Boddicker, Nicholas

Abstract Purpose: To determine the relationship between germline pathogenic variants (PV) in cancer predisposition genes and the risk of ductal carcinoma in situ (DCIS). Experimental Design: Germline PV frequencies in breast cancer predisposition genes (ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, PALB2, RAD51C, and RAD51D) were compared between DCIS cases and unaffected controls and between DCIS and invasive ductal breast cancer (IDC) cases from a clinical testing cohort (n = 9,887), a population-based cohort (n = 3,876), and the UK Biobank (n = 2,421). The risk of contralateral breast cancer (CBC) for DCIS cases with PV was estimated in the population-based cohort. Results: Germline PV were observed in 6.5% and 4.6% of women with DCIS in the clinical testing and population-based cohorts, respectively. BRCA1, BRCA2, and PALB2 PV frequencies were significantly lower among women with DCIS than those with IDC (clinical cohort: 2.8% vs. 5.7%; population-based cohort: 1.7% vs. 3.7%), whereas the PV frequencies for ATM and CHEK2 were similar. ATM, BRCA1, BRCA2, CHEK2, and PALB2 PV were significantly associated with an increased risk of DCIS (OR > 2.0), but only BRCA2 PV were associated with high risk (OR > 4) in both cohorts. The cumulative incidence of CBC among carriers of PV in high-penetrance genes with DCIS was 23% over 15 years. Conclusions: The enrichment of PV in ATM, BRCA1, BRCA2, CHEK2, and PALB2 among women with DCIS suggests that multigene panel testing may be appropriate for women with DCIS. Elevated risks of CBC in carriers of PV in high-penetrance genes with DCIS confirmed the utility of testing for surgical decision-making.

Abstract Background: Breast cancer has been associated with monogenic, polygenic, and epidemiologic (clinical, reproductive, and lifestyle) risk factors, but studies evaluating the combined effects of these factors have been limited. Methods: We extended previous work in breast cancer risk modeling, incorporating pathogenic variants (PV) in six breast cancer predisposition genes and a 105-SNP polygenic risk score (PRS), to include an epidemiologic risk score (ERS) in a sample of non-Hispanic White women drawn from prospective cohorts and population-based case–control studies, with 23,518 cases and 22,832 controls, from the Cancer Risk Estimates Related to Susceptibility (CARRIERS) Consortium. Results: The model predicts 4.4-fold higher risk of breast cancer for postmenopausal women with no predisposition PV and median PRS, but with the highest versus lowest ERS. Overall, women with CHEK2 PVs had >20% lifetime risk of breast cancer. However, 15.6% of women with CHEK2 PVs and a family history of breast cancer, and 45.1% of women with CHEK2 PVs but without a family history of breast cancer, had low (<20%) predicted lifetime risk and thus were below the threshold for MRI screening. CHEK2 PV carriers at the 10th percentile of the joint distribution of ERS and PRS, without a family history of breast cancer, had a predicted lifetime risk similar to the general population. Conclusions: These results illustrate that an ERS, alone and combined with the PRS, can contribute to clinically relevant risk stratification. Impact: Integrating monogenic, polygenic, and epidemiologic risk factors in breast cancer risk prediction models may inform personalized screening and prevention efforts.

Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNAs) and somatic mutations that are central to disease prognosis, risk stratification, and mechanisms of therapy resistance. Fluorescence in situ hybridization (FISH) panels are widely used in clinical applications as the gold standard for screening prognostic chromosomal abnormalities in CLL. DNA sequencing is an alternative approach to identifying CNAs but is not an established method for clinical CNA screening. We sequenced DNA from 509 individuals with CLL or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, using a targeted sequencing panel of 59 recurrently mutated genes in CLL and additional amplicons across regions affected by clinically relevant CNAs [i.e., del(17p), del(11q), del(13q), and trisomy 12]. We used the PatternCNV algorithm to call CNA and compared the concordance of calling clinically relevant CNAs by targeted sequencing to that of FISH. We found a high accuracy of calling CNAs via sequencing compared to FISH. With FISH as the gold standard, the specificity of targeted sequencing was >95%, sensitivity was >86%, positive predictive value was >90%, and negative predictive value was >84% across the clinically relevant CNAs. Using targeted sequencing, we were also able to identify other common CLL-associated CNAs, including del(6q), del(14q), and gain 8q, as well as complex karyotype, defined as the presence of 3 or more chromosomal abnormalities, in 26 patients. In a single and cost-effective assay that can be performed on stored DNA samples, targeted sequencing can simultaneously detect CNAs, somatic mutations, and complex karyotypes, which are all important prognostic features in CLL.

