Identification of mutation patterns and circulating tumour DNA-derived prognostic markers in advanced breast cancer patients.

BACKGROUND: The correlations between circulating tumour DNA (ctDNA)-derived genomic markers and treatment response and survival outcome in Chinese patients with advanced breast cancer (ABC) have not been extensively characterized. METHODS: Blood samples from 141 ABC patients who underwent first-line standard treatment in Peking University Cancer Hospital were collected. A next-generation sequencing based liquid biopsy assay (PredicineCARE) was used to detect somatic mutations and copy number variations (CNVs) in ctDNA. A subset of matched blood samples and tumour tissue biopsies were compared to evaluate the concordance. RESULTS: Overall, TP53 (44.0%) and PIK3CA (28.4%) were the top two altered genes. Frequent CNVs included amplifications of ERBB2 (24.8%) and FGFR1 (8.5%) and deletions of CDKN2A (3.5%). PIK3CA/TP53 and FGFR1/2/3 variants were associated with drug resistance in hormone receptor-positive (HR +) and human epidermal growth factor receptor 2-positive (HER2 +) patients. The comparison of genomic variants across matched tumour tissue and ctDNA samples revealed a moderate to high concordance that was gene dependent. Triple-negative breast cancer (TNBC) patients harbouring TP53 or PIK3CA alterations had a shorter overall survival than those without corresponding mutations (P = 0.03 and 0.008). A high ctDNA fraction was correlated with a shorter progression-free survival (PFS) (P = 0.005) in TNBC patients. High blood-based tumor mutation burden (bTMB) was associated with a shorter PFS for HER2 + and TNBC patients (P = 0.009 and 0.05). Moreover, disease monitoring revealed several acquired genomic variants such as ESR1 mutations, CDKN2A deletions, and FGFR1 amplifications. CONCLUSIONS: This study revealed the molecular profiles of Chinese patients with ABC and the clinical validity of ctDNA-derived markers, including the ctDNA fraction and bTMB, for predicting treatment response, prognosis, and disease progression. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT03792529. Registered January 3rd 2019, https://clinicaltrials.gov/ct2/show/NCT03792529 .

Multicenter Study of the Accuracy of the BD MAX Multidrug-resistant Tuberculosis Assay for Detection of Mycobacterium tuberculosis Complex and Mutations Associated With Resistance to Rifampin and Isoniazid.

BACKGROUND: Tuberculosis (TB) control is hindered by absence of rapid tests to identify Mycobacterium tuberculosis (MTB) and detect isoniazid (INH) and rifampin (RIF) resistance. We evaluated the accuracy of the BD MAX multidrug-resistant (MDR)-TB assay (BD MAX) in South Africa, Uganda, India, and Peru. METHODS: Outpatient adults with signs/symptoms of pulmonary TB were prospectively enrolled. Sputum smear microscopy and BD MAX were performed on a single raw sputum, which was then processed for culture and phenotypic drug susceptibility testing (DST), BD MAX, and Xpert MTB/RIF (Xpert). RESULTS: 1053 participants with presumptive TB were enrolled (47% female; 32% with human immunodeficiency virus). In patients with confirmed TB, BD MAX sensitivity was 93% (262/282 [95% CI, 89-95%]); specificity was 97% (593/610 [96-98%]) among participants with negative cultures on raw sputa. BD MAX sensitivity was 100% (175/175 [98-100%]) for smear-positive samples (fluorescence microscopy), and 81% (87/107 [73-88%]) in smear-negative samples. Among participants with both BD MAX and Xpert, sensitivity was 91% (249/274 [87-94%]) for BD MAX and 90% (246/274 [86-93%]) for Xpert on processed sputa. Sensitivity and specificity for RIF resistance compared with phenotypic DST were 90% (9/10 [60-98%]) and 95% (211/222 [91-97%]), respectively. Sensitivity and specificity for detection of INH resistance were 82% (22/27 [63-92%]) and 100% (205/205 [98-100%]), respectively. CONCLUSIONS: The BD MAX MDR-TB assay had high sensitivity and specificity for detection of MTB and RIF and INH drug resistance and may be an important tool for rapid detection of TB and MDR-TB globally.

