassified variants were downgraded to a more benign classification. Our findings highlight the importance of periodic variant reassessment to ensure timely and appropriate care for patients and their families.

Recontact to return new or updated PALB2 genetic results in the clinical laboratory setting.

OBJECTIVE: The purpose of this study was to recontact individuals with clinically actionable test results identified through a retrospective research study and to provide a framework for laboratories to recontact patients. METHODS: Genetic testing was conducted on 2977 individuals originally referred for BRCA1 and BRCA2 hereditary breast and ovarian cancer testing that had a negative genetic test result. A gene panel was used to identify pathogenic variants in known or newly discovered genes that could explain the underlying cause of disease; however, analysis was restricted to PALB2 for the purposes of this study. A patient recontact decision tree was developed to assist in the returning of updated genetic test results to clinics and patients. RESULTS: Novel clinically actionable pathogenic variants were identified in the PALB2 gene in 18 participants (0.6%), the majority of whom were recontacted with their new or updated genetic test results. Eight individuals were unable to be recontacted; five individuals had already learnt about their new or updated findings from genetic testing outside the context of this study; three individuals prompted cascade testing in family members; two individuals were deceased. CONCLUSION: Novel pathogenic variants in PALB2 were identified in 18 individuals through retrospective gene panel testing. Recontacting these individuals regarding these new or updated findings had a range of outcomes. The process of conveying genomic results within this framework can be effectively accomplished while upholding patient autonomy, potentially leading to advantageous outcomes for patients and their families.

Comprehensive genomic profiling for oncological advancements by precision medicine.

Considerable advancements in next generation sequencing (NGS) techniques have sparked the use of comprehensive genomic profiling (CGP) as a guiding tool for precision-centered oncological treatments. The past two decades have seen the completion of the human genome project, and the consequential invention of NGS. High-throughput sequencing technologies support the discovery and commonplace use of individualized cancer treatments, specifically immune-centered checkpoint inhibitor therapies, and oncogene and tumor suppressor gene targeted therapies. Nevertheless, CGP is not commonly used in all clinical settings. This review investigates the clinically relevant applications of CGP. Studies published between the years 2000-2023 have shown substantial evidence of the benefits of integrating CGP into routine care practice, while also making important comparisons to current-standard oncological treatment strategies. Findings of a comprehensive genomic profile includes predictive, prognostic, and diagnostic biomarkers, together with somatic mutation identification which can indicate the efficacy of immunotherapies and molecularly guided therapies. This review highlights the importance of CGP in identifying driver mutations in tumors that subsequently can be effectively targeted with molecular therapeutics and lead to drug discovery, allowing for increased precision in treating tumors selectively based on their specific genetic mutations, thereby improving patient outcomes.

Improving RNA Fusion Call Confidence and Reliability in Molecular Diagnostic Testing.

Next-generation sequencing is a superior method for detecting known and novel RNA fusions in formalin-fixed, paraffin-embedded tissue over fluorescence in situ hybridization and RT-PCR. However, confidence in fusion calling and true negatives may be compromised by poor RNA quality. Using a commercial panel of 507 genes and the recommended 3-million read threshold to accept results, two cases yielded false negatives while exceeding this recommendation during clinical validation. To develop a reliable quality control metric that better reflects internal sample quality and improves call confidence, gene expression across 361 patient tumor samples was evaluated to derive a set of 15 genes to serve as a proxy quality control (pQC). These 15 genes were assessed for their normalized expression using the sequencing data from each case and selected for robustness. A threshold of 11 pQC genes produced a 4.71% fail rate, selected for stringency as an acceptable level of repeated testing in the clinical setting, minimizing false-negative calls. To increase the chance that low-quality samples pass pQC, a revision to the library preparation method was also tested, with 75% of previously failed samples passing pQC on resequencing by increasing cDNA input. Taken together, a next-generation sequencing analysis quality control tool is presented that serves as a surrogate for housekeeping genes and improves confidence in fusion calls.

Cost-effectiveness of noninvasive fetal RhD blood group genotyping in nonalloimmunized and alloimmunized pregnancies.

