Simon Jacob

Characterization of Common UGT1A8, UGT1A9, and UGT2B7 Variants with Different Capacities to Inactivate Mutagenic 4-Hydroxylated Metabolites of Estradiol and Estrone

The oxidative metabolism of estrone (E1) and estradiol (E2) to form carcinogenic 4-hydroxy-catecholestrogens (4-OHCE) is associated with uterine and breast carcinogenesis. In this study, we conducted functional analyses of genetic variants in the UDP-glucuronosyltransferase UGT1A8, UGT1A9, and UGT2B7 enzymes primarily involved in the inactivation of 4-OHCEs. Compared with UGT2B7*2 (H268Y), UGT2B7*1 exhibited a 2-fold lower efficiency (intrinsic clearance) at conjugating 4-hydroxyestrone and 4-hydroxyestradiol at positions 3 and 4 caused by altered capacities (Vmax) and affinities (Km). The -79 G>A promoter variation, characterizing the UGT2B7*2g haplotype, leads to a 50% reduction of transcription (P < 0.001) in human endometrial carcinoma-1B cells. Furthermore, a >12-fold decreased intrinsic clearance of the *1 proteins was induced by selected amino acid substitutions in UGT1A8 (*3 C277Y) and UGT1A9 (*3 M33T). Frequencies of the low-activity alleles in Caucasians were 45% for UGT2B7*1, 5% for the -79A promoter variant, 1.2% for UGT1A8*3, and 2.2% for UGT1A9*3. Supporting a protective role in two organs sensitive to 4-OHCE-induced damages, the expression of UGT enzymes was shown by immunohistochemistry in normal breast and endometrial tissues and confirmed by Western blotting in a subset of samples. Altogether, findings suggest that specific polymorphisms in UGT genes may modulate the exposure to carcinogenic metabolites of E2 and potentially lead to an altered risk of breast and endometrial cancers in women carrying the variant alleles.

Factors associated with upgrading to malignancy at surgery of atypical ductal hyperplasia diagnosed on core biopsy

Previous studies have shown that 4-54% of breast lesions reported on core biopsies as atypical ductal hyperplasia (ADH) are upgraded on further excision to ductal carcinoma in situ (DCIS) or invasive carcinoma. We evaluated the rate of upgrading ADH to carcinoma at surgery for ADH diagnosed by percutaneous biopsy, and examined characteristics associated with malignancy. We identified 13,488 consecutive biopsies conducted at one center over a nine-year period. A total of 422 biopsies with ADH in 415 patients were included. DCIS or invasive carcinoma was found in 132 cases (31.3% upgrading). Multivariate model revealed that ipsilateral breast symptoms, mammographic lesion other than microcalcifications alone, 14G core needle biopsy, papilloma co-diagnosis, severe ADH and pathologists with lower volume of ADH diagnosis were factors statistically associated with malignancy. However, no subgroups were identified for safe clinical-only follow-up. Surgery is recommended in all cases of ADH diagnosed by percutaneous breast biopsy.

Quantitative DNA methylation analysis of laser capture microdissected formalin-fixed and paraffin-embedded tissues

We developed an assay to quantify DNA methylation in breast cancer cells isolated by laser capture microdissection (LCM). The assay uses methylation sensitive restriction enzyme (MSRE) digestion and quantitative polymerase chain reaction (qPCR). To assess the validity and precision of the assay, we prepared standard samples with expected methylation percentage (MP) for two gene promoters (PLAU (plasminogen inhibitor, urokinase) and TIMP3 (TIMP metallopeptidase inhibitor 3)) that we compared with measured MPs. We found good linearity of MSRE digestion and qPCR procedures for both promoters (beta=0.90-1.19+/-0.05-0.10 and r=0.95-0.98; all P<0.0001). Moreover, results remained similar after addition of a purification step between MSRE digestion and qPCR procedures. The validity of this technique was also confirmed by successfully replicating previously published MPs of four cell lines for PLAU and TIMP3 promoters. We assessed the consistency of our approach by comparing MPs of PLAU and TIMP3 promoters from nine breast cancer patients and two cell lines using LCM frozen tissues and their corresponding formalin-fixed paraffin-embedded tissues. We found good consistency (intraclass correlation coefficient=0.93) of MPs between frozen tissues and formalin-fixed paraffin-embedded tissues. Our data demonstrate that this assay based on digestion with MSRE and qPCR procedures is a good technique to quantify MP on limited amounts of DNA and may find clinical applications.