7011 Background: Monoclonal B-cell lymphocytosis (MBL) is a precursor to chronic lymphocytic leukemia (CLL) but also a risk factor for other lymphoid malignancies. Clonal hematopoiesis (CH), originally defined as the presence of mutations in myeloid driver genes, is a precursor to myeloid malignancies. Recently, CH that includes genes associated with lymphoid malignancies has been reportedly associated with risk of lymphoid malignancies, and particularly CLL. Little is known about the association between CH and MBL and their joint effects on risk of hematological malignancies (HM). Methods: Study participants were from the Mayo Clinic Biobank, a large-scale biorepository of patients who provided a peripheral blood sample. CH at enrollment was determined using whole-exome sequencing (50x coverage). Myeloid-CH (M-CH) was based on mutations located in 56 genes associated with myeloid malignancies. Lymphoid-CH (L-CH) was based on 235 genes associated with lymphoid malignancies. Individuals were screened for MBL using eight-color flow cytometry in two different cohorts of Biobank participants. Cohort 1 (N=3883) included participants who had available sample for MBL screening at enrollment. Cohort 2 (N=5684) were participants recontacted to provide a sample for MBL screening. Incident HM were identified using ICD codes and confirmed via medical record review. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI). Cox regression was used to estimate hazard ratios (HR), with time defined as date between MBL sample and the first of incident HM, death, or 9/30/2023. Analyses were adjusted for age at MBL screening and sex. Results: In total, 9567 individuals (39% male, median age 66 years) were screened for MBL with available CH, of whom 17% were positive for MBL, 7% were positive for M-CH, and 2% were positive for L-CH. We found no evidence of an association between M-CH and MBL (OR=1.11, 95% CI:0.91-1.34) nor L-CH and MBL (OR=1.05, 95% CI:0.70-1.55). When subset to Cohort 1, where the same sample was used for both CH and MBL screening, the results held (M-CH OR=1.11, 95% CI: 0.83-1.48; L-CH OR=0.82, 95% CI:0.40-1.53). Next, we investigated the effect of each of these precursors with incident HM. Median follow-up was 4 years, and 78 individuals developed incident HM (31 myeloid, 49 lymphoid overall, 8 CLL). When modeling both precursors, both L-CH (HR=6.23, 95% CI:2.46-15.79) and MBL (HR=3.89, 95% CI:2.17-6.99) were independently associated with incident lymphoid malignancy. When excluding CLL events, the association held (L-CH: HR=5.94, 95% CI:2.11-16.77; MBL: HR=2.95, 95% CI:1.54-5.66). Only M-CH was significantly associated with incident myeloid malignancies. Conclusions: In the largest cohort with MBL and CH precursors measured, we found no evidence of an association between them. However, both MBL and L-CH were strong independent risk factors for incident lymphoid malignancies.

10511 Background: Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n > 4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing (SGE)-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot encoded by exons 15 to 26 for pathogenic missense variants. Methods: SGE of exons 15 to 26 of BRCA2(MANE transcript ENST00000380152.8) was performed in the haploid human HAP1 cell line. Each exon along with 10bp flanking intronic sequence was studied as an independent target region. The most efficient sgRNA for each target region was selected and cloned into a Cas9 expressing plasmid. Site-saturation mutagenesis (SSM) libraries for each of the 14 target regions were designed to target every nucleotide and were generated by site directed mutagenesis using NNN-tailed PCR primers. HAP1 cells were transfected and harvested at Day5 and 14. Cell survival in response to each variant was assessed based on deep sequence analysis of the surviving endogenously targeted HAP1 cells at Day 5 and Day 14. Results: A total of 7013 SNVs were characterized as functionally abnormal (n = 955), intermediate/uncertain, or functionally normal (n = 5224) based on 95% precision for ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risk of breast cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. Conclusions: The production of functional maps for 97% of all SNVs in the BRCA2 DBD allowed for separation of nucleotide/protein-level functional aberrations. When coupled with genetic and clinical sources of evidence this study led to clinical classification of 5500 individual variants. These data will prove useful in the future, through integration with other datasets, for characterization and clinical classification of all variants in this region in individuals from all racial and ethnic backgrounds and for all BRCA2 associated forms of cancer.

