AXL/WRNIP1 Mediates Replication Stress Response and Promotes Therapy Resistance and Metachronous Metastasis in HER2+ Breast Cancer.

Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that the nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2+ breast cancer cells that are resistant to HER2-targeted therapy and metastasize to the brain. In preclinical models, knocking down or pharmacologically inhibiting AXL or WRNIP1 attenuated protection of stalled replication forks. Furthermore, deficiency or inhibition of AXL and WRNIP1 also prolonged metastatic latency and delayed relapse. Together, these findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies. SIGNIFICANCE: Nuclear AXL and WRNIP1 interact and mediate replication stress response, promote therapy resistance, and support metastatic progression, indicating that targeting the AXL/WRNIP1 axis is a potentially viable therapeutic strategy for breast cancer.

Deep Learning-Based H-Score Quantification of Immunohistochemistry-Stained Images.

Immunohistochemistry (IHC) is a well-established and commonly used staining method for clinical diagnosis and biomedical research. In most IHC images, the target protein is conjugated with a specific antibody and stained using diaminobenzidine (DAB), resulting in a brown coloration, whereas hematoxylin serves as a blue counterstain for cell nuclei. The protein expression level is quantified through the H-score, calculated from DAB staining intensity within the target cell region. Traditionally, this process requires evaluation by 2 expert pathologists, which is both time consuming and subjective. To enhance the efficiency and accuracy of this process, we have developed an automatic algorithm for quantifying the H-score of IHC images. To characterize protein expression in specific cell regions, a deep learning model for region recognition was trained based on hematoxylin staining only, achieving pixel accuracy for each class ranging from 0.92 to 0.99. Within the desired area, the algorithm categorizes DAB intensity of each pixel as negative, weak, moderate, or strong staining and calculates the final H-score based on the percentage of each intensity category. Overall, this algorithm takes an IHC image as input and directly outputs the H-score within a few seconds, significantly enhancing the speed of IHC image analysis. This automated tool provides H-score quantification with precision and consistency comparable to experienced pathologists but at a significantly reduced cost during IHC diagnostic workups. It holds significant potential to advance biomedical research reliant on IHC staining for protein expression quantification.

NLRP12 downregulates the Wnt/ß-catenin pathway via interaction with STK38 to suppress colorectal cancer.

Colorectal cancer (CRC) at advanced stages is rarely curable, underscoring the importance of exploring the mechanism of CRC progression and invasion. NOD-like receptor family member NLRP12 was shown to suppress colorectal tumorigenesis, but the precise mechanism was unknown. Here, we demonstrate that invasive adenocarcinoma development in Nlrp12-deficient mice is associated with elevated expression of genes involved in proliferation, matrix degradation, and epithelial-mesenchymal transition. Signaling pathway analysis revealed higher activation of the Wnt/ß-catenin pathway, but not NF-κB and MAPK pathways, in the Nlrp12-deficient tumors. Using Nlrp12-conditional knockout mice, we revealed that NLRP12 downregulates ß-catenin activation in intestinal epithelial cells, thereby suppressing colorectal tumorigenesis. Consistent with this, Nlrp12-deficient intestinal organoids and CRC cells showed increased proliferation, accompanied by higher activation of ß-catenin in vitro. With proteomic studies, we identified STK38 as an interacting partner of NLRP12 involved in the inhibition of phosphorylation of GSK3ß, leading to the degradation of ß-catenin. Consistently, the expression of NLRP12 was significantly reduced, while p-GSK3ß and ß-catenin were upregulated in mouse and human colorectal tumor tissues. In summary, NLRP12 is a potent negative regulator of the Wnt/ß-catenin pathway, and the NLRP12/STK38/GSK3ß signaling axis could be a promising therapeutic target for CRC.

Refining the serum miR-371a-3p test for viable germ cell tumor detection.