BACKGROUND: We sought to determine the cost-effectiveness of noninvasive fetal RhD blood group genotyping in nonalloimmunized and alloimmunized pregnancies in Canada. STUDY DESIGN AND METHODS: We developed two probabilistic state-transition (Markov) microsimulation models to compare fetal genotyping followed by targeted management versus usual care (i.e., universal Rh immunoglobulin [RhIG] prophylaxis in nonalloimmunized RhD-negative pregnancies, or universal intensive monitoring in alloimmunized pregnancies). The reference case considered a healthcare payer perspective and a 10-year time horizon. Sensitivity analysis examined assumptions related to test cost, paternal screening, subsequent pregnancies, other alloantibodies (e.g., K, Rh c/C/E), societal perspective, and lifetime horizon. RESULTS: Fetal genotyping in nonalloimmunized pregnancies (at per-sample test cost of C$247/US$311) was associated with a slightly higher probability of maternal alloimmunization (22 vs. 21 per 10,000) and a reduced number of RhIG injections (1.427 vs. 1.795) than usual care. It was more expensive (C$154/US$194, 95% Credible Interval [CrI]: C$139/US$175-C$169/US$213) and had little impact on QALYs (0.0007, 95%CrI: -0.01-0.01). These results were sensitive to the test cost (threshold achieved at C$88/US$111), and inclusion of paternal screening. Fetal genotyping in alloimmunized pregnancies (at test cost of C$328/US$413) was less expensive (-C$6280/US$7903, 95% CrI: -C$6325/US$7959 to -C$6229/US$7838) and more effective (0.19 QALYs, 95% CrI 0.17-0.20) than usual care. These cost savings remained robust in sensitivity analyses. DISCUSSION: Noninvasive fetal RhD genotyping saves resources and represents good value for the management of alloimmunized pregnancies. If the cost of genotyping is substantially decreased, the targeted intervention can become a viable option for nonalloimmunized pregnancies.

Data sharing to improve concordance in variant interpretation across laboratories: results from the Canadian Open Genetics Repository.

BACKGROUND: This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation. METHODS: Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin. RESULTS: Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants. CONCLUSIONS: The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.

Multigene panel testing for hereditary breast and ovarian cancer in the province of Ontario.

PURPOSE: The aim of this study was to determine the diagnostic yield of multigene panel testing among patients referred with hereditary breast and ovarian cancer (HBOC). METHODS: Patients who met provincial eligibility criteria were tested at the Advanced Molecular Diagnostic Laboratory at Mount Sinai Hospital, Toronto. Gene sequencing and exon-level copy number variant (CNV) analysis was performed. The referring physician had the opportunity to choose between several different gene panels based on patient phenotype. Cases were included in the analysis based on personal and family history of cancer and the type of panel ordered. RESULTS: 3251 cases that received panel testing were included in this analysis. Overall, 9.1% (295) had a positive (pathogenic or likely pathogenic) result and 27.1% (882) had an inconclusive result (variant of uncertain significance). The genes with the highest prevalence of positive results were in BRCA2 (2.2%, 71/3235), BRCA1 (1.9%, 62/3235), and CHEK2 (1.4%, 40/2916). Of the positive cases, 9.8% (29) had a pathogenic or likely pathogenic variant in a gene associated with Lynch syndrome (MSH6, MSH2, MLH1, or PMS2). CONCLUSIONS: Our overall positive yield is similar to that reported in the literature. The yield of inconclusive results was three times that of positive results. By testing more individuals in families with HBOC and through data-sharing efforts, the clinical significance of most variants may eventually be determined and panel testing for monogenic cancer predisposition syndromes will have greater utility.

Retesting of women who are negative for a BRCA1 and BRCA2 mutation using a 20-gene panel.

BACKGROUND: The value of retesting women who previously tested negative for a pathogenic variant (mutation) in BRCA1 and BRCA2 using an expanded panel of breast and ovarian cancer genes is unclear. METHODS: We studied 110 BRCA1/2-negative women who were retested using a panel of 20 breast and/or ovarian cancer susceptibility genes at the Advanced Molecular Diagnostics Laboratory (AMDL) at Mount Sinai Hospital in Toronto between March 2017 and March 2019. All patients had previously tested negative for BRCA pathogenic variants at the AMDL between January 2012 and March 2018 and were subsequently referred for retesting by their physician. RESULTS: Overall, six pathogenic variants in genes other than BRCA1 and BRCA2 were found (prevalence 5.5%). There were two pathogenic variants found in RAD51C, and one found in each of BRIP1, PALB2, PMS2 and PTEN. The prevalence of pathogenic variants was 6.5% for women affected with cancer (6 of 93), including 4.9% for women with breast cancer (4 of 82) and 22.2% for women with ovarian cancer (2 of 9). None of the 17 unaffected women had a clinically significant or pathogenic variant. There were 44 women (40%) for whom the result of the panel test was inconclusive due to the detection of a variant of uncertain significance. CONCLUSIONS: Our findings indicate that the retesting of BRCA1/2-negative individuals with an expanded panel of 20 breast and ovarian cancer genes can produce clinically relevant results, with a yield of 5.5% for pathogenic variants in genes other than BRCA1 and BRCA2.

