Tissue specific DNA methylation in normal human breast epithelium and in breast cancer.

Cancer is a heterogeneous and tissue-specific disease. Thus, the tissue of origin reflects on the natural history of the disease and dictates the therapeutic approach. It is suggested that tissue differentiation, mediated mostly by epigenetic modifications, could guide tissue-specific susceptibility and protective mechanisms against cancer. Here we studied breast specific methylation in purified normal epithelium and its reflection in breast cancers. We established genome wide methylation profiles of various normal epithelial tissues and identified 110 genes that were differentially methylated in normal breast epithelium. A number of these genes also showed methylation alterations in breast cancers. We elaborated on one of them, TRIM29 (ATDC), and showed that its promoter was hypo-methylated in normal breast epithelium and heavily methylated in other normal epithelial tissues. Moreover, in breast carcinomas methylation increased and expression decreased whereas the reverse was noted for multiple other carcinomas. Interestingly, TRIM29 regulation in breast tumors clustered according to the PAM50 classification. Thus, it was repressed in the estrogen receptor positive tumors, particularly in the more proliferative luminal B subtype. This goes in line with previous reports indicating tumor suppressive activity of TRIM29 in estrogen receptor positive luminal breast cells in contrast to oncogenic function in pancreatic and lung cancers. Overall, these findings emphasize the linkage between breast specific epigenetic regulation and tissue specificity of cancer.

Indication for relumpectomy--a useful scoring system in cases of invasive breast cancer.

BACKGROUND AND OBJECTIVES: In two-thirds of breast cancer patients undergoing reoperation no residual tumor will be found. A scoring system for selection of patients who might benefit from relumpectomy is proposed. METHODS: This study is based on 293 patients with invasive breast cancer undergoing reoperation due to margins of <2 mm. Eighteen parameters were evaluated by univariate and multivariate stepwise logistic regression. RESULTS: Univariate analysis identified nine parameters associated with a residual invasive tumor: surgical margins; lobular histological type; grade 3; multifocality; positive lymph modes; non-fine needle localization (FNL) versus FNL lumpectomy; vascular/lymphatic invasion; age <50 years; and tumor size ≥3 cm. Multivariate stepwise logistic regression study identified six out of nine parameters associated with a higher probability of finding a residual invasive tumor: margins <1 mm, multifocality, tumor size ≥3 cm, positive lymph nodes, age <50 years, and lumpectomy without previous FNL. Odds of these factors were used for scoring. CONCLUSIONS: For patients with surgical margins <2 mm and a score of <4, the probability of finding a residual invasive tumor is 0%, while the probability of finding a microfocus of <2 mm of invasive carcinoma is 3.2% and of finding residual DCIS is up to 10%.

A similar cell-specific pattern of HOXA methylation in normal and in cancer tissues.

HOX genes are developmental genes that determine anterior-posterior embryonic pattern and govern the process of differentiation. Inappropriate expression of HOX genes has been implicated in developmental abnormalities and hematopoietic malignancies. In addition, HOX genes silencing by DNA methylation has been reported in cancers and related to disease aggressiveness and outcome. On the other hand, accumulating evidence suggests that epigenetic changes at HOX genes are linked to normal development and differentiation. To better understand the relationship between HOXA methylation and cancer, we analyzed the methylation pattern of HOXA genes in human primary breast and colon carcinomas, normal tissues, and normal white blood cells. Genome-wide methylation arrays of breast cancers and white blood cells demonstrated similar methylation patterns. Quantitative methylation analysis of seven representative HOXA genes revealed various levels of methylation in both normal tissues and cancers. Analysis of epithelial-enriched normal breast tissue and stroma indicated that the stroma was the major origin of HOXA methylation. Furthermore, in selected dense breast cancers, minimal increase in methylation of several HOXA genes did not correlate with the predominance of malignant epithelial cells in these tumors. Our results suggest that methylation of the HOXA cluster may be a normal developmental and cell type specific process rather than a cancer specific mechanism.

Diagnostic performance of a novel device for real-time margin assessment in lumpectomy specimens.