Local estrogen axis in the human bone microenvironment regulates estrogen receptor-positive breast cancer cells.

BACKGROUND: Approximately 70% of all breast cancers express the estrogen receptor, and are regulated by estrogen. While the ovaries are the primary source of estrogen in premenopausal women, most breast cancer is diagnosed following menopause, when systemic levels of this hormone decline. Estrogen production from androgen precursors is catalyzed by the aromatase enzyme. Although aromatase expression and local estrogen production in breast adipose tissue have been implicated in the development of primary breast cancer, the source of estrogen involved in the regulation of estrogen receptor-positive (ER+) metastatic breast cancer progression is less clear. METHODS: Bone is the most common distant site of breast cancer metastasis, particularly for ER+ breast cancers. We employed a co-culture model using trabecular  bone tissues obtained from total hip replacement (THR) surgery specimens to study ER+ and estrogen receptor-negative (ER-) breast cancer cells within the human bone microenvironment. Luciferase-expressing ER+ (MCF-7, T-47D, ZR-75) and ER- (SK-BR-3, MDA-MB-231, MCF-10A) breast cancer cells were cultured directly on bone tissue fragments or in bone tissue-conditioned media, and monitored over time with bioluminescence imaging (BLI). Bone tissue-conditioned media were generated in the presence vs. absence of aromatase inhibitors, and testosterone. Bone tissue fragments were analyzed for aromatase expression by immunohistochemistry. RESULTS: ER+ breast cancer cells were preferentially sustained in co-cultures with bone tissues and bone tissue-conditioned media relative to ER- cells. Bone fragments analyzed by immunohistochemistry revealed expression of the aromatase enzyme. Bone tissue-conditioned media generated in the presence of testosterone had increased estrogen levels and heightened capacity to stimulate ER+ breast cancer cell proliferation. Pretreatment of cultured bone tissues with aromatase inhibitors, which inhibited estrogen production, reduced the capacity of conditioned media to stimulate ER+ cell proliferation. CONCLUSIONS: These results suggest that a local estrogen signaling axis regulates ER+ breast cancer cell viability and proliferation within the bone metastatic niche, and that aromatase inhibitors modulate this axis. Although endocrine therapies are highly effective in the treatment of ER+ breast cancer, resistance to these treatments reduces their efficacy. Characterization of estrogen signaling networks within the bone microenvironment will identify new strategies for combating metastatic progression and endocrine resistance.

Clinicopathologic features, genomic profiles and outcomes of younger vs. older Chinese hormone receptor-positive (HR+)/HER2-negative (HER2-) metastatic breast cancer patients.

Background: Poor outcomes have been widely reported for younger vs. older breast cancer patients, but whether this is due to age itself or the enrichment of aggressive clinical features remains controversial. We have evaluated the clinicopathologic characteristics and genomic profiles of real-world hormone receptor-positive (HR+)/HER2-negative (HER2-) metastatic breast cancer (MBC) patients to examine the determinants of outcome for younger vs. older patients in a single clinical subtype undergoing treatment in the same clinic. Patients and methods: This study included patients presenting at the Peking University Cancer Hospital with primary stage IV or first-line metastatic HR+/HER2- breast cancer who consented to an additional blood draw for genomic profiling prior to treatment. Plasma samples were analyzed with a targeted 152-gene NGS panel to assess somatic circulating tumor DNA (ctDNA) alterations. Genomic DNA (gDNA) extracted from peripheral blood mononuclear cells was analyzed for germline variants using a targeted 600-gene NGS panel. Kaplan-Meier survival analysis was performed to analyze disease free survival (DFS), progression free survival (PFS) and overall survival (OS) in association with clinicopathologic and genomic variables. Results: Sixty-three patients presenting with HR+/HER2- MBC were enrolled in this study. Fourteen patients were < 40 years, 19 were 40-50 years, and 30 were > 50 years at the time of primary cancer diagnosis. No significant associations were observed between age and DFS, PFS or OS. Shorter OS was associated with de novo Stage IV disease (p = 0.002), Luminal B subtype (p = 0.006), high Ki67 index (p = 0.036), resistance to adjuvant endocrine therapy (p = 0.0001) and clinical stage (p = 0.015). Reduced OS was also observed in association with somatic alterations in FGFR1 (p = 0.008), CCND2 (p = 0.012), RB1 (p = 0.029) or TP53 (p = 0.029) genes, but not in association with germline variants. Conclusion: In this group of real-world HR+/HER2- MBC breast cancer patients younger age was not associated with poor outcomes. While current guidelines recommend treatment decisions based on tumor biology rather than age, young HR+ breast cancer patients are more likely to receive chemotherapy. Our findings support the development of biomarker-driven treatment strategies for these patients.

