Molecular detection of breast cancer cells in the peripheral blood of advanced-stage breast cancer patients using multimarker real-time reverse transcription-polymerase chain reaction and a novel porous barrier density gradient centrifugation technology.

PURPOSE: The goal of this study was to develop a molecular diagnostic assay to detect circulating breast cancer cells in the peripheral blood for the purpose of staging breast cancer. Our aim was to make available an assay that was not limited by the low concentration of circulating breast cancer cells and the background gene expression that is typically found in peripheral blood. EXPERIMENTAL DESIGN: In this study, we investigated the ability of two new technologies to significantly enhance the quantification of gene expression in the peripheral blood: enrichment by a novel porous barrier density gradient centrifugation technology; and multimarker real-time reverse transcription-PCR (RT-PCR). RESULTS: Using fluorescence-labeled breast cancer cells and flow cytometry, we show that processing peripheral blood by porous barrier density gradient centrifugation results in a 300-fold enrichment of breast cancer cells. Real-time RT-PCR analysis confirmed a concomitant reduction in background expression of the CK19 and MUC1 genes after enrichment. In a pilot study, porous barrier density gradient centrifugation and multimarker real-time RT-PCR enabled our laboratory to detect breast cancer-associated gene overexpression in 13 of 20 (65%) stage IV breast cancer patients. Nine of these 14 patients overexpressed three or more markers. CONCLUSIONS: These results confirm the promise of such a molecular diagnostic assay and suggest that additional studies are needed to precisely define the clinical relevance.

A testing algorithm for determination of HER2 status in patients with breast cancer.

The HER2 gene is amplified and overexpressed in 25-30% of breast carcinomas. Assessment of HER2 status for prognosis and treatment of breast cancer patients can be performed by immunohistochemistry and/or fluorescence in situ hybridization (FISH). To develop a testing algorithm for HER2 in breast cancers, we used FISH analysis to determine the HER2 gene copy number and immunostaining to detect the p185 protein. Interlaboratory, interobserver, and intermethod variabilities of immunohistochemistry were assessed. In 24 invasive breast carcinomas, the indices of HER2 status obtained by FISH and by a reference laboratory's DAKO HercepTest (immunostain) gave an overall concordance of 96%. The reference laboratory's stained slides were re-interpreted by an in-house panel of pathologists; the interpretation differed in one case. The panel's interpretations were concordant with the FISH results in all 24 cases. Interobserver variability for the panel's immunohistochemistry interpretations was assessed using three different immunostaining methods on 70 slides. The numerical (0-1+, 2+, 3+) scores showed greater variability among observers than did the overall positive/negative results. One pathologist reported inconsistent results in >30% of the slides evaluated. Borderline scoring of 1-2+ was reported in 18 slides (23%) by at least one observer. Incongruent interobserver immunohistochemistry scores, leading to different positive and negative interpretations, were obtained with 5 slides (7%). The majority of consensus positive cases exhibited strong membrane staining (3+). The majority of consensus negative cases scored as 0. Based on these observations, we developed a testing algorithm that maximizes the benefits of FISH and immunohistochemistry, providing physicians with accurate results for appropriate clinical care.

The relationship between continuity of care and trust with stage of cancer at diagnosis.

BACKGROUND AND OBJECTIVES: While continuity of care has been associated with an increased rate of cancer screening, it is unclear if continuity leads to earlier detection of cancer. This study examined the relationship between continuity of care and trust in one's physician with stage of cancer among newly diagnosed colorectal and breast cancer patients. METHODS: A total of 119 newly diagnosed cancer patients (97 breast, 22 colorectal) were surveyed in face-to-face interviews. The relationship between continuity of care and trust with the patient's primary care physicians prior to diagnosis were examined in relationship to the patient's stage at diagnosis via Spearman correlations and chi-square analyses. A stepwise logistic regression model was computed to examine the best predictors of stage at diagnosis. RESULTS: Half of the patients reported that their cancer was found through screening. Continuity of care prior to diagnosis was related to receiving mammography. Continuity of care was not, however, significantly related to earlier detection. Trust in one's primary care physician was related to earlier detection among both the entire sample of patients with colon and breast cancer and among a subsample of women with breast cancer. In a multivariate model, only detection through screening and trust predicted stage of diagnosis. CONCLUSIONS: Continuity of care is not related to earlier detection of cancer, while trust with a regular physician was associated with earlier detection of cancer.

Adjustment of children and their mothers with breast cancer.

OBJECTIVE: To examine the adjustment of children of mothers with both active and nonactive breast cancers in comparison with a healthy community control sample. METHODS: Participants included 80 mothers and their children. Half of the mothers had breast cancer or a history of breast cancer. Children in both groups ranged in age from 8 to 19 years. Assessments included measures of maternal stressors and resources, maternal and child adjustment and posttraumatic stress, and maternal coping and illness uncertainty reported by both mothers and their children. RESULTS: Few differences were found between the groups, although there was a trend for girls of mothers with breast cancer to have a higher frequency of depressive symptoms. Children of mothers who perceived support from friends and family had fewer depressive symptoms, after we controlled for child gender. CONCLUSIONS: The social support perceived by mothers with breast cancer may serve as a protective factor for their children's psychological adjustment.