Abstract Background: Germline Pathogenic Variants (PVs) in ATM, BRCA1, BRCA2, CHEK2, and PALB2 are known to increase breast cancer (BCa) risk, but the effect of these PVs on tumor biology is not well-understood. Ki-67 is an established prognostic marker in hormone receptor (HR) positive breast cancer but the distribution of Ki-67 in breast cancer associated with germline PVs is not known. Methods: The study included patients with loco-regional (Stage I-III) HR-positive (ER+ or PR+), HER2-negative invasive ductal or lobular breast cancer evaluated at Mayo Clinic for curative intent treatment between 2012 and 2021. PV carrier status was ascertained in more than two-thirds of cases from germline whole exome sequencing (Regeneron) of patients who participated in a prospective registry at Mayo Clinic. Additional PV carriers were identified through a review of the results of clinical germline genetic testing in electronic medical records aided by natural language processing. Clinically performed Ki-67 values were abstracted from electronic medical records and analyzed as continuous and categorical variables (low: < 20% vs. high: ≥20%). Throughout the timeline of the study, a consistent method for assessment of Ki-67 was utilized at Mayo Clinic, Rochester: Immunohistochemical staining of the Ki-67 antigen in formalin-fixed paraffin-embedded tissue was performed using MIB-1 clone, the scanned slides were reviewed by a technologist and traced areas further analyzed for Ki-67 using Aperio Software. Patients who received neoadjuvant therapy prior to the assessment of Ki-67, with metastatic disease at diagnosis, with PVs in two or more genes of interest, or unknown germline PV or Ki-67 status were excluded from the analysis. Multiple primary tumors in the same patient diagnosed within one year of each other were analyzed independently. The control (non-carrier) group included randomly selected patients from the prospective registry who tested negative for PVs in the five genes over the same time period. Ki-67 values were compared between germline PV carriers and non-carriers in a multivariable logistic regression analysis adjusting for sex, race, age at diagnosis, stage at presentation, and histology of the tumor. Results: A total of 113 HR+/HER2- tumors from 111 PV carriers (ATM: 27, BRCA1: 24, BRCA2: 28, CHEK2: 22, and PALB2:12) along with 431 HR+/HER2- tumors from 422 randomly selected non-carriers were evaluated. The majority (>95%) of patients were non-Hispanic White. Germline PV carriers in each gene were diagnosed at a significantly younger age (P< 0.05) compared to non-carriers. The median Ki-67 value was 12 (Range: 1 to 93) for non-carriers, 16 (Range: 0 to 79) for ATM, 31 (Range: 4 to 86) for BRCA1, 30 (Range: 6 to 80) for BRCA2, 13 (Range: 1 to 38) for CHEK2, and 20 (Range: 3 to 46) for PALB2 PV carriers. In multivariable analysis, compared to non-carriers, we observed significantly higher odds of a high Ki67 value (≥20%) for tumors arising from germline PV carriers in BRCA1 (Odds Ratio (OR): 7.3, 95%CI: 2.3 – 23.3, p< 0.001), BRCA2 (OR: 3.0, 95%CI: 1.2 – 7.4, p=0.02) and PALB2 (OR: 3.9, 95%CI: 1.1 – 14.5, p=0.04). No such relationship was observed with either ATM (OR: 1.1, 95%CI: 0.4 – 2.9, p=0.81) or CHEK2 (OR: 0.8, 95%CI: 0.3 – 2.2, p=0.63). Conclusion: This study reveals that HR+/HER2- tumors in BRCA1, BRCA2, and PALB2 PV carriers have a significantly higher Ki-67 expression compared to non-carriers. These findings have implications for understanding prognosis and endocrine response PV with HR+ breast cancer. Further exploration of the relationships between Ki67 and germline PV status is necessary to personalize systemic therapy options in germline PV carriers. Acknowledgments: Thank you to Regeneron Genetics Center. The study was funded in part by the NIH Specialized Program of Research Excellence in Breast Cancer [P50CA116201] to Mayo Clinic, Mayo Clinic Department of Oncology Small Grants Program, and the Paul Calabresi Program at Mayo Clinic Citation Format: Robert Scheel, Grace Choong, Tara Rao, Nicholas Boddicker, Chunling Hu, Jie Na, Vinod Kaggal, Sean Murphy, Karthik Giridhar, Saba Yasir, Matthew Goetz, Fergus Couch, Siddhartha Yadav. Ki-67 Expression in Breast Cancer associated with ATM, BRCA1, BRCA2, CHEK2 and PALB2 Pathogenic Variants [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-08-07.

