Integrative multi-omic profiling of triple-negative breast cancer for identifying suitable therapies.

PURPOSE: Triple-negative breast cancer (TNBC) is a heterogenous disease that carries the poorest prognosis of all breast cancers. Although novel TNBC therapies in development are frequently targeted towards tumors carrying a specific genomic, transcriptomic, or protein biomarker, how these biomarkers are correlated is poorly understood. EXPERIMENTAL DESIGN: To better understand the molecular features of TNBC and their correlation with one another, we performed multi-modal profiling on a cohort of 95 TNBCs. Our approach involved quantifying tumor-infiltrating lymphocytes through H&E staining, assessing the abundance of retinoblastoma (Rb), androgen receptor (AR), and PD-L1 proteins through immunohistochemistry, and carrying out transcriptomic profiling using the Nanostring BC360 platform, targeted DNA sequencing on a subset of cases, as well as evaluating associations with overall survival. RESULTS: Levels of RB1 mRNA and RB protein are better correlated with markers of Rb functionality than is RB1 mutational status. Luminal AR tumors clustered into two groups with transcriptomes that cluster with either basal or mesenchymal tumors. Tumors classified as PD-L1-positive by presence of immune or tumor cells showed similar biological characteristics. HER2-low TNBC showed no distinct biological phenotype when compared to HER2-zero. The majority of TNBCs classified as basal or HER2-enriched by PAM50, the latter showing significantly improved overall survival. CONCLUSIONS: Our study contributes new insights into biomarker utility for identifying suitable TNBC therapies and the intercorrelations between genomic, transcriptomic, protein, and cellular biomarkers. Additionally, our rich data resource can be used by other researchers to explore the interplay between DNA, RNA, and protein biomarkers in TNBC.

Helping ourselves, helping others: the Young Women's Breast Cancer Study (YWS) - a multisite prospective cohort study to advance the understanding of breast cancer diagnosed in women aged 40 years and younger.

PURPOSE: Compared with older women diagnosed with breast cancer, younger women are more likely to die of breast cancer and more likely to suffer psychosocially in both the short-term and long term. The Young Women's Breast Cancer Study (YWS) is a multisite prospective cohort study established to address gaps in our knowledge about this vulnerable and understudied population. PARTICIPANTS: The YWS enrolled 1302 women newly diagnosed with stages 0-IV breast cancer at age 40 years or younger at 13 academic and community sites in North America between 2006 and 2016. Longitudinal patient-reported outcome data are complemented by clinical data abstraction and biospecimen collection at multiple timepoints. FINDINGS TO DATE: Key findings related to fertility include that nearly 40% of participants were interested in pregnancy following diagnosis; of those who reported interest, 10% pursued fertility preservation. Overall, approximately 10% of YWS participants became pregnant in the first 5 years after diagnosis; follow-up is ongoing for pregnancies after 5 years. Studies focused on psychosocial outcomes have characterised quality of life, post-traumatic stress and fear of recurrence, with findings detailing the factors associated with the substantial psychosocial burden many young women face during and following active treatment. Multiple studies have leveraged YWS biospecimens, including whole-exome sequencing of tumour analyses that revealed that select somatic alterations occur at different frequencies in young (age≤35) versus older women with luminal A breast cancer, and a study that explored clonal hematopoiesis of indeterminate potential found it to be rare in young survivors. FUTURE PLANS: With a median follow-up of approximately 10 years, the cohort is just maturing for many relevant long-term outcomes and provides outstanding opportunities to further study and build collaborations to address gaps in our knowledge, with the ultimate objective to improve care and outcomes for young women with breast cancer. TRIAL REGISTRATION NUMBER: NCT01468246.

Pure estrogen receptor antagonists potentiate capecitabine activity in ESR1-mutant breast cancer.