Genomic Complexity Predicts Resistance to Endocrine Therapy and CDK4/6 Inhibition in Hormone Receptor-Positive (HR+)/HER2-Negative Metastatic Breast Cancer.

PURPOSE: Clinical biomarkers to identify patients unlikely to benefit from CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy (ET) are lacking. We implemented a comprehensive circulating tumor DNA (ctDNA) analysis to identify genomic features for predicting and monitoring treatment resistance. EXPERIMENTAL DESIGN: ctDNA was isolated from 216 plasma samples collected from 51 patients with hormone receptor-positive (HR+)/HER2-negative (HER2-) metastatic breast cancer (MBC) on a phase II trial of palbociclib combined with letrozole or fulvestrant (NCT03007979). Boosted whole-exome sequencing (WES) was performed at baseline and clinical progression to evaluate genomic alterations, mutational signatures, and blood tumor mutational burden (bTMB). Low-pass whole-genome sequencing was performed at baseline and serial timepoints to assess blood copy-number burden (bCNB). RESULTS: High bTMB and bCNB were associated with lack of clinical benefit and significantly shorter progression-free survival (PFS) compared with patients with low bTMB or low bCNB (all P < 0.05). Dominant APOBEC signatures were detected at baseline exclusively in cases with high bTMB (5/13, 38.5%) versus low bTMB (0/37, 0%; P = 0.0006). Alterations in ESR1 were enriched in samples with high bTMB (P = 0.0005). There was a high correlation between bTMB determined by WES and bTMB determined using a 600-gene panel (R = 0.98). During serial monitoring, an increase in bCNB score preceded radiographic progression in 12 of 18 (66.7%) patients. CONCLUSIONS: Genomic complexity detected by noninvasive profiling of bTMB and bCNB predicted poor outcomes in patients treated with ET and CDK4/6i and identified early disease progression before imaging. Novel treatment strategies including immunotherapy-based combinations should be investigated in this population.

Dynamic changes in gene alterations during chemotherapy in metastatic castrate resistant prostate cancer.

Docetaxel chemotherapy is a standard treatment option for metastatic castrate resistant prostate cancer (mCRPC) patients. To date, the genomic perturbations underlying the emergence of resistance in mCRPC patients during chemotherapy treatment have not been fully characterized. Previous studies have established that AR, TP53, RB1 and PTEN gene alterations are frequent at this stage of progression and that TP53, RB1 and PTEN, but not AR alterations are associated with poor outcome. However, the clonal dynamics of these key driver cancer genes during chemotherapy in mCRPC patients have not been described. Toward this goal, we performed a retrospective analysis of serially profiled cell-free DNA (cfDNA) alterations in blood samples collected from mCRPC patients before and after starting chemotherapy who were followed for response and clinical outcomes. While AR alterations and measures of mutational load were significantly reduced in patients with stable or decreased PSA levels after 3 cycles of chemotherapy, reductions in RB1, TP53 and PTEN alterations were relatively modest, which may represent the persistence of a clonal signature associated with the emergence of treatment-induced lineage plasticity (TILP) underlying resistance. The ability to monitor these driver gene clonal dynamics during chemotherapy may have utility in the clinical setting.

Circulating Cell-Free DNA-Based Detection of Tumor Suppressor Gene Copy Number Loss and Its Clinical Implication in Metastatic Prostate Cancer.