Utilization of the 2017 diagnostic criteria for hEDS by the Toronto GoodHope Ehlers-Danlos syndrome clinic: A retrospective review.

The new 2017 diagnostic criteria for hypermobile Ehlers-Danlos Syndrome (hEDS) provide a framework for diagnosing hEDS but are more stringent than the previous Villefranche criteria. Our clinical experience at the GoodHope EDS clinic was that the 2017 criteria left many highly symptomatic patients without a diagnosis of hEDS. We conducted a retrospective cohort study to confirm our clinic experience and assess the accuracy of the 2017 diagnostic criteria for hEDS in patients who had a previous hEDS diagnosis based on the Villefranche criteria. Our study found that 15% (n = 20 of 131) of patients with a prior diagnosis of hEDS met the 2017 diagnostic criteria, and many of the traits used to distinguish hEDS were not significantly more frequent in patients who met 2017 criteria versus those who did not. In both groups objective systemic manifestations were found less frequently than subjective systemic manifestations. Beighton score (BS) as assessed by primary care practitioner was found to be higher than assessment by EDS practitioner in 81% (n = 74 of 91) of cases. Generalized joint hypermobility was confirmed in only 46% (n = 51 of 111) of patients who had a previous diagnosis of hEDS. Higher BS did not correlate with increased number of systemic manifestations in our cohort. Common comorbidities of hEDS were found with similar frequency in those who met 2017 criteria and those who did not. Based on our cohort, the 2017 hEDS diagnostic criteria require refinement to improve its diagnostic accuracy.

A phase II study of ENMD-2076 in advanced soft tissue sarcoma (STS).

ENMD-2076, an aurora-A kinase inhibitor with anti-angiogenic properties, has shown activity in solid and hematologic malignancies. We investigated oral ENMD-2076 in an open-label, single-arm phase II study using 275 mg daily on a 28-day cycle in patients with advanced soft-tissue sarcomas (STS) receiving ≤1 line of prior therapy. Primary endpoint was 6-month progression-free survival (PFS) with ≤15% indicating no interest, and ≥40% indicating further interest in ENMD-2076. Secondary/exploratory endpoints included clinical benefit (CBR ≥6-months) and objective response (ORR) rates, PFS, OS, safety, and whole-exome sequencing (WES) for potentially associated biomarkers. Overall, 23/25 (92%) patients receiving ENMD-2076 were efficacy evaluable with median follow-up of 14 months (range 2.2-39.5). Common subtypes were leiomyosarcoma (n = 10), undifferentiated pleomorphic sarcoma (n = 3), angiosarcoma (n = 3), and alveolar soft-part sarcoma (n = 3). The 6-month PFS was 20.8% (95% CI:3.2-38.4) with a CBR of 17% (95% CI:1.55-33.23) and ORR of 9% (95% CI:3.08-20.46). Median PFS was 2.5 months (95% CI:2.20-4.47) and OS was 14.1 months (95% CI:6.07-20.07). The most common high-grade treatment-related adverse event was hypertension (60%). WES identified PTPRB mutations in 3/4 patients (p = 0.018) benefiting from ENMD-2076. Although this study failed to meet its primary endpoint, occasional responses and prolonged stable disease was noted. ENMD-2076 evaluation in PTPRB mutated tumors and/or angiosarcoma is warranted.

Correction: Variant classification changes over time in BRCA1 and BRCA2.

In the original version of this Article, the affiliation details for Drs. Jordan Lerner-Ellis and George Charames did not include the Department of Pathology and Laboratory Medicine at the University of Toronto. In addition, Drs. Jordan Lerner-Ellis and George Charames were incorrectly affiliated with the Institute of Health Policy, Management and Evaluation at the University of Toronto. These errors have now been corrected in both the PDF and HTML versions of the Article.

Variant classification changes over time in BRCA1 and BRCA2.