The ESR1 ligand binding domain activating mutations are the most prevalent genetic mechanism of acquired endocrine resistance in metastatic hormone receptor-positive breast cancer. These mutations confer endocrine resistance that remains estrogen receptor (ER) dependent. We hypothesized that in the presence of the ER mutations, continued ER blockade with endocrine therapies that target mutant ER is essential for tumor suppression even with chemotherapy treatment. Here, we conducted comprehensive pre-clinical in vitro and in vivo experiments testing the efficacy of adding fulvestrant to fluorouracil (5FU) and the 5FU pro-drug, capecitabine, in models of wild-type (WT) and mutant ER. Our findings revealed that while this combination had an additive effect in the presence of WT-ER, in the presence of the Y537S ER mutation there was synergy. Notably, these effects were not seen with the combination of 5FU and selective estrogen receptor modulators, such as tamoxifen, or in the absence of intact P53. Likewise, in a patient-derived xenograft (PDX) harboring a Y537S ER mutation the addition of fulvestrant to capecitabine potentiated tumor suppression. Moreover, multiplex immunofluorescence revealed that this effect was due to decreased cell proliferation in all cells expressing ER and was not dependent on the degree of ER expression. Taken together, these results support the clinical investigation of the combination of ER antagonists with capecitabine in patients with metastatic hormone receptor-positive breast cancer who have experienced progression on endocrine therapy and targeted therapies, particularly in the presence of an ESR1 activating mutation.

Second primary non-breast cancers in young breast cancer survivors.

PURPOSE: We evaluated the incidence, timing, and risk factors for second primary non-breast cancers (SPNBC) among young breast cancer (BC) survivors. METHODS: This study included participants of the Young Women's BC Study (YWS) who were diagnosed with stage 0-III BC between 2006 and 2016 and age 40 or younger at diagnosis (N = 1,230). Patient characteristics, treatment information, and clinical events were collected via serial surveys. Tumor and treatment data were obtained from medical record review. Five- and 10-year risks of SPNBCs were estimated via the cumulative incidence function, considering death, metastasis, or second primary BC as competing events. Fine and Gray subdistribution hazard models estimated subdistribution hazard ratios (sHRs) and 95% confidence intervals (CI) for SPNBC risk based on risk factors including demographics, germline genetics, primary BC characteristics, and treatments. RESULTS: Among 1,230 women, over a median follow-up of 10.1 years, 47 patients (4%) developed an SPNBC. Types of malignancy included melanoma (n = 10), thyroid (n = 10), ovarian (n = 4), sarcoma (n = 4), uterine (n = 3), rectal (n = 3), bladder (n = 2), cervical (n = 2), head/neck (n = 2), lung (n = 2), lymphoma (n = 2), pancreatic (n = 2), and renal (n = 1). Five and 10-year cumulative incidence were 1.4% and 3.2%, respectively. Median time between primary BC and SPNBC was 7.3 years. No patient factors, primary tumor characteristics, or treatments were statistically significantly associated with SPNBC in univariable or multivariable models. CONCLUSION: In this population, five-year cumulative incidence was higher than that reported among healthy women under 50 years of age, highlighting the importance of long-term surveillance for new non-breast cancers in young adult BC survivors.

Clinicogenomic characterization of inflammatory breast cancer.

Background: Inflammatory breast cancer (IBC) is a rare and poorly characterized type of breast cancer with an aggressive clinical presentation. The biological mechanisms driving the IBC phenotype are relatively undefined-partially due to a lack of comprehensive, large-scale genomic studies and limited clinical cohorts. Patients and Methods: A retrospective analysis of 2457 patients with metastatic breast cancer who underwent targeted tumor-only DNA-sequencing was performed at Dana-Farber Cancer Institute. Clinicopathologic, single nucleotide variant (SNV), copy number variant (CNV) and tumor mutational burden (TMB) comparisons were made between clinically confirmed IBC cases within a dedicated IBC center versus non-IBC cases. Results: Clinicopathologic differences between IBC and non-IBC cases were consistent with prior reports-including IBC being associated with younger age at diagnosis, higher grade, and enrichment with hormone receptor (HR)-negative and HER2-positive tumors. The most frequent somatic alterations in IBC involved TP53 (72%), ERBB2 (32%), PIK3CA (24%), CCND1 (12%), MYC (9%), FGFR1 (8%) and GATA3 (8%). A multivariate logistic regression analysis revealed a significant enrichment in TP53 SNVs in IBC; particularly in HER2-positive and HR-positive disease which was associated with worse outcomes. Tumor mutational burden (TMB) did not differ substantially between IBC and non-IBC cases and a pathway analysis revealed an enrichment in NOTCH pathway alterations in HER2-positive disease. Conclusion: Taken together, this study provides a comprehensive, clinically informed landscape of somatic alterations in a large cohort of patients with IBC. Our data support higher frequency of TP53 mutations and a potential enrichment in NOTCH pathway activation-but overall; a lack of major genomic differences. These results both reinforce the importance of TP53 alterations in IBC pathogenesis as well as their influence on clinical outcomes; but also suggest additional analyses beyond somatic DNA-level changes are warranted.