Current liquid biopsy assays lack sufficient sensitivity to detect copy number loss, which limits the interrogation of critical tumor suppressor gene deletions during cancer progression and treatment. Here we describe a liquid biopsy assay with improved sensitivity for detection of copy number loss in blood samples with low levels of circulating tumor DNA, and demonstrate its utility by profiling PTEN, RB1, and TP53 genetic loss in metastatic prostate cancer patients.

Quantitative Multiplex Immunoassay for Profiling Bone Turnover Biomarkers in Human Bone Tissue Culture Supernatants.

Bone is a metabolically dynamic tissue that is continuously built up and broken down through anabolic and catabolic processes regulated by a variety of systemic and local signaling molecules. Here, we describe quantitative multiplex immunoassay analysis of supernatants collected from cultured human bone tissue fragments to profile local factors associated with the bone turnover process.

Validation of a hierarchical deterministic record-linkage algorithm using data from 2 different cohorts of human immunodeficiency virus-infected persons and mortality databases in Brazil.

Loss to follow-up is a major source of bias in cohorts of patients with human immunodeficiency virus (HIV) and could lead to underestimation of mortality. The authors developed a hierarchical deterministic linkage algorithm to be used primarily with cohorts of HIV-infected persons to recover vital status information for patients lost to follow-up. Data from patients known to be deceased in 2 cohorts in Rio de Janeiro, Brazil, and data from the Rio de Janeiro State mortality database for 1999-2006 were used to validate the algorithm. A fully automated procedure yielded a sensitivity of 92.9% and specificity of 100% when no information was missing. When the automated procedure was combined with clerical review, in a scenario of 5% death prevalence and 20% missing mothers' names, sensitivity reached 96.5% and specificity 100%. In a practical application, the algorithm significantly increased death rates and decreased the rate of loss to follow-up in the cohorts. The finding that 23.9% of matched records did not give HIV or acquired immunodeficiency syndrome as the cause of death reinforces the need to search all-cause mortality databases and alerts for possible underestimation of death rates. These results indicate that the algorithm is accurate enough to recover vital status information on patients lost to follow-up in cohort studies.

Automated Cell Segmentation for Quantitative Phase Microscopy.

Automated cell segmentation and tracking is essential for dynamic studies of cellular morphology, movement, and interactions as well as other cellular behaviors. However, accurate, automated, and easy-to-use cell segmentation remains a challenge, especially in cases of high cell densities, where discrete boundaries are not easily discernable. Here, we present a fully automated segmentation algorithm that iteratively segments cells based on the observed distribution of optical cell volumes measured by quantitative phase microscopy. By fitting these distributions to known probability density functions, we are able to converge on volumetric thresholds that enable valid segmentation cuts. Since each threshold is determined from the observed data itself, virtually no input is needed from the user. We demonstrate the effectiveness of this approach over time using six cell types that display a range of morphologies, and evaluate these cultures over a range of confluencies. Facile dynamic measures of cell mobility and function revealed unique cellular behaviors that relate to tissue origins, state of differentiation, and real-time signaling. These will improve our understanding of multicellular communication and organization.

Xpert MTB/RIF Ultra for detection of Mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre diagnostic accuracy study.