PURPOSE: To report BRCA1 and BRCA2 (BRCA1/2) variant reassessments and reclassifications between 2012 and 2017 at the Advanced Molecular Diagnostics Laboratory (AMDL) in Toronto, Canada, which provides BRCA1/2 testing for patients in Ontario, and to compare AMDL variant classifications with submissions in ClinVar. METHODS: Variants were assessed using a standardized variant assessment tool based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology's guidelines and tracked in an in-house database. Variants were shared through the Canadian Open Genetics Repository and submitted to ClinVar for comparison against other laboratories. RESULTS: AMDL identified 1209 BRCA1/2 variants between 2012 and 2017. During this period, 32.9% (398/1209) of variants were reassessed and 12.4% (150/1209) were reclassified. The majority of reclassified variants were downgraded (112/150, 74.7%). Of the reclassified variants, 63.3% (95/150) were reclassified to benign, 20.7% (31/150) to likely benign, 10.0% (15/150) to variant of uncertain significance, 2.0% (3/150) to likely pathogenic, and 4.0% (6/150) to pathogenic. Discordant ClinVar submissions were found for 40.4% (488/1209) of variants. CONCLUSION: BRCA1/2 variants may be reclassified over time. Reclassification presents ethical and practical challenges related to recontacting patients. Data sharing is essential to improve variant interpretation, to help patients receive appropriate care based on their genetic results.

Proteome-wide onco-proteogenomic somatic variant identification in ER-positive breast cancer.

BACKGROUND: Recent advances in mass spectrometric instrumentation and bioinformatics have critically contributed to the field of proteogenomics. Nonetheless, whether that integrative approach has reached the point of maturity to effectively reveal the flow of genetic variants from DNA to proteins still remains elusive. The objective of this study was to detect somatically acquired protein variants in breast cancer specimens for which full genome and transcriptome data was already available (BASIS cohort). METHODS: LC-MS/MS shotgun proteomic results of 21 breast cancer tissues were coupled to DNA sequencing data to identify variants at the protein level and finally were used to associate protein expression with gene expression levels. RESULTS: Here we report the observation of three sequencing-predicted single amino acid somatic variants. The sensitivity of single amino acid variant (SAAV) detection based on DNA sequencing-predicted single nucleotide variants was 0.4%. This sensitivity was increased to 0.6% when all the predicted variants were filtered for MS "compatibility" and was further increased to 2.9% when only proteins with at least one wild type peptide detected were taken into account. A correlation of mRNA abundance and variant peptide detection revealed that transcripts for which variant proteins were detected ranked among the top 6.3% most abundant transcripts. The variants were detected in highly abundant proteins as well, thus establishing transcript and protein abundance and MS "compatibility" as the main factors affecting variant onco-proteogenomic identification. CONCLUSIONS: While proteomics fails to identify the vast majority of exome DNA variants in the resulting proteome, its ability to detect a small subset of SAAVs could prove valuable for precision medicine applications.

Epithelioid fibrous histiocytoma: molecular characterization of ALK fusion partners in 23 cases.

Epithelioid fibrous histiocytoma is a rare and distinctive cutaneous neoplasm. Most cases harbor ALK rearrangement and show ALK overexpression, which distinguish this neoplasm from conventional cutaneous fibrous histiocytoma and variants. SQSTM1 and VCL have previously been shown to partner with ALK in one case each of epithelioid fibrous histiocytoma. The purpose of this study was to examine a large cohort of epithelioid fibrous histiocytomas by next-generation sequencing to characterize the nature and prevalence of ALK fusion partners. A retrospective archival review was performed to identify cases of epithelioid fibrous histiocytoma (2012-2016). Immunohistochemistry was performed to confirm ALK expression. Targeted next-generation sequencing was applied on RNA extracted from formalin-fixed paraffin-embedded tissue to identify the fusion partners. Twenty-three cases fulfilled inclusion criteria. The mean patient age was 39 years (range, 8-74), there was no sex predilection, and >75% of cases involved the lower extremities. The most common gene fusions were SQSTM1-ALK (N=12; 52%) and VCL-ALK (N=7; 30%); the other four cases harbored novel fusion partners (DCTN1, ETV6, PPFIBP1, and SPECC1L). The pattern of ALK immunoreactivity was usually granular cytoplasmic (N=12; 52%) or granular cytoplasmic and nuclear (N=10; 43%); the case containing an ETV6 fusion partner showed nuclear staining alone. There was no apparent relationship between tumor morphology and the ALK fusion partner. In summary, SQSTM1 and VCL are the most common ALK fusion partners in epithelioid fibrous histiocytoma; DCTN1, ETV6, PPFIBP1, and SPECC1L represent rare fusion partners. The proteins encoded by these genes play diverse roles in scaffolding, cell adhesion, signaling, and transcription (among others) without clear commonalities. These findings expand the oncogenic promiscuity of many of these ALK fusion genes, which drive neoplasia in tumors of diverse lineages with widely varied clinical behavior. This is the first documented account of ETV6-ALK and SPECC1L-ALK translocations in neoplasms.