Second Primary Breast Cancer in Young Breast Cancer Survivors.

Importance: Among women diagnosed with primary breast cancer (BC) at or younger than age 40 years, prior data suggest that their risk of a second primary BC (SPBC) is higher than that of women who are older when they develop a first primary BC. Objective: To estimate cumulative incidence and characterize risk factors of SPBC among young patients with BC. Design, Setting, and Participants: Participants were enrolled in the Young Women's Breast Cancer Study, a prospective study of 1297 women aged 40 years or younger who were diagnosed with stage 0 to III BC from August 2006 to June 2015. Demographic, genetic testing, treatment, and outcome data were collected by patient surveys and medical record review. A time-to-event analysis was used to account for competing risks when determining cumulative incidence of SPBC, and Fine-Gray subdistribution hazard models were used to evaluate associations between clinical factors and SPBC risk. Data were analyzed from January to May 2023. Main Outcomes and Measures: The 5- and 10- year cumulative incidence of SPBC. Results: In all, 685 women with stage 0 to III BC (mean [SD] age at primary BC diagnosis, 36 [4] years) who underwent unilateral mastectomy or lumpectomy as the primary surgery for BC were included in the analysis. Over a median (IQR) follow-up of 10.0 (7.4-12.1) years, 17 patients (2.5%) developed an SPBC; 2 of these patients had cancer in the ipsilateral breast after lumpectomy. The median (IQR) time from primary BC diagnosis to SPBC was 4.2 (3.3-5.6) years. Among 577 women who underwent genetic testing, the 10-year risk of SPBC was 2.2% for women who did not carry a pathogenic variant (12 of 544) and 8.9% for carriers of a pathogenic variant (3 of 33). In multivariate analyses, the risk of SPBC was higher among PV carriers vs noncarriers (subdistribution hazard ratio [sHR], 5.27; 95% CI, 1.43-19.43) and women with primary in situ BC vs invasive BC (sHR, 5.61; 95% CI, 1.52-20.70). Conclusions: Findings of this cohort study suggest that young BC survivors without a germline pathogenic variant have a low risk of developing a SPBC in the first 10 years after diagnosis. Findings from germline genetic testing may inform treatment decision-making and follow-up care considerations in this population.

Conception and pregnancy among women with a live birth after breast cancer treatment: A survey study of young breast cancer survivors.

BACKGROUND: Breast cancer (BC) is the most common malignancy in women of reproductive age. This study sought to explore the postcancer conception and pregnancy experience of young BC survivors to inform counseling. METHODS: In the Young Women's Breast Cancer Study (NCT01468246), a multicenter, prospective cohort, participants diagnosed at age ≤40 years with stage 0-III BC who reported ≥1 postdiagnosis live birth were sent an investigator-developed survey. RESULTS: Of 119 eligible women, 94 (79%) completed the survey. Median age at diagnosis was 32 years (range, 17-40) and at first postdiagnosis delivery was 38 years (range, 29-47). Most had stage I or II (77%) and HR+ (78%) BC; 51% were nulligravida at diagnosis. After BC treatment, most (62%) conceived naturally, though 38% used assisted reproductive technology, 74% of whom first attempted natural conception for a median of 9 months (range, 2-48). Among women with a known inherited pathogenic variant (n = 20), two underwent preimplantation genetic testing. Of 59 women on endocrine therapy before pregnancy, 26% did not resume treatment. Hypertensive disorders of pregnancy (20%) was the most common obstetrical condition. Nine percent of newborns required neonatal intensive care unit admission and 9% had low birth weight. CONCLUSION: Among women with live births after BC treatment, most conceived naturally and having a history of BC did not appear to negatively impact pregnancy complications, though the high rate of hypertensive disorders of pregnancy warrants further investigation. The prolonged period of attempting natural conception for some survivors suggests the potential need for improved understanding and counseling surrounding family planning goals after BC.