Abstract Chronic Lymphocytic Leukemia (CLL) exhibits a substantial familial risk that extends beyond CLL to other B-cell lymphomas (including follicular lymphoma and diffuse large B-cell lymphoma). Monoclonal B-cell Lymphocytosis (MBL), a precursor to CLL, is also associated with a significant risk for hematologic malignancies overall and for lymphoid malignancies, specifically. Moreover, previous research has shown an association between the CLL polygenic risk score (CLL-PRS) and common lymphoma subtypes. This study aims to further evaluate CLL-PRS associations with hematological cancer overall and by lineage while also considering MBL status a strong risk factor for lymphoid malignancies. Utilizing the Mayo Clinic Biobank, we selected individuals aged 40 years or older, residing in proximal counties, who had no prior hematologic malignancies, and with available biospecimens. MBL screening was done using 8-color flow cytometry, and MBL with CLL (CD5+/CD20dim) or atypical CLL (CD5+/CD20+) immunophenotype were included. CLL-PRS was calculated from 41 known susceptibility variants, with weights being the log of the odds ratios from the largest CLL genome-wide association study. Incident hematologic malignancies were ascertained through ICD codes and confirmed via medical record review. Hazard ratios (HR) and 95% confidence intervals (CIs) for associations with hematologic malignancies were estimated through Cox regression. Of 7,332 participants, 1,150 (16%) screened positive for MBL; median age was 67 years (range 40-101), and 37% male. Median follow-up was 2.9 years (range 0-13), and 56 hematologic cancers were identified. After adjusting for age and sex, the CLL-PRS was associated with increased risk of developing hematologic (HR=1.49, 95%CI=1.12-1.98, P=0.006) which was all attributable to lymphoid cancers (HR=1.79, 95%CI=1.26-2.56, P=0.001). When adjusting for baseline MBL status in the models, the CLL-PRS associations remained significant but were attenuated for hematological (HR=1.33, 95%CI=0.99-1.79, P=0.058) and lymphoid (HR=1.56, 95%CI=1.07-2.26, P=0.020) malignancies, while MBL status was also significantly associated with risk of hematologic (HR=2.76, 95%CI=1.55-4.91, P=0.001) and lymphoid (HR=3.61, 95%CI=1.78-3.32, P<0.001) malignancies. In this prospective cohort study of individuals screened for MBL, CLL-PRS was associated with an elevated risk of hematologic malignancies overall and within the lymphoid lineage, underscoring a shared genetic etiology across many B-cell lymphomas. However, the associations between CLL-PRS and hematologic malignancies were attenuated when accounting for MBL status or when analyzing among MBL-positive individuals, highlighting the role of MBL status in these associations. Further research with expanded cohorts is needed to unravel these complex genetic and clinical interactions. Citation Format: Raphael Mwangi, Dennis P. Robinson, Sara J. Achenbach, Geffen Kleinstern, Aaron D. Norman, Kari G. Rabe, Janet E. Olson, Neil E. Kay, Rosalie Griffin, Nicholas J. Boddicker, Esteban Braggio, Sameer A. Parikh, Curtis A. Hanson, Celine M. Vachon, James R. Cerhan, Tait D. Shanafelt, Susan L. Slager. Genetic associations with hematologic malignancies: An evaluation of CLL polygenic risk score and MBL status in the Mayo Clinic Biobank Cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6152.