BACKGROUND: Margin status in breast lumpectomy procedures is a prognostic factor for local recurrence and the need to obtain clear margins is often a cause for repeated surgical procedures. A recently developed device for real-time intraoperative margin assessment (MarginProbe; Dune Medical Devices, Caesarea, Israel), was clinically tested. The work presented here looks at the diagnostic performance of the device. METHODS: The device was applied to freshly excised lumpectomy and mastectomy specimens at specific tissue measurement sites. These measurement sites were accurately marked, cut out, and sent for histopathologic analysis. Device readings (positive or negative) were compared with histology findings (namely malignant, containing any microscopically detected tumor, or nonmalignant) on a per measurement site basis. The sensitivity and specificity of the device was computed for the full dataset and for additional relevant subgroups. RESULTS: A total of 869 tissue measurement sites were obtained from 76 patients, 753 were analyzed, of which 165 were cancerous and 588 were nonmalignant. Device performance on relatively homogeneous sites was: sensitivity 1.00 (95% CI: 0.85-1), specificity 0.87 (95% CI: 0.83-0.90). Performance for the full dataset was: sensitivity 0.70 (95% CI: 0.63-0.77), specificity 0.70 (95% CI: 0.67-0.74). Device sensitivity was estimated to change from 56% to 97% as the cancer feature size increased from 0.7 mm to 6.6 mm. Detection rate of samples containing pure DCIS clusters was not different from rates of samples containing IDC. CONCLUSIONS: The device has high sensitivity and specificity in distinguishing between normal and cancer tissue even down to small cancer features.

A prospective, randomized, controlled, multicenter study of a real-time, intraoperative probe for positive margin detection in breast-conserving surgery.

BACKGROUND: This randomized, double-arm trial was designed to study the benefit of a novel device (MarginProbe, Dune Medical Devices, Caesarea, Israel) in intraoperative margin assessment for breast-conserving surgery (BCS) and the associated reduction in reoperations. METHODS: In the device group, the probe was applied to the lumpectomy specimen and additional tissue was excised according to device readings. Study arms were compared by reoperation rates and by correct surgical reaction confirmed by histology. RESULTS: Three hundred patients were enrolled. Device use was associated with improved correct surgical reaction, defined as additional re-excision in all histologically detected positive margins, with tumor within 1 mm of inked margin. The repeat lumpectomy rate was significantly reduced by 56% in the device arm: 5.6% versus 12.7% in the control arm. There were no differences in excised tissue volume or cosmetic outcome. CONCLUSIONS: Intraoperative use of the MarginProbe for positive margin detection is safe and effective in BCS and decreases the rate of repeat operations.

Functional analysis of individual cells and microenvironment of breast cancer-draining lymph nodes.

The development of distant metastases is the major cause of death in breast cancer (BC). In many BC cases, metastases are present in patients with no metastasis-positive lymph nodes (LN). Hence, there is a need to improve prognosis by a better prediction of the nodal status and tumor spread. The current study is designed to develop and utilize new functional characteristics of the cells and microenvironment of BC-draining LN, which may help to improve the estimation of LN metastatic involvement. Innovative devices and methodologies were developed for collecting, transferring, and analyzing LN at an individual-cell resolution. Using these devices, a suspension of living cells were prepared from the LN and processed for various assays, including immunophenotypic analysis, activation status, and invasion activity. The functional profile of tumor-activated LN cells showed an increase in the intracellular enzymatic reaction rate, accompanied by a homogeneous distribution of transferrin receptor as well as by a significant increase in matrix metalloproteinase proteolytic activity. Moreover, the proportion of cells exhibiting such a profile was significantly higher in tumor-containing LN than in tumor-free LN. Thus, the live and postfixation features of LN cells and their microenvironment, correlated with the functional status of the LN, may serve to improve their predictive value in breast cancer examination.

Intracellular esterase activity in living cells may distinguish between metastatic and tumor-free lymph nodes.

BACKGROUND: One of the major clinical problems in breast cancer detection is the relatively high incidence of occult lymph node metastases undetectable by standard procedures. Since the ascertainment of breast cancer stage determines the following treatment, such a "hypo-diagnosis" leads to inadequate therapy, and hence is detrimental for the outcome and survival of the patients. The purpose of our study was to investigate functional metabolic characteristics of living cells derived from metastatic and tumor-free lymph nodes of breast cancer (BC) patients. METHODS: Our methodology is based on the ability of living cells to hydrolyze fluorescein diacetate (FDA) by intracellular esterases and on the association of FDA hydrolysis rates with a specific cell status, both in physiological and pathological conditions. RESULTS: The present study demonstrates a significant difference in the ability to utilize FDA by lymph node cells derived from metastatic and tumor-free lymph nodes in general average, as well as in the metastatic and tumor-free lymph nodes of individual patients. Cells from metastatic lymph nodes had a higher capacity for FDA hydrolysis, and increased this activity after additional activation by autologous tumor tissue (tt). The association between increased FDA hydrolysis rate and activated T lymphocytes and antigen-presenting cells (APC) was shown. CONCLUSION: The results of the present study may contribute to predicting the risk of involvement of seemingly "tumor-free" axillary lymph nodes in occult metastatic processes, and to reducing false-negative results of axillary examination.

Electrical impedance scanning as a new breast cancer risk stratification tool for young women.