Circulating miR-371a-3p has excellent performance in the detection of viable (non-teratoma) germ cell tumor (GCT) pre-orchiectomy; however, its ability to detect occult disease is understudied. To refine the serum miR-371a-3p assay in the minimal residual disease setting we compared performance of raw (Cq) and normalized (∆Cq, RQ) values from prior assays, and validated interlaboratory concordance by aliquot swapping. Revised assay performance was determined in a cohort of 32 patients suspected of occult retroperitoneal disease. Assay superiority was determined by comparing resulting receiver-operator characteristic (ROC) curves using the Delong method. Pairwise t-tests were used to test for interlaboratory concordance. Performance was comparable when thresholding based on raw Cq vs. normalized values. Interlaboratory concordance of miR-371a-3p was high, but reference genes miR-30b-5p and cel-miR-39-3p were discordant. Introduction of an indeterminate range of Cq 28-35 with a repeat run for any indeterminate improved assay accuracy from 0.84 to 0.92 in a group of patients suspected of occult GCT. We recommend that serum miR-371a-3p test protocols are updated to (a) utilize threshold-based approaches using raw Cq values, (b) continue to include an endogenous (e.g., miR-30b-5p) and exogenous non-human spike-in (e.g., cel-miR-39-3p) microRNA for quality control, and (c) to re-run any sample with an indeterminate result.

Evaluation of miR-371a-3p to predict viable germ cell tumor in patients with pure seminoma receiving retroperitoneal lymph node dissection.

INTRODUCTION AND OBJECTIVE: Conventional serum tumor markers (STMs) for testicular germ cell tumors (GCTs) offer limited performance with particularly poor sensitivity in cases of minimal residual disease and pure seminoma. While growing evidence has indicated miR-371a-3p to be a superior biomarker, its utility in detecting pure seminoma at recurrence has not been extensively explored. This study's objective was to explore miR-371a-3p's utility in detecting metastatic pure seminoma at retroperitoneal lymph node dissection (RPLND). METHODS: RNA was isolated from patient serum samples collected pre-RPLND. Fifteen patients were assigned to our 'benign' (n = 6) or 'seminoma' (n = 9) group based on pathological confirmation of viable seminoma. Five of the patients received chemotherapy before RPLND (PC-RPLND), and 10 were chemotherapy naïve. MiR-371a-3p expression was quantified via RT-quantitative polymerase chain reaction. The Cq values were statistically evaluated to obtain performance measurements. RESULTS: Median relative expression of miR-371a-3p was higher in the Seminoma group than the Benign, but this difference was not statistically significant (Rq = 3705 and 241, respectively, p = 0.2844). Of the 10 chemotherapy naïve patients, nine had viable seminoma at RPLND, and seven had elevated miR-371a-3p expression. Among the five postchemotherapy (PC) patients, zero had viable GCT at RPLND, and two had elevated miR-371a-3p expression. The primary RPLND group presented 78% sensitivity and 100% specificity. Specificity in the PC-RPLND group was 60%. An optimal Rq threshold of 28.62 was determined by Youden's J statistic, yielding 78% sensitivity and 67% specificity. Receiver operating characteristic analysis provided an AUC of 0.704 (95% CI: 0.43-0.98, p = 0.1949). Despite modest performance, miR-371a-3p exhibited improved sensitivity and specificity compared with conventional STMs. CONCLUSIONS: MiR-371a-3p outperformed STMs in the primary RPLND settings. However, miR-371a-3p was not a robust predictor of pathology in the PC setting. These results suggest that pure seminoma at RPLND is a clinical context, wherein the miRNA assay may require further refinement.

Characterization of Desmoglein 3 (DSG3) as a Sensitive and Specific Marker for Esophageal Squamous Cell Carcinoma.

Although P40 and P63 are both sensitive and specific for routine esophageal squamous cell carcinoma (SCC) diagnosis, we recently showed that P40 and P63 immunoreactivities were significantly lower in well-differentiated SCC than those in higher grade tumors. Therefore, a novel esophageal SCC marker, ideally performing better in well-differentiated SCC, is still needed. We characterized desmoglein 3 (DSG3) immunohistochemistry in esophageal SCC, esophageal adenocarcinoma, small-cell lung carcinoma, and large B-cell lymphoma, alongside P40 and CK5/6. The World Health Organization classification was used to grade tumors as well-differentiated (WD), moderately differentiated (MD), or poorly differentiated (PD). There were 20 WD, 26 MD, and 17 PD components among 39 esophageal SCC cases. All esophageal SCC components showed significant DSG3 immunoreactivity (mean, 80%; range, 30%-100%), and the proportions of DSG3 immunoreactive cells were higher in the WD and MD components than in the PD components. No esophageal adenocarcinoma cases showed more than 10% DSG3 immunoreactivity with only weak cytoplasmic staining. With a 5% immunoreactivity cutoff, DSG3 positivity was 100% in all 63 SCC components, 18% in adenocarcinoma cases, and 0% in small-cell lung carcinoma or large B-cell lymphoma cases. The overall DSG3 specificity was 94%. To the best of our knowledge, this is the first study to characterize DSG3 as a sensitive and specific marker for esophageal SCC.

