Allison W. Kurian

Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

Background To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. Methodology/Principal Findings We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. Conclusions/Significance For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of ‘liquid biopsies’ to better model drug discovery.

Clinical Evaluation of a Multiple-Gene Sequencing Panel for Hereditary Cancer Risk Assessment

PURPOSE Multiple-gene sequencing is entering practice, but its clinical value is unknown. We evaluated the performance of a customized germline-DNA sequencing panel for cancer-risk assessment in a representative clinical sample. METHODS Patients referred for clinical BRCA1/2 testing from 2002 to 2012 were invited to donate a research blood sample. Samples were frozen at -80° C, and DNA was extracted from them after 1 to 10 years. The entire coding region, exon-intron boundaries, and all known pathogenic variants in other regions were sequenced for 42 genes that had cancer risk associations. Potentially actionable results were disclosed to participants. RESULTS In total, 198 women participated in the study: 174 had breast cancer and 57 carried germline BRCA1/2 mutations. BRCA1/2 analysis was fully concordant with prior testing. Sixteen pathogenic variants were identified in ATM, BLM, CDH1, CDKN2A, MUTYH, MLH1, NBN, PRSS1, and SLX4 among 141 women without BRCA1/2 mutations. Fourteen participants carried 15 pathogenic variants, warranting a possible change in care; they were invited for targeted screening recommendations, enabling early detection and removal of a tubular adenoma by colonoscopy. Participants carried an average of 2.1 variants of uncertain significance among 42 genes. CONCLUSION Among women testing negative for BRCA1/2 mutations, multiple-gene sequencing identified 16 potentially pathogenic mutations in other genes (11.4%; 95% CI, 7.0% to 17.7%), of which 15 (10.6%; 95% CI, 6.5% to 16.9%) prompted consideration of a change in care, enabling early detection of a precancerous colon polyp. Additional studies are required to quantify the penetrance of identified mutations and determine clinical utility. However, these results suggest that multiple-gene sequencing may benefit appropriately selected patients.

Survival Analysis of Cancer Risk Reduction Strategies for BRCA1/2 Mutation Carriers

PURPOSE Women with BRCA1/2 mutations inherit high risks of breast and ovarian cancer; options to reduce cancer mortality include prophylactic surgery or breast screening, but their efficacy has never been empirically compared. We used decision analysis to simulate risk-reducing strategies in BRCA1/2 mutation carriers and to compare resulting survival probability and causes of death. METHODS We developed a Monte Carlo model of breast screening with annual mammography plus magnetic resonance imaging (MRI) from ages 25 to 69 years, prophylactic mastectomy (PM) at various ages, and/or prophylactic oophorectomy (PO) at ages 40 or 50 years in 25-year-old BRCA1/2 mutation carriers. RESULTS With no intervention, survival probability by age 70 is 53% for BRCA1 and 71% for BRCA2 mutation carriers. The most effective single intervention for BRCA1 mutation carriers is PO at age 40, yielding a 15% absolute survival gain; for BRCA2 mutation carriers, the most effective single intervention is PM, yielding a 7% survival gain if performed at age 40 years. The combination of PM and PO at age 40 improves survival more than any single intervention, yielding 24% survival gain for BRCA1 and 11% for BRCA2 mutation carriers. PM at age 25 instead of age 40 offers minimal incremental benefit (1% to 2%); substituting screening for PM yields a similarly minimal decrement in survival (2% to 3%). CONCLUSION Although PM at age 25 plus PO at age 40 years maximizes survival probability, substituting mammography plus MRI screening for PM seems to offer comparable survival. These results may guide women with BRCA1/2 mutations in their choices between prophylactic surgery and breast screening.