Comprehensive genomic characterization of HER2-low and HER2-0 breast cancer.

The molecular underpinnings of HER2-low and HER2-0 (IHC 0) breast tumors remain poorly defined. Using genomic findings from 1039 patients with HER2-negative metastatic breast cancer undergoing next-generation sequencing from 7/2013-12/2020, we compare results between HER2-low (n = 487, 47%) and HER2-0 tumors (n = 552, 53%). A significantly higher number of ERBB2 alleles (median copy count: 2.05) are observed among HER2-low tumors compared to HER2-0 (median copy count: 1.79; P = 2.36e-6), with HER2-0 tumors harboring a higher rate of ERBB2 hemideletions (31.1% vs. 14.5%). No other genomic alteration reaches significance after accounting for multiple hypothesis testing, and no significant differences in tumor mutational burden are observed between HER2-low and HER2-0 tumors (median: 7.26 mutations/megabase vs. 7.60 mutations/megabase, p = 0.24). Here, we show that the genomic landscape of HER2-low and HER2-0 tumors does not differ significantly, apart from a higher ERBB2 copy count among HER2-low tumors, and a higher rate of ERBB2 hemideletions in HER2-0 tumors.

Circulating Tumor DNA and Late Recurrence in High-Risk Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer.

PURPOSE: To examine the prevalence and dynamics of circulating tumor DNA (ctDNA) and its association with metastatic recurrence in patients with high-risk early-stage hormone receptor-positive breast cancer (HR+ BC) more than 5 years from diagnosis. METHODS: We enrolled 103 patients with high-risk stage II-III HR+ BC diagnosed more than 5 years prior without clinical evidence of recurrence. We performed whole-exome sequencing (WES) on primary tumor tissue to identify somatic mutations tracked via a personalized, tumor-informed ctDNA test to detect minimal residual disease (MRD). We collected plasma at the time of consent and at routine visits every 6-12 months. Patients were followed for clinical recurrence. RESULTS: In total, 85 of 103 patients had sufficient tumor tissue; of them, 83 of 85 (97.6%) patients had successful whole-exome sequencing. Personalized ctDNA assays were designed targeting a median of 36 variants to test 219 plasma samples. The median time from diagnosis to first sample was 8.4 years. The median follow-up was 10.4 years from diagnosis and 2.0 years from first sample. The median number of plasma samples per patient was two. Eight patients (10%) had positive MRD testing at any time point. Six patients (7.2%) developed distant metastatic recurrence, all of whom were MRD-positive before overt clinical recurrence, with median ctDNA lead time of 12.4 months. MRD was not identified in one patient (1.2%) with local recurrence. Two of eight MRD-positive patients had not had clinical recurrence at last follow-up. CONCLUSION: In this prospective study, in patients with high-risk HR+ BC in the late adjuvant setting, ctDNA was identified a median of 1 year before all cases of distant metastasis. Future studies will determine if ctDNA-guided intervention in patients with HR+ BC can alter clinical outcomes.

Somatic and Germline Genomic Alterations in Very Young Women with Breast Cancer.

PURPOSE: Young age at breast cancer diagnosis correlates with unfavorable clinicopathologic features and worse outcomes compared with older women. Understanding biological differences between breast tumors in young versus older women may lead to better therapeutic approaches for younger patients. EXPERIMENTAL DESIGN: We identified 100 patients ≤35 years old at nonmetastatic breast cancer diagnosis who participated in the prospective Young Women's Breast Cancer Study cohort. Tumors were assigned a surrogate intrinsic subtype based on receptor status and grade. Whole-exome sequencing of tumor and germline samples was performed. Genomic alterations were compared with older women (≥45 years old) in The Cancer Genome Atlas, according to intrinsic subtype. RESULTS: Ninety-three tumors from 92 patients were successfully sequenced. Median age was 32.5 years; 52.7% of tumors were hormone receptor-positive/HER2-negative, 28.0% HER2-positive, and 16.1% triple-negative. Comparison of young to older women (median age 61 years) with luminal A tumors (N = 28 young women) revealed three significant differences: PIK3CA alterations were more common in older patients, whereas GATA3 and ARID1A alterations were more common in young patients. No significant genomic differences were found comparing age groups in other intrinsic subtypes. Twenty-two patients (23.9%) in the Young Women's Study cohort carried a pathogenic germline variant, most commonly (13 patients, 14.1%) in BRCA1/2. CONCLUSIONS: Somatic alterations in three genes (PIK3CA, GATA3, and ARID1A) occur at different frequencies in young versus older women with luminal A breast cancer. Additional investigation of these genes and associated pathways could delineate biological susceptibilities and improve treatment options for young patients with breast cancer. See related commentary by Yehia and Eng, p. 2209.