Histologic patterns of polyethylene glycol-liposomal doxorubicin-related cutaneous eruptions.

Polyethylene glycol (PEG)-liposomal doxorubicin (Stealth R, Doxil) is a formulation of doxorubicin, which is encapsulated in liposomes formulated with PEG. It is favored in the palliative setting over doxorubicin because of its generally favorable side effect profile. Adverse reactions are predominantly skin eruptions. We report 3 cases of women with breast cancer undergoing treatment with liposomal doxorubicin who developed palmar-plantar erythrodysesthesia and diffuse morbilliform eruptions. Biopsies in the 2 cases demonstrated vacuolar interface dermatitis with epidermal dysmaturation and the third case suggested a drug eruption. Additionally, we report a woman with metastatic breast cancer who developed a similar morbilliform eruption soon after completing a regimen of liposomal doxorubicin. The biopsy revealed an atypical squamous proliferation showing epidermal dysmaturation with focal evidence of interface damage. Both clinician and pathologist alike should be cognizant of this cutaneous eruption, as well as the histologic patterns.

Dietary fat reduction and breast cancer outcome: interim efficacy results from the Women's Intervention Nutrition Study.

BACKGROUND: Preclinical and observational studies suggest a relationship between dietary fat intake and breast cancer, but the association remains controversial. We carried out a randomized, prospective, multicenter clinical trial to test the effect of a dietary intervention designed to reduce fat intake in women with resected, early-stage breast cancer receiving conventional cancer management. METHODS: A total of 2437 women were randomly assigned between February 1994 and January 2001 in a ratio of 40:60 to dietary intervention (n = 975) or control (n = 1462) groups. An interim analysis was performed after a median follow-up of 60 months when funding for the intervention ceased. Mean differences between dietary intervention and control groups in nutrient intakes and anthropometric variables were compared with t tests. Relapse-free survival was examined using Kaplan-Meier analysis, stratified log-rank tests, and Cox proportional hazards models. Statistical tests were two-sided. RESULTS: Dietary fat intake was lower in the intervention than in the control group (fat grams/day at 12 months, 33.3 [95% confidence interval {CI} = 32.2 to 34.5] versus 51.3 [95% CI = 50.0 to 52.7], respectively; P<.001), corresponding to a statistically significant (P = .005), 6-pound lower mean body weight in the intervention group. A total of 277 relapse events (local, regional, distant, or ipsilateral breast cancer recurrence or new contralateral breast cancer) have been reported in 96 of 975 (9.8%) women in the dietary group and 181 of 1462 (12.4%) women in the control group. The hazard ratio of relapse events in the intervention group compared with the control group was 0.76 (95% CI = 0.60 to 0.98, P = .077 for stratified log rank and P = .034 for adjusted Cox model analysis). Exploratory analyses suggested a differential effect of the dietary intervention based on hormonal receptor status. CONCLUSIONS: A lifestyle intervention reducing dietary fat intake, with modest influence on body weight, may improve relapse-free survival of breast cancer patients receiving conventional cancer management. Longer, ongoing nonintervention follow-up will address original protocol design plans, which called for 3 years of follow-up after completion of recruitment.

Altered promoter usage characterizes monoallelic transcription arising with ERBB2 amplification in human breast cancers.

Analysis of a collection of human breast cancers (n = 150), enriched in ERBB2-positive cases (n = 57) and involving tumor genotyping relative to population-matched blood genotyping (n = 749) for a common ERBB2 single nucleotide polymorphism Ala(G)1170Pro(C), revealed that ERBB2 amplification in breast cancer is invariably monoallelic. Analysis of paired breast cancer and blood samples from informative (G1170C heterozygotic) ERBB2-positive (n = 12) and ERBB2-negative (n = 17) cases not only confirmed monoallelic amplification and ERBB2 transcriptional overexpression but also revealed that most low ERBB2 expressing breast cancers (12/17) exhibit unbalanced allelic transcription, showing 3-fold to nearly 5,000-fold preferential expression from one of two inherited alleles. To explore cis-acting transcriptional mechanisms potentially selected during ERBB2 amplification, levels of four different ERBB2 transcript variants (5.2, 4.7, 2.1, and 1.4 kb) were correlated with total (4.6 kb) ERBB2 mRNA levels in ERBB2-positive (n = 14) versus ERBB2-negative (n = 43) primary breast cancers. Relative expression of only the 2.1 kb extracellular domain-encoding splice variant and a 4.7 kb mRNA variant that uses an alternative start site were significantly increased in association with ERBB2-positivity, implicating altered promoter usage and selective transcript regulation within the ERBB2 amplicon. Altogether, these findings provide new mechanistic insights into the development of ERBB2-positive breast cancer and strong rationale for delineating candidate cis-acting regulatory elements that may link allele-specific ERBB2 transcription in premalignant breast epithelia with subsequent development of breast cancers bearing monoallelic ERBB2 amplicons.