BACKGROUND: The Xpert MTB/RIF assay is an automated molecular test that has improved the detection of tuberculosis and rifampicin resistance, but its sensitivity is inadequate in patients with paucibacillary disease or HIV. Xpert MTB/RIF Ultra (Xpert Ultra) was developed to overcome this limitation. We compared the diagnostic performance of Xpert Ultra with that of Xpert for detection of tuberculosis and rifampicin resistance. METHODS: In this prospective, multicentre, diagnostic accuracy study, we recruited adults with pulmonary tuberculosis symptoms presenting at primary health-care centres and hospitals in eight countries (South Africa, Uganda, Kenya, India, China, Georgia, Belarus, and Brazil). Participants were allocated to the case detection group if no drugs had been taken for tuberculosis in the past 6 months or to the multidrug-resistance risk group if drugs for tuberculosis had been taken in the past 6 months, but drug resistance was suspected. Demographic information, medical history, chest imaging results, and HIV test results were recorded at enrolment, and each participant gave at least three sputum specimen on 2 separate days. Xpert and Xpert Ultra diagnostic performance in the same sputum specimen was compared with culture tests and drug susceptibility testing as reference standards. The primary objectives were to estimate and compare the sensitivity of Xpert Ultra test with that of Xpert for detection of smear-negative tuberculosis and rifampicin resistance and to estimate and compare Xpert Ultra and Xpert specificities for detection of rifampicin resistance. Study participants in the case detection group were included in all analyses, whereas participants in the multidrug-resistance risk group were only included in analyses of rifampicin-resistance detection. FINDINGS: Between Feb 18, and Dec 24, 2016, we enrolled 2368 participants for sputum sampling. 248 participants were excluded from the analysis, and 1753 participants were distributed to the case detection group (n=1439) and the multidrug-resistance risk group (n=314). Sensitivities of Xpert Ultra and Xpert were 63% and 46%, respectively, for the 137 participants with smear-negative and culture-positive sputum (difference of 17%, 95% CI 10 to 24); 90% and 77%, respectively, for the 115 HIV-positive participants with culture-positive sputum (13%, 6·4 to 21); and 88% and 83%, respectively, across all 462 participants with culture-positive sputum (5·4%, 3·3 to 8·0). Specificities of Xpert Ultra and Xpert for case detection were 96% and 98% (-2·7%, -3·9 to -1·7) overall, and 93% and 98% for patients with a history of tuberculosis. Xpert Ultra and Xpert performed similarly in detecting rifampicin resistance. INTERPRETATION: For tuberculosis case detection, sensitivity of Xpert Ultra was superior to that of Xpert in patients with paucibacillary disease and in patients with HIV. However, this increase in sensitivity came at the expense of a decrease in specificity. FUNDING: Government of Netherlands, Government of Australia, Bill & Melinda Gates Foundation, Government of the UK, and the National Institute of Allergy and Infectious Diseases.

The impact of antiretroviral therapy and isoniazid preventive therapy on tuberculosis incidence in HIV-infected patients in Rio de Janeiro, Brazil.

BACKGROUND: Tuberculosis is a common complication and leading cause of death in HIV infection. Antiretroviral therapy (ART) lowers the risk of tuberculosis, but may not be sufficient to control HIV-related tuberculosis. Isoniazid preventive therapy (IPT) reduces tuberculosis incidence significantly, but is not widely used. METHODS: We analysed tuberculosis incidence in 11 026 HIV-infected patients receiving medical care at 29 public clinics in Rio de Janeiro, Brazil, between 1 September 2003 and 1 September 2005. Data were collected through a retrospective medical record review. We determined rates of tuberculosis in patients who received neither ART nor IPT, only ART, only IPT, or both ART and IPT. RESULTS: The overall tuberculosis incidence was 2.28 cases/100 person-years (PY) [95% confidence interval (CI) 2.06-2.52]. Among patients who received neither ART nor IPT, incidence was 4.01/100 PY. Patients who received ART had an incidence of 1.90/100 PY (95% CI 1.66-2.17) and those treated with IPT had a rate of 1.27/100 PY (95% CI 0.41-2.95). The incidence among patients who received ART and IPT was 0.80/100 PY (95% CI 0.38-1.47). Multivariate Cox proportional hazards modeling revealed a 76% reduction in tuberculosis risk among patients receiving both ART and IPT (adjusted relative hazard 0.24; P < 0.001) after adjusting for age, previous tuberculosis diagnosis, and CD4 cell counts at baseline. CONCLUSION: The use of both IPT and ART in HIV-infected patients is associated with significantly reduced tuberculosis incidence. In conjunction with expanded access to ART, the wider use of IPT in patients with HIV will improve tuberculosis control in high burden areas.

The intraductal approach to the breast: raison d'être.

Opportunities for the detection, prediction, and treatment of breast cancer exist at three biological levels: systemically via the blood, at the whole organ level, and within the individual ductal lobular structures of the breast. This review covers the evaluation of approaches targeted to the ductal lobular units, where breast cancer begins. Studies to date suggest the presence of 5 to 12 independent ductal lobular systems per breast, each harboring complex cellular fluids contributed by local and systemic processes. New techniques for accessing and interrogating these systems offer the potential to gauge the microenvironment of the breast and distill biological risk profiles.