Validation of a new classifier for the automated analysis of the human epidermal growth factor receptor 2 (HER2) gene amplification in breast cancer specimens

Amplification of the human epidermal growth factor receptor 2 (HER2) is a prognostic marker for poor clinical outcome and a predictive marker for therapeutic response to targeted therapies in breast cancer patients. With the introduction of anti-HER2 therapies, accurate assessment of HER2 status has become essential. Fluorescence in situ hybridization (FISH) is a widely used technique for the determination of HER2 status in breast cancer. However, the manual signal enumeration is time-consuming. Therefore, several companies like MetaSystem have developed automated image analysis software. Some of these signal enumeration software employ the so called “tile-sampling classifier”, a programming algorithm through which the software quantifies fluorescent signals in images on the basis of square tiles of fixed dimensions. Considering that the size of tile does not always correspond to the size of a single tumor cell nucleus, some users argue that this analysis method might not completely reflect the biology of cells. For that reason, MetaSystems has developed a new classifier which is able to recognize nuclei within tissue sections in order to determine the HER2 amplification status on nuclei basis. We call this new programming algorithm “nuclei-sampling classifier”. In this study, we evaluated the accuracy of the “nuclei-sampling classifier” in determining HER2 gene amplification by FISH in nuclei of breast cancer cells. To this aim, we randomly selected from our cohort 64 breast cancer specimens (32 nonamplified and 32 amplified) and we compared results obtained through manual scoring and through this new classifier. The new classifier automatically recognized individual nuclei. The automated analysis was followed by an optional human correction, during which the user interacted with the software in order to improve the selection of cell nuclei automatically selected. Overall concordance between manual scoring and automated nuclei-sampling analysis was 98.4% (100% for nonamplified cases and 96.9% for amplified cases). However, after human correction, concordance between the two methods was 100%. We conclude that the nuclei-based classifier is a new available tool for automated quantitative HER2 FISH signals analysis in nuclei in breast cancer specimen and it can be used for clinical purposes.

Physical activity, mammographic density, and age-related lobular involution among premenopausal and postmenopausal women.

Objective:Physical activity may protect against breast cancer by modulating breast tissue composition. We evaluated the association of physical activity with two visual assessments of breast tissue composition—percentage of mammographic density (a radiologic observation) and age-related lobular involution (a histologic assessment). Methods:Among 164 premenopausal and postmenopausal women with breast cancer, physical activity (household, occupational, and recreational) performed during the year preceding the diagnosis was evaluated using a validated questionnaire. Percentage of mammographic density was assessed in the contralateral breast by a computer-assisted method. Age-related lobular involution was assessed in normal breast tissue on H&E-stained slides. Multivariate generalized linear models were used to assess associations by quartiles of physical activity. Results:Overall, we observed no significant association between total physical activity and percentage of mammographic density or degree of lobular involution. However, occupational physical activity was significantly positively associated with the predominant type I/no type III lobules among premenopausal women (last quartile: prevalence ratio [PR], 5.92; Ptrend = 0.04). Although total physical activity was positively associated with the predominant type I/no type III lobules among premenopausal women (last quartile: PR, 2.61; Ptrend = 0.08), an inverse association was observed among postmenopausal women (last quartile: PR, 0.44; Ptrend = 0.01). Higher levels of household physical activity were significantly associated with higher prevalence of lower mammographic density and complete involution among postmenopausal women (last quartile: PR, 1.21; Ptrend = 0.01). Conclusions:Physical activity may be associated with less dense and more involuted breasts. Physical activitys effect on mammographic density or age-related lobular involution may mediate, in part, its protective effect against breast cancer.

Low Frequency of Cancer Occurrence in Same Breast Quadrant Diagnosed with Lobular Neoplasia at Percutaneous Needle Biopsy

PURPOSE To determine the type of mammographic abnormality leading to needle biopsy of lobular neoplasia (LN) and define the clinical evolution of low-risk LN lesions diagnosed at needle biopsy but not surgically removed. MATERIALS AND METHODS This study was approved by the institutional review board, and the requirement to obtain informed consent was waived. Among 16 945 needle biopsies performed between April 1998 and August 2008, LN was determined to be the most suspicious lesion in 352 samples (2.1%) (pleomorphic and necrotic forms were excluded). Among 299 pure LN lesions that were not surgically removed, follow-up was available for 276 lesions in 275 women. RESULTS Needle biopsy was performed because of mammographic calcifications in 215 of the 276 lesions (77.9%) and because of mammographic masses in 35 (12.7%). The mean follow-up was 5.0 years ± 2.4 (range, 0.6-12.2 years). All 275 women underwent one mammographic follow-up, 205 (74.5%) underwent a second mammographic follow-up, and 147 (53.5%) underwent a third mammographic follow-up. Cancer was diagnosed in 27 of the 275 cases (9.8%) after a mean of 3.9 years ± 2.6 (range, 1.2-10.8 years). Only three cancers (1.1%) occurred in the same breast quadrant as the one originally diagnosed with LN at needle biopsy. CONCLUSION Lumpectomy of pure LN lesions may not prevent malignancy in most cases. Consequently, women with pure LN of a low-risk type diagnosed at needle biopsy are strongly encouraged to undergo a yearly breast clinical examination and yearly mammographic follow-up to detect an eventual cancer in its early stages.