Data sharing as a national quality improvement program: reporting on BRCA1 and BRCA2 variant-interpretation comparisons through the Canadian Open Genetics Repository (COGR).

PurposeThe purpose of this study was to develop a national program for Canadian diagnostic laboratories to compare DNA-variant interpretations and resolve discordant-variant classifications using the BRCA1 and BRCA2 genes as a case study.MethodsBRCA1 and BRCA2 variant data were uploaded and shared through the Canadian Open Genetics Repository (COGR; http://www.opengenetics.ca). A total of 5,554 variant observations were submitted; classification differences were identified and comparison reports were sent to participating laboratories. Each site had the opportunity to reclassify variants. The data were analyzed before and after the comparison report process to track concordant- or discordant-variant classifications by three different models.ResultsVariant-discordance rates varied by classification model: 38.9% of variants were discordant when using a five-tier model, 26.7% with a three-tier model, and 5.0% with a two-tier model. After the comparison report process, the proportion of discordant variants dropped to 30.7% with the five-tier model, to 14.2% with the three-tier model, and to 0.9% using the two-tier model.ConclusionWe present a Canadian interinstitutional quality improvement program for DNA-variant interpretations. Sharing of variant knowledge by clinical diagnostic laboratories will allow clinicians and patients to make more informed decisions and lead to better patient outcomes.

Genetic biomarkers associated with response to palliative radiotherapy in patients with painful bone metastases.

BACKGROUND: Palliative radiotherapy (RT) is effective in patients with painful bone metastases. Genetic factors may identify subgroup of patients who responded to RT. To identify DNA biomarkers associated with response to palliative RT. METHODS: Patients who received a single 8 Gy dose of RT for painful bone metastases were categorised into responders (n=36), non-responders (NR) (n=71). Saliva samples were sequenced to identify single-nucleotide variants (SNVs) in genes with known disease-causing variants from inflammation, radiation response, and DNA damage pathways. In univariate analysis, Cochran-Armitage trend tests were used to identify SNVs that associated with pain response (P<0.005), and the Penalized LASSO method with minimum Bayesian Information Criterion was used to identify multi-SNVs that jointly predict pain response to RT. The corresponding estimated effect of the multi-SNVs were used to drive the prognostic score for each patient. Based on it, patients were divided into 3 equal size risk groups. RESULTS: Forty-one significant variants were identified in univariate analysis. Multivariable analysis selected 14 variants to generate prognostic scores, adjusting for gender and primary cancer site. Eighty-nine percent of patients in the high prognostic group responded to palliative radiation therapy (P=0.0001). Estimated effect sizes of the variants ranged from 0.108-2.551. The most statistically significant variant was a deletion at position 111992032 in the ataxin gene ATXN2 (P=0.0001). Five variants were non-synonymous, including AOAH rs7986 (P=0.0017), ZAN rs539445 (P=0.00078) and rs542137 (P=0.00078), RAG1 rs3740955 (P=0.0014), and GBGT1 rs75765336 (P=0.0026). CONCLUSIONS: SNVs involved in mechanisms including DNA repair, inflammation, cellular adhesion, and cell signalling have significant associations with radiation response. SNVs with predictive power may stratify patient populations according to likelihood of responding to treatment, therefore enabling more efficient identification of beneficial strategies for pain management and improved resource utilisation.

Genetic biomarkers associated with changes in quality of life and pain following palliative radiotherapy in patients with bone metastases.