Serum Small RNA Sequencing and miR-375 Assay Do Not Identify the Presence of Pure Teratoma at Postchemotherapy Retroperitoneal Lymph Node Dissection.

Existing tumor markers for testicular germ cell tumor (TGCT) cannot detect the presence of pure teratoma. Serum miRNAs have strong performance detecting other subtypes of TGCT. Previous reports suggest high levels of miR-375 expression in teratoma tissue. The purpose of this study was to explore the role of serum miRNA, including miR-375, in detecting the presence of teratoma at postchemotherapy retroperitoneal lymph node dissection (PC-RPLND). We prospectively collected presurgical serum from 40 TGCT patients undergoing PC-RPLND (21 with teratoma at RPLND and 19 with no evidence of disease). We examined the utility of serum miR-375-3p and miR-375-5p by quantitative polymerase chain reaction, and searched for other putative serum miRNAs with small RNA sequencing. The area under the receiver operating characteristic curve (AUC) and univariate analyses were utilized to evaluate test characteristics and predictors of teratoma. Both serum miR-375-3p and miR-375-5p exhibited poor performance (miR-375-3p: 86% sensitivity, 32% specificity, AUC: 0.506; miR-375-5p: 55% sensitivity, 67% specificity, AUC: 0.556). Teratoma at orchiectomy was the only predictor of PC-RPLND teratoma. Small RNA sequencing identified three potentially discriminatory miRNAs, but further validation demonstrated no utility. Our results confirm prior reports that serum miR-375 cannot predict teratoma, and suggest that there may not exist a predictive serum miRNA for teratoma.

Real-World Application of Pre-Orchiectomy miR-371a-3p Test in Testicular Germ Cell Tumor Management.

PURPOSE: Current serum tumor markers for testicular germ cell tumor are limited by low sensitivity. Growing evidence supports the use of circulating miR-371a-3p as a superior marker for malignant (viable) germ cell tumor management. We evaluated the real-world application of serum miR-371a-3p levels in detecting viable germ cell tumor among patients undergoing partial or radical orchiectomy. MATERIALS AND METHODS: Serum samples were collected from 69 consecutive patients before orchiectomy. Performance characteristics of serum miR-371a-3p were compared with conventional serum tumor markers (⍺-fetoprotein/ß-human chorionic gonadotropin/lactate dehydrogenase) between patients with viable germ cell tumor and those without viable germ cell tumor on orchiectomy pathology. Relative miR-371a-3p levels were correlated with clinical course. The Kruskal-Wallis test and linear and ordinal regression models were used for analysis. RESULTS: For detecting viable germ cell tumor, combined conventional serum tumor markers had a specificity of 100%, sensitivity of 58% and AUC of 0.79. The miR-371a-3p test showed a specificity of 100%, sensitivity of 93% and AUC of 0.978. Median relative expression of miR-371a-3p in viable germ cell tumor cases was more than 6,800-fold higher than in those lacking viable germ cell tumor. miR-371a-3p levels correlated with composite stage (p=0.006) and, among composite stage I cases, independently associated with embryonal carcinoma percentage (p=0.0012) and tumor diameter (p <0.0001). Six patients underwent orchiectomy after chemotherapy and were correctly predicted to have presence or absence of viable germ cell tumor by the miR-371a-3p test. CONCLUSIONS: If validated, the miR-371a-3p test can be used in conjunction with conventional serum tumor markers to aid clinical decision making. A positive miR-371a-3p test in patients after preoperative chemotherapy or with solitary testes could potentially guide subsequent orchiectomy or observation.

Clinical Significance of Annexin A2 Expression in Breast Cancer Patients.