BACKGROUND: Electrical impedance scanning (EIS) measures changes in breast tissue associated with breast cancer (Br-Ca) development. The T-Scan(tm2000 (ED is designed to use EIS to identify women ages 30-39 with elevated risk of breast cancer (i.e., T-Scan+ women). AIM: To estimate the relative probability of breast cancer in a T-Scan+ woman compared to a randomly selected young woman. METHODS: A prospective, two-cohort trial was conducted in pre-menopausal women. The Specificity (S(p))-Cohort evaluated T-Scan specificity in 1,751 asymptomatic women ages 30-39. The Sensitivity)S(n))-Cohort evaluated T-Scan sensitivity in 390 women ages 45-30 scheduled for biopsy. Specificity, sensitivity, and conservative estimate of disease prevalence were used to calculate relative probability. RESULTS: In the S(p)-Cohort, 93 of 1,751 women were T-Scan+ (S(p) = 94.7%; 95% CI: 93.7-95.7%). In the S(n)-Cohort, 23 of 87 biopsy-proven cancers were T-Scan+ (S(n) = 26.4%; 95% CI: 17.4-35.4%). Given S(p) = 94.7%, S(n) = 26.4% and prevalence of 1.5 cancers/1,000 women (ages 30-39), the relative probability of a T-Scan+ woman having Br-Ca is 4.95: (95% CI: 3.16-7.14). CONCLUSION: EIS can identify a subset of young women with a relative probability of breast cancer almost five times greater than in the population of young women at-large. T-Scan+ women have a sufficiently high risk of Br-Ca to warrant further surveillance or imaging.

A device for real-time, intraoperative margin assessment in breast-conservation surgery.

BACKGROUND: This trial was designed to study performance of a novel handheld probe (Dune Medical Devices, Caesarea, Israel) in intraoperative detection of positive margins and its potential benefit toward minimizing the positive margin rate. METHODS: The probe was intraoperatively applied to 57 lumpectomy specimens. Surgeons were blinded to device output, and surgical decisions were not affected by probe data. Probe readings were compared with histological analysis per margin and per patient. RESULTS: Nineteen of 22 (86%) pathology-positive patients were intraoperatively detected with device use. Per-margin sensitivity was .71, and specificity was .68, maintained within a range of positive margin definitions (0-.4 cm). CONCLUSIONS: The device is an effective tool for intraoperative detection of positive margins with the potential for significant positive margin rate reduction.

Gastrointestinal carcinomas occurring in breast cancer patients.

Breast cancer patients are reported to have a higher rate of second primary malignancies. We retrospectively reviewed the coexistence of breast and gastrointestinal (GI) tumors in the same patients and the characteristics of the tumors. The charts of all patients more than 35 years of age who were diagnosed with breast cancer and hospitalized for various reasons between 1985 and 2003 were reviewed and those who also had a diagnosis of GI malignancy were then selected. Age and tumor characteristics were evaluated. Out of all the patients, 2,650 had a diagnosis of breast cancer, while 40 (1.5%) also had GI malignancies. Among a comparable group of 70,784 consecutive female patients without breast cancer, 1,292 patients (1.8%) had a diagnosis of GI malignancy. The location of GI tumors in patients with both tumors was as follows: stomach, 6 (15%); right colon, 8 (20%); left colon, 7 (17.5%); sigma, 9 (22.5%); and rectum, 10 (25%). Seventeen of the patients (51.5%) had Dukes C and D tumors, 14 (42.5%) Dukes B, and 2 (6%) Dukes A or in situ. The stage of the others was not identified. The mean age at diagnosis of breast cancer was 68.5 years (range 48-88 years). In 23 (57.5%), GI cancer was diagnosed after breast cancer, in 7 (17.5%) it was diagnosed within 3 months of diagnosing breast cancer, and in 8 (20%) it was diagnosed prior to the diagnosis of breast cancer. Five patients suffered from an additional primary cancer: three endometrial, one lung, one esophageal, and one patient had two additional tumors in the endometrium and thyroid. We conclude that the rate of GI malignancies in breast cancer patients is slightly lower than in comparable patients without breast cancer. GI malignancies tend to be diagnosed later and are found more often in the distal colon.

Ductal carcinoma in situ of the breast in men: a review.

Breast carcinoma in men is rare and comprises approximately 1% of all breast cancer cases. In contrast to the increase in the detection rate of ductal carcinoma in situ (DCIS) in women resulting from the wide use of screening mammography programs, the rate of male DCIS is still small and represents approximately 5% of all male breast cancers. Considerable debate exists concerning the nature of this entity, including the clinical course, pathologic findings, treatment, and prognosis. In this review, the relevant literature dealing with male DCIS is summarized in an attempt to more precisely define the features of this disease.