Clinicopathological features and BRCA1 and BRCA2 mutation status in a prospective cohort of young women with breast cancer.

BACKGROUND: Breast cancer in young women is more likely to have higher risk features and be associated with germline BRCA1/BRCA2 mutations. We present the clinicopathologic features of breast cancers in a prospective cohort of young women, and associations between surrogate molecular subtype and BRCA1/BRCA2 mutation status. METHODS: Histopathological features, biomarker status, tumour stage and BRCA status were collected. Invasive tumours were categorised as luminal A-like (ER + and/or PR + , HER2-, grade 1/2), luminal B-like (ER + and/or PR + , HER2 + , or ER + and/or PR + , HER2-, and grade 3), HER2-enriched (ER/PR-, HER2 + ) or triple-negative. RESULTS: In all, 57.3% (654/1143) of invasive tumours were high grade. In total, 32.9% were luminal A-like, 42.4% luminal B-like, 8.3% HER2-enriched, and 16.4% triple-negative. Among different age groups, there were no differences in molecular phenotype, stage, grade or histopathology. 11% (131) of tumours were from BRCA mutation carriers; 64.1% BRCA1 (63.1% triple-negative), and 35.9% BRCA2 (55.3% luminal B-like). DISCUSSION: The opportunity to provide comparisons across young age groups, BRCA mutation status, surrogate molecular phenotype, and the identification of more aggressive hormone receptor-positive phenotypes in this population provides direction for future work to further understand and improve disparate outcomes for young women with luminal B-like cancers, particularly BRCA2-associated cancers, with potential implications for tailored prevention and treatment.

Impact of Cancer History on Outcomes Among Hospitalized Patients with COVID-19.

BACKGROUND: Early reports suggested increased mortality from COVID-19 in patients with cancer but lacked rigorous comparisons to patients without cancer. We investigated whether a current cancer diagnosis or cancer history is an independent risk factor for death in hospitalized patients with COVID-19. PATIENTS AND METHODS: We identified patients with a history of cancer admitted to two large hospitals between March 13, 2020, and May 10, 2020, with laboratory-confirmed COVID-19 and matched them 1:2 to patients without a history of cancer. RESULTS: Men made up 56.2% of the population, with a median age of 69 years (range, 30-96). The median time since cancer diagnosis was 35.6 months (range, 0.39-435); 80% had a solid tumor, and 20% had a hematologic malignancy. Among patients with cancer, 27.8% died or entered hospice versus 25.6% among patients without cancer. In multivariable analyses, the odds of death/hospice were similar (odds ratio [OR], 1.09; 95% confidence interval [CI], 0.65-1.82). The odds of intubation (OR, 0.46; 95% CI, 0.28-0.78), shock (OR, 0.54; 95% CI, 0.32-0.91), and intensive care unit admission (OR, 0.51; 95% CI, 0.32-0.81) were lower for patients with a history of cancer versus controls. Patients with active cancer or who had received cancer-directed therapy in the past 6 months had similar odds of death/hospice compared with cancer survivors (univariable OR, 1.31; 95% CI, 0.66-2.60; multivariable OR, 1.47; 95% CI, 0.69-3.16). CONCLUSION: Patients with a history of cancer hospitalized for COVID-19 had similar mortality to matched hospitalized patients with COVID-19 without cancer, and a lower risk of complications. In this population, patients with active cancer or recent cancer treatment had a similar risk for adverse outcomes compared with survivors of cancer. IMPLICATIONS FOR PRACTICE: This study investigated whether a current cancer diagnosis or cancer history is an independent risk factor for death or hospice admission in hospitalized patients with COVID-19. Active cancer, systemic cancer therapy, and a cancer history are not independent risk factors for death from COVID-19 among hospitalized patients, and hospitalized patients without cancer are more likely to have severe COVID-19. These findings provide reassurance to survivors of cancer and patients with cancer as to their relative risk of severe COVID-19, may encourage oncologists to provide standard anticancer therapy in patients at risk of COVID-19, and guide triage in future waves of infection.

Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer.

PURPOSE: Existing cell-free DNA (cfDNA) methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. We developed a test for tracking hundreds of patient-specific mutations to detect MRD with a 1,000-fold lower error rate than conventional sequencing. EXPERIMENTAL DESIGN: We compared the sensitivity of our approach to digital droplet PCR (ddPCR) in a dilution series, then retrospectively identified two cohorts of patients who had undergone prospective plasma sampling and clinical data collection: 16 patients with ER+/HER2- metastatic breast cancer (MBC) sampled within 6 months following metastatic diagnosis and 142 patients with stage 0 to III breast cancer who received curative-intent treatment with most sampled at surgery and 1 year postoperative. We performed whole-exome sequencing of tumors and designed individualized MRD tests, which we applied to serial cfDNA samples. RESULTS: Our approach was 100-fold more sensitive than ddPCR when tracking 488 mutations, but most patients had fewer identifiable tumor mutations to track in cfDNA (median = 57; range = 2-346). Clinical sensitivity was 81% (n = 13/16) in newly diagnosed MBC, 23% (n = 7/30) at postoperative and 19% (n = 6/32) at 1 year in early-stage disease, and highest in patients with the most tumor mutations available to track. MRD detection at 1 year was strongly associated with distant recurrence [HR = 20.8; 95% confidence interval, 7.3-58.9]. Median lead time from first positive sample to recurrence was 18.9 months (range = 3.4-39.2 months). CONCLUSIONS: Tracking large numbers of individualized tumor mutations in cfDNA can improve MRD detection, but its sensitivity is driven by the number of tumor mutations available to track.

Prospective study of family history and colorectal cancer risk by tumor LINE-1 methylation level.

BACKGROUND: Beyond known familial colorectal cancer (CRC) syndromes, the mechanisms underlying the elevated CRC risk associated with CRC family history remain largely unknown. A recent retrospective study suggests familial clustering of CRC with hypomethylation in long interspersed nucleotide element 1 (LINE-1). We tested the hypothesis that CRC family history might confer a higher risk of LINE-1 methylation-low CRC. METHODS: Using the Nurses' Health Study and the Health Professionals Follow-up Study, we prospectively examined the association between CRC family history and the risk of rectal and colon cancer (N = 1224) according to tumor LINE-1 methylation level by duplication method Cox proportional hazards regression. We examined microsatellite instability (MSI) status to exclude the influence of Lynch syndrome. All statistical tests were two-sided. RESULTS: The association between CRC family history and non-MSI CRC risk differed statistically significantly by LINE-1 methylation level (P (heterogeneity) = .02). CRC family history was associated with a statistically significantly higher risk of LINE-1 methylation-low non-MSI cancer (multivariable hazard ratio [HR] = 1.68, 95% confidence interval [CI] = 1.19 to 2.38 for 1 vs 0 first-degree relatives with CRC; multivariable HR = 3.48, 95% CI = 1.59 to 7.6 for ≥2 vs 0 first-degree relatives with CRC; P (trend) < .001). In contrast, CRC family history was not statistically significantly associated with LINE-1 methylation-high non-MSI cancer (P (trend) = .35). CONCLUSIONS: This molecular pathological epidemiology study shows that CRC family history is associated with a higher risk of LINE-1 methylation-low CRC, suggesting previously unrecognized heritable predisposition to epigenetic alterations. Additional studies are needed to evaluate tumor LINE-1 methylation as a molecular biomarker for familial cancer risk assessment.

Expression profiling of archival tumors for long-term health studies.