CDH1 Truncating Mutations in the E-Cadherin Gene: An Indication for Total Gastrectomy to Treat Hereditary Diffuse Gastric Cancer

Background:Approximately 1% to 3% of all gastric cancers are associated with families exhibiting an autosomal dominant pattern of susceptibility. E-cadherin (CDH1) truncating mutations have been shown to be present in approximately 30% of families with hereditary diffuse gastric cancer (HDGC) and are associated with a significantly increased risk of gastric cancer and lobular breast cancer. Methods:Individuals from a large kindred with HDGC who were identified to have a CDH1 mutation prospectively underwent comprehensive screening with stool occult blood testing, standard upper gastrointestinal endoscopy with random gastric biopsies, high-magnification endoscopy with random gastric biopsies, endoscopic ultrasonography, CT, and PET scans to evaluate the stomach for occult cancer. Subsequently, they each underwent total gastrectomy with d-2 node dissection and Roux-en-y esophagojejunostomy. The stomach and resected lymph nodes were evaluated pathologically. Results:Six patients were identified as CDH1 carriers from a single family. There were 2 men and 4 women. The mean age was 54 years (range, 51–57 years). No patient had any signs or symptoms of gastric cancer. Exhaustive preoperative stomach evaluation was normal in each case, and the stomach and adjacent lymph nodes appeared normal at surgery. However, each patient (6 of 6, 100%) was found to have multiple foci of T1 invasive diffuse gastric adenocarcinoma (pure signet-ring cell type). No patient had lymph node or distant metastases. Each was staged as T1N0M0. Each patient recovered uneventfully without morbidity or mortality. Conclusions:CDH1 mutations in individuals from families with HDGC are associated with gastric cancer in a highly penetrant fashion. CDH1 mutations are an indication for total gastrectomy in these patients. This mutation will identify patients with cancer before other detectable symptoms or signs of the disease.

Use of and mortality after bilateral mastectomy compared with other surgical treatments for breast cancer in California, 1998-2011.

IMPORTANCE Bilateral mastectomy is increasingly used to treat unilateral breast cancer. Because it may have medical and psychosocial complications, a better understanding of its use and outcomes is essential to optimizing cancer care. OBJECTIVE To compare use of and mortality after bilateral mastectomy, breast-conserving therapy with radiation, and unilateral mastectomy. DESIGN, SETTING, AND PARTICIPANTS Observational cohort study within the population-based California Cancer Registry; participants were women diagnosed with stages 0-III unilateral breast cancer in California from 1998 through 2011, with median follow-up of 89.1 months. MAIN OUTCOMES AND MEASURES Factors associated with surgery use (from polytomous logistic regression); overall and breast cancer-specific mortality (from propensity score weighting and Cox proportional hazards analysis). RESULTS Among 189,734 patients, the rate of bilateral mastectomy increased from 2.0% (95% CI, 1.7%-2.2%) in 1998 to 12.3% (95% CI, 11.8%-12.9%) in 2011, an annual increase of 14.3% (95% CI, 13.1%-15.5%); among women younger than 40 years, the rate increased from 3.6% (95% CI, 2.3%-5.0%) in 1998 to 33% (95% CI, 29.8%-36.5%) in 2011. Bilateral mastectomy was more often used by non-Hispanic white women, those with private insurance, and those who received care at a National Cancer Institute (NCI)-designated cancer center (8.6% [95% CI, 8.1%-9.2%] among NCI cancer center patients vs 6.0% [95% CI, 5.9%-6.1%] among non-NCI cancer center patients; odds ratio [OR], 1.13 [95% CI, 1.04-1.22]); in contrast, unilateral mastectomy was more often used by racial/ethnic minorities (Filipina, 52.8% [95% CI, 51.6%-54.0%]; OR, 2.00 [95% CI, 1.90-2.11] and Hispanic, 45.6% [95% CI, 45.0%-46.2%]; OR, 1.16 [95% CI, 1.13-1.20] vs non-Hispanic white, 35.2% [95% CI, 34.9%-35.5%]) and those with public/Medicaid insurance (48.4% [95% CI, 47.8%-48.9%]; OR, 1.08 [95% CI, 1.05-1.11] vs private insurance, 36.6% [95% CI, 36.3%-36.8%]). Compared with breast-conserving surgery with radiation (10-year mortality, 16.8% [95% CI, 16.6%-17.1%]), unilateral mastectomy was associated with higher all-cause mortality (hazard ratio [HR], 1.35 [95% CI, 1.32-1.39]; 10-year mortality, 20.1% [95% CI, 19.9%-20.4%]). There was no significant mortality difference compared with bilateral mastectomy (HR, 1.02 [95% CI, 0.94-1.11]; 10-year mortality, 18.8% [95% CI, 18.6%-19.0%]). Propensity analysis showed similar results. CONCLUSIONS AND RELEVANCE Use of bilateral mastectomy increased significantly throughout California from 1998 through 2011 and was not associated with lower mortality than that achieved with breast-conserving surgery plus radiation. Unilateral mastectomy was associated with higher mortality than were the other 2 surgical options.