Increasing evidence suggests that AnxA2 contributes to invasion and metastasis of breast cancer. However, the clinical significance of AnxA2 expression in breast cancer has not been reported. The expression of AnxA2 in cell lines, tumor tissues, and serum samples of breast cancer patients were analyzed by immunoblotting, immunohistochemistry, and enzyme-linked immunosorbent assay, respectively. We found that AnxA2 was significantly upregulated in tumor tissues and serum samples of breast cancer patients compared with normal controls. The high expression of serum AnxA2 was significantly associated with tumor grades and poor survival of the breast cancer patients. Based on molecular subtypes, AnxA2 expression was significantly elevated in tumor tissues and serum samples of triple-negative breast cancer (TNBC) patients compared with other breast cancer subtypes. Our analyses on breast cancer cell lines demonstrated that secretion of AnxA2 is associated with its tyrosine 23 (Tyr23) phosphorylation in cells. The expression of non-phosphomimetic mutant of AnxA2 in HCC1395 cells inhibits its secretion from cells compared to wild-type AnxA2, which further suggest that Tyr23 phosphorylation is a critical step for AnxA2 secretion from TNBC cells. Our analysis of AnxA2 phosphorylation in clinical samples further confirmed that the phosphorylation of AnxA2 at Tyr23 was high in tumor tissues of TNBC patients compared to matched adjacent non-tumorigenic breast tissues. Furthermore, we observed that the diagnostic value of serum AnxA2 was significantly high in TNBC compared with other breast cancer subtypes. These findings suggest that serum AnxA2 concentration could be a potential diagnostic biomarker for TNBC patients.

Correction to: Serum exosomal-annexin A2 is associated with African-American triple-negative breast cancer and promotes angiogenesis.

After publication of the original article [1], we were notified that the wrong version of Fig. 2b has been published.

Glycine by MR spectroscopy is an imaging biomarker of glioma aggressiveness.

BACKGROUND: High-grade gliomas likely remodel the metabolic machinery to meet the increased demands for amino acids and nucleotides during rapid cell proliferation. Glycine, a non-essential amino acid and intermediate of nucleotide biosynthesis, may increase with proliferation. Non-invasive measurement of glycine by magnetic resonance spectroscopy (MRS) was evaluated as an imaging biomarker for assessment of tumor aggressiveness. METHODS: We measured glycine, 2-hydroxyglutarate (2HG), and other tumor-related metabolites in 35 glioma patients using an MRS sequence tailored for co-detection of glycine and 2HG in gadolinium-enhancing and non-enhancing tumor regions on 3T MRI. Glycine and 2HG concentrations as measured by MRS were correlated with tumor cell proliferation (MIB-1 labeling index), expression of mitochondrial serine hydroxymethyltransferase (SHMT2), and glycine decarboxylase (GLDC) enzymes, and patient overall survival. RESULTS: Elevated glycine was strongly associated with presence of gadolinium enhancement, indicating more rapidly proliferative disease. Glycine concentration was positively correlated with MIB-1, and levels higher than 2.5 mM showed significant association with shorter patient survival, irrespective of isocitrate dehydrogenase status. Concentration of 2HG did not correlate with MIB-1 index. A high glycine/2HG concentration ratio, >2.5, was strongly associated with shorter survival (P < 0.0001). GLDC and SHMT2 expression were detectable in all tumors with glycine concentration, demonstrating an inverse correlation with GLDC. CONCLUSIONS: The data suggest that aggressive gliomas reprogram glycine-mediated one-carbon metabolism to meet the biosynthetic demands for rapid cell proliferation. MRS evaluation of glycine provides a non-invasive metabolic imaging biomarker that is predictive of tumor progression and clinical outcome. KEY POINTS: 1. Glycine and 2-hydroxyglutarate in glioma patients are precisely co-detected using MRS at 3T.2. Tumors with elevated glycine proliferate and progress rapidly.3. A high glycine/2HG ratio is predictive of shortened patient survival.

Serum exosomal-annexin A2 is associated with African-American triple-negative breast cancer and promotes angiogenesis.