Orthonasal and retronasal perception of some green leaf volatiles used in beverage flavors.

Orthonasal perception of six green leaf volatiles (GLVs) classified only hexanal, (E)-2-hexenal, and (Z)-3-hexen-1-yl formate as green. (Z)-3-Hexen-1-yl hexanoate and (Z)-3-hexen-1-yl 3-methylbutyrate were more floral, and (Z)-3-hexen-1-yl acetate fell between the two groups. For retronasal perception, classification along a bipolar green-fruity scale is proposed for describing these GLVs. Data from grouping and dissimilarity tests as well as from sensory profiling show green character for these compounds in standard beverage bases having low Brix or high acidity. As the Brix value increases (or acidity decreases) within the limits encountered in commercial beverages, the character becomes fruity. Several tastant-dependent changes in intensity scores for retronasal descriptors were shown to occur for mixtures of GLVs. The GLVs did not affect intensity scores for gustatory descriptors.

Sweetener/sweetness-induced changes in flavor perception and flavor release of fruity and green character in beverages.

Green leaf volatile (GLV) mixtures, commercial orange flavors, and commercial strawberry flavors were applied to beverage bases in which concentrations of citric acid as well as a sweetener (sucrose or aspartame/acesulfame-K) were varied. Sensory profiling showed that flavor-specific fruity character increased as perceptible sweetness increased, independent of whether the sweetness resulted from sucrose (a change from 9 to 12 Brix) or aspartame/acesulfame-K (a change from 0.2 to 0.4 Brix). Sweetness was affected only by the tastants in the base and not by the flavors, although flavor-specific interactions between sweetener type and sweetener level occurred. Flavor release from the sucrose bases was compared to flavor release from bases containing aspartame/acesulfame-K by static headspace measurements and by MS-Nose measurements using an artificial throat. These measurements showed greater flavor volatility from bases having low Brix (fewer soluble solids). This negative Brix effect was also evident in the sensory data for perception of some GLV green notes. The headspace data could not support a positive Brix effect, the typical salting out, which would correspond to the observed perceptual enhancement of fruity notes.

Quantitative analysis of breast cancer tissue microarrays shows that both high and normal levels of HER2 expression are associated with poor outcome.

Using a tissue microarray cohort of 300 breast cancers and 84 samples of normal breast epithelium, we analyzed HER2/neu expression and compared traditional clinical (manual) scoring with a recently developed system for the quantitative measurement of immunohistochemical stains (AQUA). As expected, both methods identified a population (10-15%) of high-HER2-expressing tumors with poor 30-year disease-related survival. Using AQUA analysis, we found that normal epithelium expresses a low but detectable level of HER2 and that 17.5% of tumors exhibit similar low-level HER2 expression. This low group was not definable by manual scoring. Surprisingly, HER2-normal tumors were as aggressive as HER2-overexpressing tumors. Our studies suggest that in situ quantitative measurement of HER2 stratifies breast tumors into three expression levels: normal, intermediate, and high, where both normal and high levels are associated with a worse outcome.

The Fourth International Symposium on the Intraductal Approach to Breast Cancer, Santa Barbara, California, 10-13 March 2005.

Intraductal approaches encompass procedures and technologies that are designed to access and interrogate the ductal-alveolar systems of the human breast, and include nipple aspiration, ductal lavage, random periareolar fine needle aspiration, and ductoscopy. These approaches are being used to collect and analyze fluids and cells to develop methods for breast cancer detection and risk assessment; to introduce imaging technologies to explore the mammary tree for abnormalities; to administer therapeutic and/or preventive agents directly to the breast tissue; and to explore the biology of the normal mammary gland. The latest research findings in these areas, presented at The 4th International Symposium on the Intraductal Approach to Breast Cancer in 2005, are summarized in this report.

Patterns of reduced nipple aspirate fluid production and ductal lavage cellularity in women at high risk for breast cancer.