Clinical Actionability of Multigene Panel Testing for Hereditary Breast and Ovarian Cancer Risk Assessment

IMPORTANCE The practice of genetic testing for hereditary breast and/or ovarian cancer (HBOC) is rapidly evolving owing to the recent introduction of multigene panels. While these tests may identify 40% to 50% more individuals with hereditary cancer gene mutations than does testing for BRCA1/2 alone, whether finding such mutations will alter clinical management is unknown. OBJECTIVE To define the potential clinical effect of multigene panel testing for HBOC in a clinically representative cohort. DESIGN, SETTING, AND PARTICIPANTS Observational study of patients seen between 2001 and 2014 in 3 large academic medical centers. We prospectively enrolled 1046 individuals who were appropriate candidates for HBOC evaluation and who lacked BRCA1/2 mutations. INTERVENTIONS We carried out multigene panel testing on all participants, then determined the clinical actionability, if any, of finding non-BRCA1/2 mutations in these and additional comparable individuals. MAIN OUTCOMES AND MEASURES We evaluated the likelihood of (1) a posttest management change and (2) an indication for additional familial testing, considering gene-specific consensus management guidelines, gene-associated cancer risks, and personal and family history. RESULTS Among 1046 study participants, 40 BRCA1/2-negative patients (3.8%; 95% CI, 2.8%-5.2%) harbored deleterious mutations, most commonly in moderate-risk breast and ovarian cancer genes (CHEK2, ATM, and PALB2) and Lynch syndrome genes. Among these and an additional 23 mutation-positive individuals enrolled from our clinics, most of the mutations (92%) were consistent with the spectrum of cancer(s) observed in the patient or family, suggesting that these results are clinically significant. Among all 63 mutation-positive patients, additional disease-specific screening and/or prevention measures beyond those based on personal and family history alone would be considered for most (33 [52%] of 63; 95% CI, 40.3%-64.2%). Furthermore, additional familial testing would be considered for those with first-degree relatives (42 [72%] of 58; 95% CI, 59.8%-82.2%) based on potential management changes for mutation-positive relatives. This clinical effect was not restricted to a few of the tested genes because most identified genes could change clinical management for some patients. CONCLUSIONS AND RELEVANCE In a clinically representative cohort, multigene panel testing for HBOC risk assessment yielded findings likely to change clinical management for substantially more patients than does BRCA1/2 testing alone. Multigene testing in this setting is likely to alter near-term cancer risk assessment and management recommendations for mutation-affected individuals across a broad spectrum of cancer predisposition genes.

Occurrence of breast cancer subtypes in adolescent and young adult women

IntroductionBreast cancers are increasingly recognized as heterogeneous based on expression of receptors for estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2). Triple-negative tumors (ER-/PR-/HER2-) have been reported to be more common among younger women, but occurrence of the spectrum of breast cancer subtypes in adolescent and young adult (AYA) women aged between 15 and 39 years is otherwise poorly understood.MethodsData regarding all 5,605 AYA breast cancers diagnosed in California during the period 2005 to 2009, including ER and PR status (referred to jointly as hormone receptor (HR) status) and HER2 status, was obtained from the population-based California Cancer Registry. Incidence rates were calculated by subtype (triple-negative; HR+/HER2-; HR+/HER2+; HR-/HER2+), and logistic regression was used to evaluate differences in subtype characteristics by age group.ResultsAYAs had higher proportions of HR+/HER2+, triple-negative and HR-/HER2+ breast cancer subtypes and higher proportions of patients of non-White race/ethnicity than did older women. AYAs also were more likely to be diagnosed with stage III/IV disease and high-grade tumors than were older women. Rates of HR+/HER2- and triple-negative subtypes in AYAs varied substantially by race/ethnicity.ConclusionsThe distribution of breast cancer subtypes among AYAs varies from that observed in older women, and varies further by race/ethnicity. Observed subtype distributions may explain the poorer breast cancer survival previously observed among AYAs.

