Highsmith, W. Edward

We previously identified a +ACA polymorphism at ‐600 bp in the BRCA1 promoter that creates a FAC1 transcriptional repressor binding site. Subsequently, we performed a genotyping study to determine the prevalence of this BRCA1 promoter polymorphism among unaffected individuals and patients with breast disease. The genotypic distribution of the BRCA1 promoter was determined for 997 unaffected individuals and 763 patients with breast disease from the Mayo Clinic, UNC Hospitals, and the Carolina Breast Cancer Study. The BRCA1 promoter genotypic frequency among unaffected individuals was 481/997 (48%) WT/WT, 432/997 (43%) WT/ACA, and 91/997 (9%) ACA/ACA. The BRCA1 promoter genotypic frequency among patients with breast disease was 348/763 (46%) WT/WT, 343/763 (45%) WT/ACA, and 72/763 (9%) ACA/ACA. No differences were noted in the genotypic distribution of the BRCA1 promoter among patients with DCIS, BRCA1 mutation, suspected BRCA1‐related cancer (BRCAx), or sporadic breast cancer. The allelic frequency of the +ACA BRCA1 promoter polymorphism among breast disease groups [343/415 (83%) WT/ACA and 72/415 (17%) ACA/ACA] were not different than the allelic frequency of the controls [432/523 (83%) WT/ACA and 91/523 (17%) ACA/ACA]. Our results suggest that the +ACA BRCA1 promoter polymorphism occurs commonly, and may contribute to breast cancer upon activation of the FAC1 repressor protein. Support: NIH CA78343.

10588 Background: Accurate tumor classification is increasingly critical to individualized care as patient outcomes improve with use of targeted cancer therapies and predictive biomarkers. In metastatic cancers of uncertain or unknown origin, identification of a primary site remains equivocal in a significant number of cases. Gene expression signatures may improve accuracy and specificity of tumor classification, however, large-scale, blinded validation studies that demonstrate stable performance in metastatic and poorly differentiated cases are currently lacking. Methods: Seven hundred and ninety cases (51% female, 44% metastatic, 63% grade 2 and 3, 14% limited tissue specimens) representing 28 tumor types and 50 subtypes were processed and adjudicated between Mayo Clinic, UCLA and Massachusetts General Hospital. Blinded FFPE tumor sections were submitted and tested using a 92-gene RT-PCR assay (CancerTYPE ID, bioTheranostics Inc.). Molecular predictions were evaluated for concordance with the reference diagnosis; diagnostic accuracy between clinical subsets was compared. Results: The 92-gene assay demonstrated overall sensitivities of 87% for tumor classification and 82% for subtyping. Forty-seven (5.9%) cases were considered unclassifiable. Comparative analysis of performance between metastatic (n=329) and primary (n=414) tumors showed no statistically significant difference in accuracy (85% vs 88%, p=0.16). Similarly, no significant decreases in performance were observed across histological grades (p=0.58) and when comparing limited tissue to excisional biopsy specimens (p=0.16). Strong precision was demonstrated for accurate identification of a primary tumor in tissues biopsied from common metastatic sites, reported as positive predictive values of 100% for lung, brain and peritoneum, 92% for ovary and 80% for liver. Conclusions: Results from this multisite, blinded study validate the diagnostic accuracy of the 92-gene assay for classifying a diverse set of tumors, and support its clinical utility in the diagnosis of metastatic tumors to determine tissue of origin, and in the differential diagnosis of metastatic vs new primary disease.

1501 Background: Despite BC risk reduction of 50-65% by preventive endocrine therapy (ET), very few at-risk women choose to take them. A woman’s perceived BC risk correlates with uptake of ET. A PRS comprised of 77 BC genetic susceptibility loci (Single Nucleotide Polymorphisms (SNP) improves the accuracy of risk prediction for BC. We examined the impact of the addition of individualized PRS BC risk prediction to standard risk calculator estimates on intent to take BC prevention medication. Methods: Eligible women had ≥5% 10 yr BC Tyrer-Cuzick risk (IBIS) or 5 year Gail score ≥3%, with no history of BC or hereditary BC syndrome. Standard BC risk estimates (IBIS or Gail) were incorporated into the counselling on BC preventive ET. A self-reported questionnaire at baseline quantified intention to take ET and explored factors associated with this decision. Blood samples were obtained and genotyped for 77 SNPs, individualized PRS were calculated then incorporated into IBIS and Gail predictions for 5 yr, 10 yr, & lifetime BC risk. At a second visit, PRS risk & prevention recommendations were revisited. Post visit questionnaires assessed change in intent to take ET. Multivariable linear regression was performed to assess impact of baseline variables on change in intent to take medication. Results: From 2016 to 2017, 151 women in Canada & USA were enrolled, median age: 56.1 (range 36-76.4), 35.6% were premenopausal, 98.7% were Caucasian. Median 5yr, 10yr, & lifetime IBIS risk estimates were 3.8% (2.0-11.5), 7.9% (5.0-23.1), and 25.3% (5.5 to 92.2). PRS increased BC risk estimates in 84 (55.6%) and reduced BC risk estimates in 67 (44%) women. After PRS risk counselling, intention to take ET significantly changed (p<0.001): 41.9% of those with increased PRS were more inclined, and 46.7% of women with decreased PRS were less inclined to take ET. On multivariable regression, increase in PRS (p<0.0001) and less concern about ET side effects (p<0.0001) were associated with greater intent to take ET. Conclusions: In high risk women, PRS significantly changed BC risk estimates & intent to take preventive ET. Further assessments of the impact of PRS scores on compliance with ET are warranted. Clinical trial information: NCT02517593.