BACKGROUND: Limited information is available on biomarker(s) for triple-negative breast cancer (TNBC) that can address the higher incidence and aggressiveness of TNBC in African-American (AA) women. Our previous studies have demonstrated annexin A2 (AnxA2) association with exosomes which promotes angiogenesis and metastasis. Therefore, our goal was to examine the expression and function of exosomal-annexin A2 (exo-AnxA2) derived from the serum samples of breast cancer patients. METHODS: The expression of serum exo-AnxA2 and its association with clinicopathological features of the breast cancer patients were determined. The role of serum exo-AnxA2 to promote angiogenesis was determined by an in vivo Matrigel plug assay. RESULTS: Our results show that the expression of serum exo-AnxA2 in breast cancer patients (n = 169; 83.33 ± 2.040 ng/mL, P < 0.0001) is high compared to non-cancer females (n = 68; 34.21 ± 2.238 ng/mL). High expression of exo-AnxA2 levels in breast cancer was significantly associated with tumor grade (P < 0.0001), poor overall survival (hazard ratio (HR) 2.802; 95% confidence intervals (CI) = 1.030-7.620; P = 0.0353), and poor disease-free survival (HR 7.934; 95% CI = 1.778-35.398; P = 0.0301). The expression of serum exo-AnxA2 levels was significantly elevated in TNBC (n = 68; 109.1 ± 2.905 ng/mL; P < 0.0001) in comparison to ER+ (n = 50; 57.35 ± 1.545 ng/mL), HER2+ (n = 59; 78.25 ± 1.146 ng/mL), and non-cancer females (n = 68; 34.21 ± 2.238 ng/mL). Exo-AnxA2 showed diagnostic values with a maximum AUC as 1.000 for TNBC, 0.8304 for ER+, and 0.9958 for HER2+ compared to non-cancer females. The expression of serum exo-AnxA2 was significantly elevated in AA women with TNBC (n = 29; 118.9 ± 4.086 ng/mL, P < 0.0001) in comparison to Caucasian-American TNBC (n = 27; 97.60 ± 3.298 ng/mL) patients. Our in vivo results suggest a role of serum exo-AnxA2 in angiogenesis and its association with aggressiveness of TNBC in AA women. CONCLUSIONS: Our results demonstrated that the expression of serum exo-AnxA2 is high in AA women with TNBC and promotes angiogenesis. These findings suggest that exo-AnxA2 holds promise as a potential prognosticator of TNBC and may lead to an effective therapeutic option.

Identification of breast cancer DNA methylation markers optimized for fine-needle aspiration samples.

BACKGROUND: Random periareolar fine-needle aspiration (RP-FNA) is increasingly used in trials of breast cancer prevention for biomarker assessments. DNA methylation markers may have value as surrogate endpoint biomarkers, but this requires identification of biologically relevant markers suitable for paucicellular, lymphocyte-contaminated clinical samples. METHODS: Unbiased whole-genome 5-aza-2'-deoxycytidine (5AZA)-induced gene expression assays, followed by several phases of qualitative and quantitative methylation-specific PCR (MSP) testing, were used to identify novel breast cancer DNA methylation markers optimized for clinical FNA samples. RESULTS: The initial 5AZA experiment identified 453 genes whose expression was potentially regulated by promoter region methylation. Informatics filters excluded 273 genes unlikely to yield useful DNA methylation markers. MSP assays were designed for 271 of the remaining genes and, ultimately, 33 genes were identified that were differentially methylated in clinical breast cancer samples, as compared with benign RP-FNA samples, and never methylated in lymphocytes. A subset of these markers was validated by quantitative multiplex MSP in extended clinical sample sets. Using a novel permutation method for analysis of quantitative methylation data, PSAT1, GNE, CPNE8, and CXCL14 were found to correlate strongly with specific clinical and pathologic features of breast cancer. In general, our approach identified markers methylated in a smaller subpopulation of tumor cells than those identified in published methylation array studies. CONCLUSIONS: Clinically relevant DNA methylation markers were identified using a 5AZA-induced gene expression approach. IMPACT: These breast cancer-relevant, FNA-optimized DNA methylation markers may have value as surrogate endpoint biomarkers in RP-FNA studies.

The clinical consequences of hemizygosity across 2 MB of 10q23 are restricted to Cowden syndrome.

Cowden syndrome is caused by germline mutations in PTEN and clinically characterized by hamartomas, macrocephaly, classic dermatologic stigmata, and an estimated 85 % lifetime risk of female breast cancer. A young woman with macrocephaly, tricholemmomas, AV malformations, and mammary papillomatosis was found to be hemizygous for PTEN in her germline DNA. Using MLPA, comparative genomic hybridization, and DNA sequencing, we identified a 2-Mb deletion in chromosome 10 spanning 344-kb centromeric and 1.7-Mb telomeric of PTEN. Her father who has a clinical history including macrocephaly, Hashimoto's thyroiditis, colonic polyposis, acral keratoses, and goiter was also found to have the same deletion. In benign breast tissue from the hemizygous female, PTEN protein expression was significantly reduced in luminal and stromal cells but present in the myoepithelium. Compared with a typical papilloma of the breast which had intense cytoplasmic PTEN staining, the majority of the patient's papilloma had significantly decreased PTEN expression while some cells had mislocalized perinuclear PTEN expression. In addition to PTEN, 22 other protein-coding genes were deleted including two predicted haploinsufficient genes and five additional genes that have previously been associated with hereditary predispositions to certain diseases. However, because all significant clinical features of the proband and her father are common to patients with genetic alterations in PTEN, the other 22 hemizygous protein-coding genes appear to be haplosufficient.