Abstract High-count monoclonal B-cell lymphocytosis (HCMBL) is a precursor condition to chronic lymphocytic leukemia (CLL). We have shown that among individuals with HCMBL, the CLL-International Prognostic Index (CLL-IPI) is prognostic for time-to-first therapy (TTFT). Little is known about the prognostic impact of somatically mutated genes among individuals with HCMBL. We sequenced DNA from 371 individuals with HCMBL using a targeted sequencing panel of 59 recurrently mutated genes in CLL to identify high-impact mutations. We compared the sequencing results with that of our treatment-naïve CLL cohort (N = 855) and used Cox regression to estimate hazard ratios and 95% confidence intervals (CIs) for associations with TTFT. The frequencies of any mutated genes were lower in HCMBL (52%) than CLL (70%). At 10 years, 37% of individuals with HCMBL with any mutated gene had progressed requiring treatment compared with 10% among individuals with HCMBL with no mutations; this led to 5.4-fold shorter TTFT (95% CI, 2.6-11.0) among HCMBL with any mutated gene vs none, independent of CLL-IPI. When considering individuals with low risk of progression according to CLL-IPI, those with HCMBL with any mutations had 4.3-fold shorter TTFT (95% CI, 1.6-11.8) vs those with none. Finally, when considering both CLL-IPI and any mutated gene status, we observed individuals with HCMBL who were high risk for both prognostic factors had worse prognosis than patients with low-risk CLL (ie, 5-year progression rate of 32% vs 21%, respectively). Among HCMBL, the frequency of somatically mutated genes at diagnosis is lower than that of CLL. Accounting for both the number of mutated genes and CLL-IPI can identify individuals with HCMBL with more aggressive clinical course.

Abstract Many germline BRCA2 missense variants of uncertain significance (VUS) remain to be clinically classified as pathogenic or benign. Here we identify 70 missense VUS in the DNA binding domain (DBD) of BRCA2 that partially reduced homologous recombination DNA repair activity of the protein in a homology directed DNA repair cell-based assay. CRISPR/cas9-based knockin of these variants reduced cell survival of haploid cells and conferred increased sensitivity to the PARP inhibitor Olaparib. Sequence alignment analysis found that the variants were highly conserved across species from pufferfish to human. Structural analysis showed that these variants are located predominantly in unstructured regions of the DBD. Case-control association studies showed that the hypomorphic variants conferred moderate risks of breast (OR:2.85; 95%CI:1.43-5.88) and ovarian cancer (OR:3.37; 95%CI:1.30-8.54) that were significantly different from risks associated with known pathogenic and benign variants. Cumulative lifetime risks of breast and ovarian cancer among carriers were estimated at 25% and 4.7%, respectively. Age at diagnosis of breast cancer among hypomorphs carriers was 55 years of age as opposed to 48 years of age for carriers of pathogenic variants. In addition, the combination of functional and other genetic data in an ACMG/AMP-like variant classification model reclassified 39 variants as likely pathogenic. This novel group of low penetrance missense variants further calibrate BRCA2 variant classification methods and have significant implications for the management of variant carriers. Citation Format: Huaizhi (Gilbert) Huang, Chunling Hu, Jie Na, Mohamed Abozaid, Amik Munankarmy, Tara Rao, Carolyn A. Dunn Lumby, Paulo Cilas Morais Lyra, Ronan E. Couch, Benjamin R. Persons, Eric C. Polley, Rachid Karam, Tina Pesaran, Siddhartha Yadav, Alvaro N.A. Monteiro, Nicholas J. Boddicker, Susan M. Domchek, Marcy E. Richardson, Fergus J. Couch. Functional and clinical characterization of hypomorphic missense variants in the BRCA2 cancer predisposition gene [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR09.

Abstract Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of surviving endogenously targeted haploid cells. A total of 7013 SNVs were characterized as functionally abnormal (n=955), intermediate/uncertain, or functionally normal (n=5224) based on 95% agreement with ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risks of breast and ovarian cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). In addition, 86% of tumors associated with abnormal missense SNVs displayed loss of heterozygosity (LOH), whereas 26% of tumors with normal variants had LOH. The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers and the remaining functional scores can be used directly for clinical classification and interpretation of many additional variants.