PURPOSE: More than 20 million archival tissue samples are stored annually in the United States as formalin-fixed, paraffin-embedded (FFPE) blocks, but RNA degradation during fixation and storage has prevented their use for transcriptional profiling. New and highly sensitive assays for whole-transcriptome microarray analysis of FFPE tissues are now available, but resulting data include noise and variability for which previous expression array methods are inadequate. EXPERIMENTAL DESIGN: We present the two largest whole-genome expression studies from FFPE tissues to date, comprising 1,003 colorectal cancer (CRC) and 168 breast cancer samples, combined with a meta-analysis of 14 new and published FFPE microarray datasets. We develop and validate quality control (QC) methods through technical replication, independent samples, comparison to results from fresh-frozen tissue, and recovery of expected associations between gene expression and protein abundance. RESULTS: Archival tissues from large, multicenter studies showed a much wider range of transcriptional data quality relative to smaller or frozen tissue studies and required stringent QC for subsequent analysis. We developed novel methods for such QC of archival tissue expression profiles based on sample dynamic range and per-study median profile. This enabled validated identification of gene signatures of microsatellite instability and additional features of CRC, and improved recovery of associations between gene expression and protein abundance of MLH1, FASN, CDX2, MGMT, and SIRT1 in CRC tumors. CONCLUSIONS: These methods for large-scale QC of FFPE expression profiles enable study of the cancer transcriptome in relation to extensive clinicopathological information, tumor molecular biomarkers, and long-term lifestyle and outcome data.

Postmenopausal hormone therapy is associated with a reduced risk of colorectal cancer lacking CDKN1A expression.

Experimental studies have shown that estrogen- or progesterone-activated signaling leads to growth inhibition effects on colon cancer cells through the upregulation of several cell-cycle regulators. However, epidemiologic studies evaluating hormone therapy use and colorectal cancer risk by the status of cell-cycle regulators are lacking. In this study, we used data from the prospective Nurses' Health Study to evaluate whether the association between hormone therapy use and colorectal cancer risk differs by the molecular pathologic status of microsatellite instability (MSI) and expression of cell-cycle-related tumor biomarkers, including CDKN1A (p21, CIP1), CDKN1B (p27, KIP1), and TP53 (p53) by immunohistochemistry. Duplication Cox regression analysis was used to determine an association between hormone therapy use, cancer risk, and specific tumor biomarkers in 581 incident colon and rectal cancer cases that occurred during 26 years of follow-up among 105,520 postmenopausal women. We found a difference between hormone therapy use and colorectal cancer risk according to CDKN1A expression (P(heterogeneity) = 0.01). Current hormone therapy use was associated with a reduced risk for CDKN1A-nonexpressed [multivariate relative risk (RR), 0.61; 95% confidence interval (CI), 0.46-0.82] but not for CDKN1A-expressed (RR, 1.32; 95% CI, 0.76-2.31) tumors. The lower risk for CDKN1A-nonexpressed but not for CDKN1A-expressed cancers was also present among current users of estrogen-alone therapy. We found no significant difference in the relations between hormone therapy use and cancer risk according to MSI, CDKN1B, or TP53 status. Together, our molecular pathological epidemiology findings suggest a preventive effect of hormone therapy against colorectal carcinogenesis that depends, in part, on loss of cyclin-dependent kinase inhibitor CDKN1A.

Patterns of adjuvant chemotherapy use in a population-based cohort of patients with resected stage II or III colon cancer.

PURPOSE: Previous studies have examined predictors for initiation of adjuvant chemotherapy in stages II and III colon cancer. However, little is known regarding the use of specific chemotherapy regimens or treatment duration. PATIENTS AND METHODS: We studied treatment records for 2,560 patients with stage II or III colon cancer who received adjuvant chemotherapy between January 2004 and April 2010 at US cancer care facilities participating in a nationwide, commercially available chemotherapy order entry system that captures patient demographics, stage, and details of chemotherapy treatment. Multivariate analyses of prospectively recorded patient and provider characteristics identified predictors of specific therapeutic approaches. RESULTS: The addition of oxaliplatin to fluoropyrimidine-based adjuvant therapy increased during the study period (P trend < .001), and this combination represented 78% and 90% of adjuvant chemotherapy in stage II or III disease, respectively, by 2007. Older patients, those with diminished performance status, and those treated in a private practice setting were significantly less likely to receive oxaliplatin. Thirty percent of patients discontinued adjuvant therapy after less than 3 months. Older age, treatment without oxaliplatin, and receipt of treatment from a physician with a low volume of patients were each independently associated with premature discontinuation [corrected]. Six percent of patients received bevacizumab as part of their adjuvant regimen. CONCLUSION: After 2004, oxaliplatin and fluoropyrimidine-based therapy rapidly became the predominant adjuvant treatment for both stage II and stage III colon cancer in this large US cohort. Both increasing patient age and lower volume of an oncologist's practice were associated with early termination of adjuvant therapy.