Sporadic breast cancer patients' germline DNA exhibit an AT-rich microsatellite signature.

Using a custom CGH-like oligonucleotide array to measure the global microsatellite content in the genomes of 72 cancer, cancer-free, and high risk patient and cell line samples (56 germline DNA and 16 in tumor or tumor cell line DNA) we found a unique, reproducible, and statistically significant pattern of 18 motif-specific microsatellite families (out of 962 possible 1-6 mer repeats) in breast cancer patient germline and tumor DNA, but not in germline DNA of cancer-free volunteer controls or in breast cancer patients with BRCA1/2 mutations. These high-similarity A/T rich repetitive motifs were also more pronounced in the germlines and tumors of colon cancer tumor patients (3/6 samples) and microsatellite unstable colon cancer cell lines; however, germline DNA of sporadic breast cancer patients exhibited the largest global content shift for those motifs with extreme AT/GC ratios. These results indicate that global microsatellite variability is complex, suggest the existence of a previously unknown genomic destabilization mechanism in breast cancer patients' germline DNA, and warrant further testing of such microsatellite variability as a predictor of future breast cancer development.

DNA methylation in benign breast epithelium in relation to age and breast cancer risk.

BACKGROUND: Many established breast cancer risk factors are related to the timing and duration of exposure to reproductive hormones, which are known to drive breast epithelial cell proliferation. The epigenetic molecular clock hypothesis suggests that CpG island methylation records the cell division history of benign epithelium. In proliferative epithelium, such as breast, this may provide an individualized cell-based measure of cancer risk. METHODS: Methylation of cyclin D2, APC, HIN1, RASSF1A, and RAR-beta2 was measured by quantitative multiplex methylation-specific PCR in 290 benign and malignant breast epithelial cell samples obtained by palpation-directed fine-needle aspiration biopsy from 164 women. Univariate, multivariate, and unsupervised cluster analysis was used to establish the relationship between TSG methylation and a personal history of breast cancer, predicted breast cancer risk, and specific breast cancer risk factors. RESULTS: RASSF1A methylation was highly correlated with breast cancer risk [odds ratio (OR), 5.28; 95% confidence interval (95% CI), 1.95-14.32; P = 0.001], atypical cytology (OR, 4.11; 95% CI, 1.30-12.98; P = 0.016), and benign breast disease requiring biopsy (OR, 6.12; 95% CI, 1.41-26.51; P = 0.016). RASSF1A methylation increased linearly between ages 32 and 55. Increasing parity was associated with decreased APC methylation. CONCLUSIONS: TSG methylation increases in benign breast epithelium with increasing age. Because it is independently related to a personal history of benign or malignant breast disease and to predicted breast cancer risk, it may have value for breast cancer risk stratification and as a surrogate endpoint marker in prevention trials.

RASSF1A polymorphism A133S is associated with early onset breast cancer in BRCA1/2 mutation carriers.

The tumor suppressor gene RASSF1A regulates cell cycle progression, apoptosis, and microtubule stability and is inactivated by promoter methylation in approximately 50% of breast cancers. It has been shown previously that the polymorphism A133S in RASSF1A reduces its ability to regulate cell cycle progression and this polymorphism is associated with an increased risk of breast cancer. We analyzed the frequency of RASSF1A A133S in 190 Caucasian women without breast cancer and 653 patients with breast cancer including 138 BRCA1 and BRCA2 (BRCA1/2) mutation carriers, 395 non-BRCA1/2 mutations carriers, and 120 untested for BRCA1/2 mutations. Patients with breast cancer had a higher frequency of A133S than the controls [P = 0.017; odds ratios (OR), 1.71; 95% confidence intervals (95% CI), 1.10-2.66]. There is also a higher frequency of A133S in patients with higher familial breast cancer risk (P = 0.029; OR, 1.76; 95% CI, 1.06-2.92) and patients carrying BRCA1/2 mutations (P = 0.037, OR, 1.82; 95% CI, 1.04-3.18). Importantly, we found that the co-occurrence of a BRCA1 or BRCA2 mutation and A133S in RASSF1A was associated with earlier onset of breast cancer compared with those individuals with either a BRCA1/2 mutation or the A133S polymorphism alone (36.0 versus 42.0 years old, P = 0.002). Our data suggest that the presence of the RASSF1A A133S polymorphism is associated with breast cancer pathogenesis in general and modifies breast cancer age of onset in BRCA1/2 mutations carriers. Our results warrant a large-scale study to examine the effect of the A133S polymorphism in the development of breast and other types of cancers.