Mondor's disease of the axilla: a rare complication of sentinel node biopsy.

Three cases of Mondor's disease of the axilla following sentinel lymph node biopsy (SLNB) are described. In all cases we used the combination of blue dye and radiocolloid, and complete axillary dissection was not performed. The numbers of lymph nodes removed in each case were five, four, and two, respectively. All the events of Mondor's disease resolved spontaneously or following a short therapy of anti-inflammatory agents.

The characteristics of malignant breast tumors in hormone replacement therapy users versus nonusers.

BACKGROUND: The purpose of this study was to investigate the characteristics of breast cancer in hormone replacement therapy (HRT) users vs. nonusers. METHODS: We investigated the characteristics of all patients between the ages of 50 and 75 years with breast tumors. Then, an age-adjusted group of 55 nonusers was chosen to match and compare with HRT users. RESULTS: Of the 243 patients available for evaluation, 55 (22.6%) used HRT. Disease stages in HRT users vs. nonusers were as follows: ductal carcinoma in situ (DCIS), 20% and 17.1%; stage I, 45.5% and 41.7%; stage II, 30.9% and 26.2%; stage III, 3.6% and 13.4%; and stage IV, 0% and 1.6% (P =.27). In the age-adjusted cohort, stages in nonusers were as follows: DCIS, 7.3%; stage I, 47.3%; stage II, 25.5%; stage III, 20%; and stage IV, 0% (P =.03). Tumor grades in HRT users vs. nonusers were as follows: grade I, 30.4% and 15.7%; grade II, 52.2% and 52.2%; and grade III, 17.4% and 32.1% (P =.035). Grades in cohort nonusers were as follows: I, 13.2%; II, 52.8%; and III, 34% (P =.05). In the invasive tumors, the positive estrogen receptor (ER) rates were 81.6% and 85.7% (P =.89); positive progesterone receptor (PR) rates were 53.1% and 54% (P =.95); and Her 2-neu positive rates were 18.4% and 17.6% (P =.95), respectively. No significant difference was found in intratumor DCIS, vascular invasion, and Ki-67 (P =.14,.9, and.79, respectively). The rate of lobular and favorable histological types was higher in the HRT user group: 26.6% vs. 15%. CONCLUSIONS: Breast tumors in HRT users vs. nonusers were of a significantly lower stage and grade and accounted for a higher number of favorable histological types, but all other parameters were similar in the two groups.

Breast hamartoma: fine-needle aspiration cytologic finding.

BACKGROUND: Breast hamartoma is an unusual, well-circumscribed, tumor-like mass entering into the differential diagnosis of benign breast disease. To the authors' knowledge, the cytology of these lesions has not been well described. Although fine-needle aspiration is a well established procedure for the detection of breast carcinoma, its utility in classifying benign breast disease is less clear. METHODS: Fine-needle aspirates from eight patients with histologically proven hamartomas were reviewed. None of the cases had a preoperative fine-needle aspiration diagnosis of hamartoma. Cytologic characteristics were retrospectively evaluated in a semiquantitative manner and compared with the histologic findings. RESULTS: The aspirates were moderately cellular and contained sheets of both bland ductal cells and lobular units. Adipose tissue was present in varying amounts. Bipolar stromal nuclei were readily apparent, whereas intact stromal fragments were less prominent. Cytologic atypia was uniformly absent. CONCLUSIONS: The cytology of breast hamartomas shows considerable overlap with other benign breast disease and is unlikely to be interpreted as malignant. The findings of intact lobular units and a relative paucity of stroma in an aspirate from a well circumscribed breast lesion may suggest the diagnosis of hamartoma.

The Usefulness of MIBI Scanning to Detect Underlying Carcinoma in Women with Acute Mastitis.

The efficacy of Tc-99m sestamibi scintimammography in the detection of breast carcinoma has been proven in previous studies. In this study we evaluated the influence of active mammary inflammation on Tc-99m sestamibi scintimammography and its ability to detect breast malignancy in the presence of this condition. Twenty-one women with acute nonpuerperal mastitis underwent breast scintimammography using Tc-99m sestamibi. Their mean age was 49.6 years. In 15 women the scan was positive, 2 suffered from inflammatory carcinoma of the breast, and all the other women had acute mastitis. Six patients had a negative scan, and in all of them, acute mastitis was diagnosed. Total accuracy, sensitivity and specificity in the detection of breast mastitis were 62%, 68%, and 88%, respectively. Scintimammography in patients with acute inflammation of the breast is frequently positive in the absence of malignant condition, therefore it should not be used during acute mastitis for the detection of breast cancer.