Advantages and disadvantages of technologies for HER2 testing in breast cancer specimens.

OBJECTIVES Human epidermal growth factor receptor 2 (HER2) plays a central role as a prognostic and predictive marker in breast cancer specimens. Reliable HER2 evaluation is central to determine the eligibility of patients with breast cancer to targeted anti-HER2 therapies such as trastuzumab and lapatinib. Presently, several methods exist for the determination of HER2 status at different levels (protein, RNA, and DNA level). METHODS In this review, we discuss the main advantages and disadvantages of the techniques developed so far for the evaluation of HER2 status in breast cancer specimens. RESULTS Each technique has its own advantages and disadvantages. It is therefore not surprising that no consensus has been reached so far on which technique is the best for the determination of HER2 status. CONCLUSIONS Currently, emphasis must be put on standardization of procedures, internal and external quality control assessment, and competency evaluation of already existing methods to ensure accurate, reliable, and clinically meaningful test results. Development of new robust and accurate diagnostic assays should also be encouraged. In addition, large clinical trials are warranted to identify the technique that most reliably predicts a positive response to anti-HER2 drugs.

Does breast cancer tumor size really matter that much

Tumor size should be taken into consideration when planning treatments, but final decisions should also be made on the basis of the biological characteristics of the tumor in order to achieve a personalized approach to each individual cancer and to offer the best possible treatment to each patient.

Association between expression of inflammatory markers in normal breast tissue and mammographic density among premenopausal and postmenopausal women.

Objective: Inflammatory markers may be associated with breast cancer risk. We assessed the association between expression levels of proinflammatory (interleukin 6, tumor necrosis factor-&agr;, C-reactive protein, cyclooxygenase 2, leptin, serum amyloid A1, interleukin 8, and signal transducer and activator of transcription 3) and anti-inflammatory markers (transforming growth factor-&bgr;, interleukin 10, and lactoferrin) in normal breast tissue with mammographic density, a strong breast cancer risk indicator, among 163 breast cancer patients. Methods: The expression of inflammatory markers was visually evaluated on immunohistochemistry stained slides. The percent mammographic density (PMD) was estimated by a computer-assisted method in the contralateral cancer-free breast. We used generalized linear models to estimate means of PMD by median expression levels of the inflammatory markers while adjusting for age and waist circumference. Results: Higher expression levels (above median) of the proinflammatory marker interleukin 6 were associated with higher PMD among all women (24.1% vs 18.5%, P = 0.007). Similarly, higher expression levels (above median) of the proinflammatory markers (interleukin 6, tumor necrosis factor-&agr;, C-reactive protein, and interleukin 8) were associated with higher PMD among premenopausal women (absolute difference in the PMD of 8.8% [P = 0.006], 7.7% [P = 0.022], 6.7% [P = 0.037], and 16.5% [P = 0.032], respectively). Higher expression levels (above median) of the anti-inflammatory marker transforming growth factor-&bgr; were associated with lower PMD among all (18.8% vs 24.3%, P = 0.005) and postmenopausal women (14.5% vs 20.7%, P = 0.013). Conclusions: Our results provide support for the hypothesized role of inflammatory markers in breast carcinogenesis through their effects on mammographic density. Inflammatory markers could be targeted in future breast cancer prevention interventions.

Association between local inflammation and breast tissue age-related lobular involution among premenopausal and postmenopausal breast cancer patients

Increased levels of pro-inflammatory markers and decreased levels of anti-inflammatory markers in the breast tissue can result in local inflammation. We aimed to investigate whether local inflammation in the breast tissue is associated with age-related lobular involution, a process inversely related to breast cancer risk. Levels of eleven pro- and anti-inflammatory markers were assessed by immunohistochemistry in normal breast tissue obtained from 164 pre- and postmenopausal breast cancer patients. Involution status of the breast (degree of lobular involution and the predominant lobule type) was microscopically assessed in normal breast tissue on hematoxylin-eosin stained mastectomy slides. Multivariate generalized linear models were used to assess the associations. In age-adjusted analyses, higher levels of pro-inflammatory markers IL-6, TNF-α, CRP, COX-2, leptin, SAA1 and IL-8; and anti-inflammatory marker IL-10, were inversely associated with the prevalence of complete lobular involution (all P≤0.04). Higher levels of the pro-inflammatory marker COX-2 were also associated with lower prevalence of predominant type 1/no type 3 lobules in the breast, an indicator of complete involution, in age-adjusted analysis (P = 0.017). Higher tissue levels of inflammatory markers, mainly the pro-inflammatory ones, are associated with less involuted breasts and may consequently be associated with an increased risk of developing breast cancer.