Atypia and DNA methylation in nipple duct lavage in relation to predicted breast cancer risk.

BACKGROUND: Tumor suppressor gene (TSG) methylation is identified more frequently in random periareolar fine needle aspiration samples from women at high risk for breast cancer than women at lower risk. It is not known whether TSG methylation or atypia in nipple duct lavage (NDL) samples is related to predicted breast cancer risk. METHODS: 514 NDL samples obtained from 150 women selected to represent a wide range of breast cancer risk were evaluated cytologically and by quantitative multiplex methylation-specific PCR for methylation of cyclin D2, APC, HIN1, RASSF1A, and RAR-beta2. RESULTS: Based on methylation patterns and cytology, NDL retrieved cancer cells from only 9% of breasts ipsilateral to a breast cancer. Methylation of >/=2 genes correlated with marked atypia by univariate analysis, but not multivariate analysis, that adjusted for sample cellularity and risk group classification. Both marked atypia and TSG methylation independently predicted abundant cellularity in multivariate analyses. Discrimination between Gail lower-risk ducts and Gail high-risk ducts was similar for marked atypia [odds ratio (OR), 3.48; P = 0.06] and measures of TSG methylation (OR, 3.51; P = 0.03). However, marked atypia provided better discrimination between Gail lower-risk ducts and ducts contralateral to a breast cancer (OR, 6.91; P = 0.003, compared with methylation OR, 4.21; P = 0.02). CONCLUSIONS: TSG methylation in NDL samples does not predict marked atypia after correcting for sample cellularity and risk group classification. Rather, both methylation and marked atypia are independently associated with highly cellular samples, Gail model risk classifications, and a personal history of breast cancer. This suggests the existence of related, but independent, pathogenic pathways in breast epithelium.

Role of physical activity in modulating breast cancer risk as defined by APC and RASSF1A promoter hypermethylation in nonmalignant breast tissue.

Physical activity reduces breast cancer risk. Promoter hypermethylation of the tumor suppressor genes APC and RASSF1A, which is potentially reversible, is associated with breast cancer risk. We conducted a cross-sectional study in 45 women without breast cancer to determine the association of physical activity with promoter hypermethylation of APC and RASSF1A in breast tissue. We used quantitative methylation-specific PCR to test the methylation status of APC and RASSF1A, and questionnaires to assess study covariates and physical activity (measured in metabolic equivalent hours per week). In univariate analyses, the study covariate, benign breast biopsy number, was positively associated with promoter hypermethylation of APC (P = 0.01) but not RASSF1A. Mulitvariate logistic regression indicated that, although not significant, physical activities for a lifetime [odds ratio (OR), 0.57; 95% confidence interval (95% CI), 0.22-1.45; P = 0.24], previous 5 years (OR, 0.62; 95% CI, 0.34-1.12; P = 0.11), and previous year (OR, 0.72; 95% CI, 0.43-1.22; P = 0.22) were inversely related to promoter hypermethylation of APC but not RASSF1A for all physical activity measures. Univariate logistic regression indicated that physical activities for a lifetime, previous 5 years, and previous year were inversely associated with benign breast biopsy number, and these results were approaching significance for lifetime physical activity (OR, 0.41; 95% CI, 0.16-1.01; P = 0.05) and significant for physical activity in the previous 5 years (OR, 0.57; 95% CI, 0.34-0.94; P = 0.03). The study provides indirect evidence supporting the hypothesis that physical activity is inversely associated with promoter hypermethylation of tumor suppressor genes, such as APC, in nonmalignant breast tissue.