BACKGROUND: Patients with bone metastases undergoing palliative radiation therapy (RT) may experience changes in both the functional and symptomatic aspects of quality of life (QOL). The European Organization of Cancer Research and Treatment (EORTC) QOL Questionnaire Core-15 Palliative (QLQ-C15-PAL) is a validated questionnaire employed to assess QOL specifically in palliative patients. Our study aimed to identify single-nucleotide variant (SNV) genetic biomarkers associated with changes in QOL and pain. METHODS: Fifty-two patients who received a single 8-Gy RT for painful bone metastases completed the EORTC QOL-C15-PAL questionnaire prior to randomization and at 42-day post RT. Saliva samples obtained at day of RT were sequenced, and SNVs from genes involved in inflammation, radiation response, immune response, DNA damage, or QOL were assessed for association with changes in global QOL or the pain scale items using the Cochran-Armitage trend test. The penalized LASSO method with minimum Bayesian information criterion was used to select a multi-SNV model out of significant SNVs (P<0.005) and to produce prognostic scores for patients that categorized them into risk groups of low, middle, and high. RESULTS: The multivariable model predicting global QOL included 14 SNVs, of which HS1BP3 rs35579164 G:C and ABCA1 rs2230805 C>T had the largest positive and negative effect sizes, respectively (HS1BP3: 8.21, ABCA1: -3.44). The model for the response of QOL pain item included 8 SNVs, of which PLAUR rs4760 A>G and ELAC2rs11545302 had the largest positive and negative effect sizes, respectively (PLAUR: 5.23; ELAC: -3.84). The patients' risk groups were highly predictive of QOL response (P<0.0001) and pain item response (P<0.0001). In logistic regression analysis accounting for baseline factors of gender and primary cancer site, the global QOL risk group predicts pain response after RT [OR: 2.1, 95% confidence interval (CI): 1.2-3.9, P=0.015], but the QOL pain item risk group did not (OR: 0.93; 95% CI: 0.5-1.6, P=0.79). The multi-SNVs model included SNVs from genes involved in metabolism, membrane transport, cell cycle control, ciliary structure, and gene expression regulation. CONCLUSIONS: SNVs were significantly associated with changes in global QOL of global domain and pain item in patients with bone metastases. Identification of genetic biomarkers predictive of QOL items may allow patients and health care providers anticipate and better address the needs of the palliative cancer patient population.

Genetic biomarkers associated with pain flare and dexamethasone response following palliative radiotherapy in patients with painful bone metastases.

BACKGROUND: In patients who receive palliative radiation therapy (RT) for painful bone metastases, 40% experience a transient increase in pain known as a pain flare. Prophylactic dexamethasone has been shown to reduce pain flare incidence to 25%. We aimed to identify DNA biomarkers associated with pain flare and dexamethasone response. METHODS: Daily pain levels were recorded by 81 patients who received a single 8 Gy RT for painful bone metastases, of which 50 also received prophylactic dexamethasone. To identify single-nucleotide variants (SNVs), patient saliva samples obtained at day of RT were sequenced for 4,813 disease-associated genes, then filtered for genes associated with inflammation, radiation or immune response, and DNA damage. Significant SNVs (P<0.005) identified by the Cochran-Armitage trend test underwent the Penalized LASSO method with minimum Bayesian Information Criterion to select a multi-SNV model that jointly predicted pain flare, and pain flare despite prophylactic dexamethasone (dexamethasone response). The corresponding estimated effects of the multi-SNVs were used to drive the prognostic score of developing pain flare for each patient, who were divided into three risk groups of roughly equal sizes. RESULTS: Risk groups were significantly predictive of pain flare (P<0.0001) and dexamethasone response (P<0.0001). The high-risk patient groups had a 78% chance of developing pain flare, and pain flare despite dexamethasone [OR =24.6, 95% confidence interval (CI): 1.8-342.7, P=0.02]. The multivariable model for pain flare included 15 variants, with effect sizes ranging from -4.97 (NBPF1 rs3872309 C>T) to 5.54 (DNM2 10940838 A>C). The multivariable model for dexamethasone response included 6 variants, with effect sizes ranging from -1.03 (NBPF1 rs3872309 C>T) to 0.85 (TSEN54 rs62088470 C>G). CONCLUSIONS: Significant SNVs associated with pain flare were found in genes with functions in biosynthesis (DHODH, PECR), lipid excretion and metabolism (UGT2A1/2, VLDLR), and intracellular signalling (DNM2, SEC23A). Significant SNVs associated with dexamethasone response were from genes involved in extracellular matrix (HAS1, ADAMTS16) and cytoskeleton regulation (GAS2L2). Identification of SNVs predictive of pain flare and dexamethasone response enables targeted prophylactic therapy according to a patient's predisposed response.