Context.— The College of American Pathologists proficiency testing program has been instrumental in identifying problems in clinical testing. Objective.— To describe how this program was used to identify a single-nucleotide polymorphism that affects clinical testing for spinocerebellar ataxia type 3. Design.— A proficiency testing sample with discordant results for spinocerebellar ataxia type 3 analysis was further evaluated by targeted Sanger sequencing and genotype polymerase chain reaction using multiple DNA polymerases. Results.— Of 28 laboratories responding in the spinocerebellar ataxia type 3 Proficiency Survey, 18 reported an incorrect homozygous result and 10 reported the expected heterozygous result. A heterozygous single-nucleotide polymorphism complementary to the 3′ end of a published forward primer was identified in the proficiency testing sample, which may have led to allele dropout. However, this primer was used by only 3 of 18 laboratories (16%) reporting a homozygous result. A new forward primer of identical sequence, except for the 3′ end being complementary to the single-nucleotide polymorphism, showed the expected heterozygous pattern. The possibility of DNA polymerase 3′-5′ exonuclease activity contributing to allele dropout was investigated by testing 9 additional polymerases with and without exonuclease activity. No clear pattern emerged, but enzymes with and without 3′-5′ exonuclease activity yielded both homozygous and expected heterozygous results with the published forward primer. Conclusions.— Proactive systematic primer sequence checking is recommended because single-nucleotide polymorphism interference may result in allele dropout and impact clinical testing. Allele dropout is also influenced by other factors, including DNA polymerase exonuclease activity.

AbstractBACKGROUNDDroplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need.METHODSWe validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies.RESULTSFor the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals.CONCLUSIONSStandard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.

AbstractBackground: Pathogenic germline mutations in the CDKN2A tumor suppressor gene are rare and associated with highly penetrant familial melanoma and pancreatic cancer in non-Hispanic whites (NHW). To date, the prevalence and impact of CDKN2A rare coding variants (RCV) in racial minority groups remain poorly characterized. We examined the role of CDKN2A RCVs on the risk of pancreatic cancer among minority subjects.Methods: We sequenced CDKN2A in 220 African American (AA) pancreatic cancer cases, 900 noncancer AA controls, and 183 Nigerian controls. RCV frequencies were determined for each group and compared with that of 1,537 NHW patients with pancreatic cancer. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for both a case–case comparison of RCV frequencies in AAs versus NHWs, and case–control comparison between AA cases versus noncancer AA controls plus Nigerian controls. Smaller sets of Hispanic and Native American cases and controls also were sequenced.Results: One novel missense RCV and one novel frameshift RCV were found among AA patients: 400G&gt;A and 258_278del. RCV carrier status was associated with increased risk of pancreatic cancer among AA cases (11/220; OR, 3.3; 95% CI, 1.5–7.1; P = 0.004) compared with AA and Nigerian controls (17/1,083). Further, AA cases had higher frequency of RCVs: 5.0% (OR, 13.4; 95% CI, 4.9–36.7; P &lt; 0.001) compared with NHW cases (0.4%).Conclusions: CDKN2A RCVs are more common in AA than in NHW patients with pancreatic cancer and associated with moderately increased pancreatic cancer risk among AAs.Impact: RCVs in CDKN2A are frequent in AAs and are associated with risk for pancreatic cancer. Cancer Epidemiol Biomarkers Prev; 27(11); 1364–70. ©2018 AACR.

Fibrillary GN (FGN) is a rare primary glomerular disease. Histologic and histochemical features of FGN overlap with those of other glomerular diseases, and no unique histologic biomarkers for diagnosing FGN have been identified. We analyzed the proteomic content of glomeruli in patient biopsy specimens and detected DnaJ heat shock protein family (Hsp40) member B9 (DNAJB9) as the fourth most abundant protein in FGN glomeruli. Compared with amyloidosis glomeruli, FGN glomeruli exhibited a >6-fold overexpression of DNAJB9 protein. Sanger sequencing and protein sequence coverage maps showed that the DNAJB9 protein deposited in FGN glomeruli did not have any major sequence or structural alterations. Notably, we detected DNAJB9 in all patients with FGN but not in healthy glomeruli or in 19 types of non-FGN glomerular diseases. We also observed the codeposition of DNAJB9 and Ig- γ . Overall, these findings indicate that DNAJB9 is an FGN marker with 100% sensitivity and 100% specificity. The magnitude and specificity of DNAJB9 overabundance in FGN also suggests that this protein has a role in FGN pathogenesis. With this evidence, we propose that DNAJB9 is a strong biomarker for rapid diagnosis of FGN in renal biopsy specimens.