Lifetime risks of specific breast cancer subtypes among women in four racial/ethnic groups

IntroductionBreast cancer comprises clinically distinct subtypes, but most risk statistics consider breast cancer only as a single entity. To estimate subtype-specific lifetime breast cancer risks, we took advantage of population-based data for which information regarding tumor expression of estrogen receptor (ER), progesterone receptor (PR) and HER2/neu (HER2) was newly available.MethodsWe included women whose breast cancer was diagnosed in the state of California from 2006 to 2007 and was reported to the National Cancer Institutes Surveillance, Epidemiology and End Results Program (N = 40,936). We calculated absolute lifetime and age-specific probabilities (percent, 95% confidence interval) of developing breast cancer subtypes defined by ER, PR, and HER2 status - luminal (ER and/or PR-positive, HER2-negative), HER2-positive (ER and PR-positive or negative, HER2-positive), and triple-negative (ER-negative, PR-negative, and HER2-negative) - separately for white, black, Hispanic, and Asian women.ResultsThe luminal breast cancer subtype predominates across racial/ethnic groups, with lifetime risk lowest in Hispanic women (4.60%, 4.41-4.80%) and highest in white women (8.10%, 7.94-8.20%). HER2-positive breast cancer varies less by race (1.56-1.91%). Lifetime risk of triple-negative breast cancer is highest in black women (1.98%, 1.80-2.17%), compared to 0.77% (0.67-0.88%) for Asians, 1.04% (0.96-1.13%) for Hispanics and 1.25% (1.20-1.30%) for whites. Across racial/ethnic groups, nearly half of all luminal breast cancers occur after age 70.ConclusionsThese absolute risk estimates may inform health policy and resource planning across diverse populations, and can help patients and physicians weigh the probabilities of developing specific breast cancer subtypes against competing health risks.

Biomedical terahertz imaging with a quantum cascade laser

We present biomedical imaging using a single frequency terahertz imaging system based on a low threshold quantum cascade laser emitting at 3.7THz (λ=81μm). With a peak output power of 4mW, coherent terahertz radiation and detection provide a relatively large dynamic range and high spatial resolution. We study image contrast based on water/fat content ratios in different tissues. Terahertz transmission imaging demonstrates a distinct anatomy in a rat brain slice. We also demonstrate malignant tissue contrast in an image of a mouse liver with developed tumors, indicating potential use of terahertz imaging for probing cancerous tissues.

BRCA1 and BRCA2 mutations across race and ethnicity: distribution and clinical implications

Purpose of review To summarize evidence on the prevalence and spectrum of BRCA1 and BRCA2 BRCA1/2 mutations across racial and ethnic groups and discuss implications for clinical practice. Recent findings The prevalence of BRCA1/2 mutations is comparable among breast cancer patients of African, Asian, white, and Hispanic descent: approximately 1–4% per gene. Among ovarian cancer patients in North America, BRCA1/2 mutations are present in 13–15%. Between racial/ethnic groups, there are important differences in the spectrum of BRCA1 compared with BRCA2 mutations, in BRCA1/2 variants of uncertain significance, and in the accuracy of clinical models that predict BRCA1/2 mutation carriage. Summary Given the significant prevalence of BRCA1/2 mutations across race/ethnicity, there is a need to expand and customize genetic counseling, genetic testing, and follow-up care for members of all racial/ethnic groups.