Germline polymorphisms in the one-carbon metabolism pathway and DNA methylation in colorectal cancer.

Dietary intake of one-carbon nutrients (methyl donors) and germline variants in the one-carbon metabolism genes may influence global DNA methylation level and methylation in promoter CpG islands. In this study, we evaluated the relationship between single nucleotide polymorphisms (SNPs) in the one-carbon metabolism pathway and DNA methylation status in colorectal cancer. Utilizing 182 colorectal cancers cases in two prospective cohort studies, we determined the CpG island methylator phenotype (CIMP) status on eight CIMP-specific promoters and measured LINE-1 methylation level that correlates well with genome-wide DNA methylation level. We genotyped 23 nonsynonymous SNPs in the one-carbon metabolism genes using buffy coat DNA. Most of the 23 SNPs in the one-carbon metabolism pathway were not significantly associated with CIMP-high status (> or = 6/8 methylated promoters). However, the MTHFR 429 Ala/Ala variant (rs1801131) and the TCN2 259 Arg/Arg variant (rs1801198) were associated with CIMP-high status (MTHFR 429 multivariate odds ratio (MV OR) = 7.56; 95% confidence interval (CI), 1.32-43.3; p trend = 0.10; TCN2 259 Arg/Arg variant MV OR = 3.82; 95% CI, 1.02-14.4; p trend = 0.06). The one-carbon metabolism genotypes were not significantly associated with LINE-1 methylation, although there were modest differences in mean LINE-1 methylation levels between certain genotypes. Collectively, these exploratory data provide suggestive evidence for the association of MTHFR 429 Ala/ Ala and TCN2 259 Arg/Arg and CIMP status in colorectal cancer.

Negative lymph node count is associated with survival of colorectal cancer patients, independent of tumoral molecular alterations and lymphocytic reaction.

OBJECTIVES: The number of recovered lymph nodes is associated with good prognosis among colon cancer patients undergoing surgical resection. However, little has been known on prognostic significance of lymph node count after adjusting for host immune response to tumor and tumoral molecular alterations, both of which are associated with the lymph node count and patient survival. METHODS: Among 716 colorectal cancers (stages 1-4) in two independent prospective cohorts, we examined patient survival in relation to the negative lymph node count and lymph node ratio (LNR; positive to total lymph node counts). Cox proportional hazard models were used to compute hazard ratio of deaths, adjusted for patient, specimen, and tumoral characteristics, including lymphocytic reactions, KRAS and BRAF mutations, p53 expression, microsatellite instability (MSI), the CpG island methylator phenotype (CIMP), and LINE-1 methylation. RESULTS: Compared with patients with 0-3 negative lymph nodes, patients with 7-12 and > or =13 negative nodes experienced a significant reduction in cancer-specific and overall mortality in Kaplan-Meier analysis (log-rank P<0.0001), univariate Cox regression (P(trend)<0.0001), and multivariate analysis (P(trend)<0.0003), independent of potential confounders examined. The benefit associated with the negative node count was apparent across all stages, although the effect was significantly greater in stages 1-2 than stages 3-4 (P(interaction)=0.002). In both stage 3 and stage 4, smaller LNR was associated with improved survival (log-rank P<0.0001). CONCLUSIONS: The negative lymph node count is associated with improved survival of colorectal cancer patients, independent of lymphocytic reactions to tumor and tumoral molecular features including MSI, CIMP, LINE-1 hypomethylation and BRAF mutation.