INTRODUCTION: Nipple aspiration is a noninvasive technique for obtaining breast fluids from the duct openings of the nipple for the evaluation of abnormalities associated with breast cancer. Nipple aspirate fluid (NAF) can be elicited from 48 to 94% of healthy women, and its production has been linked to an increased relative risk for breast cancer development. NAF production has been used in studies to guide the selection of ducts for ductal lavage, a procedure in which ducts are cannulated and flushed with saline to collect cells. In a previous multicenter trial to evaluate intraductal approaches in women at high-risk for breast cancer, NAF production was observed in 84% of the subjects. However, we observed a significantly lower proportion of fluid-yielding subjects in a similar series of high-risk women. The purpose of the present study was to identify variables associated with this reduction. METHOD: Nipple aspiration was performed on 33 high-risk women (defined as having a 5-year Gail model index of more than 1.7, a personal or family history of breast cancer, and/or a BRCA1 or BRCA2 germline mutation) to identify ductal orifices for lavage procedures. Lavage was performed on all fluid-yielding ducts and on nine non-fluid-yielding ducts. RESULTS: Fluid-yielding ducts were identified in 12 of 33 (36%) of the subjects in the present series, compared with 16 of 19 (84%) of the subjects undergoing identical procedures at our facility during a multicenter trial (P = 0.001). Reduced NAF yields were associated with postmenopausal status (P = 0.02), BRCA germline mutations (P = 0.004), and risk reduction therapies, including bilateral salpingo-oophorectomy (BSO) and/or selective estrogen receptor modulators (SERMs; P = 0.009). All nine (100%) of the ductal lavage specimens collected from non-fluidyielding ducts were acellular, in comparison with 3 of 13 specimens from fluid-yielding ducts (P < .001). CONCLUSION: Analysis of high-risk women in the present series revealed patterns of reduced NAF production and ductal lavage cellularity compared with a previous multicenter trial. The present series included more BRCA-positive women, many of whom had undergone BSO and/or were using SERMs. Our data suggest that endocrine mechanisms associated with these risk-reducing therapies may be related to patterns of diminished breast fluid production.

Characterization of the HER-2/neu oncogene by immunohistochemical and fluorescence in situ hybridization analysis in oral and oropharyngeal squamous cell carcinoma.

PURPOSE: The role of HER-2/neu in squamous cell carcinoma (SCC) of the head and neck is not well defined. The purpose of the current study is to measure the frequency of HER-2/neu expression, to demonstrate HER-2/neu gene amplification in the cases found to be positive for protein overexpression, and to investigate the prognostic significance of overexpression and/or amplification in SCC of the head and neck. EXPERIMENTAL DESIGN: A cohort of 77 patients with SCC of the oral cavity or oropharynx, with stage III or IV disease and uniformly treated with surgical resection and postoperative radiation, served as the primary patient population for the study. Of these, 56 patients had adequate follow-up and paraffin-embedded specimens available for analysis. Median follow-up was 6.1 years. Each of the paraffin-embedded specimens were immunohistochemically stained for HER-2/neu expression and graded for intensity of staining by a pathologist. All cases that demonstrated positive staining by immunohistochemistry were analyzed by fluorescence in situ hybridization (FISH) to assess HER-2/neu amplification status. RESULTS: Five-year survival for the 56 evaluable patients was 40%, with 25% experiencing local relapse, 18% regional relapse, and 25% distant relapse. The percentage of tumors staining positive for HER-2/neu by immunohistochemistry was 17%. There was no statistically significant correlation between HER-2/neu and T stage, N stage, tumor grade, survival, or disease-free survival. HER-2/neu expression did correlate with vascular endothelial growth factor expression. FISH analysis revealed four cases that were amplified for HER-2/neu. Of note, of the 4 amplified cases, 2 suffered regional relapse, 1 suffered distant metastasis, and all 4 expired by 5 years of follow-up. CONCLUSIONS: This is the first demonstration of HER-2/neu gene amplification by FISH in SCC of the head and neck. FISH validates a previously contested controversial role for HER-2/neu gene overexpression in SCC of the head and neck. The prognostic significance and clinical implications of HER-2/neu expression and amplification in head and neck cancer will require additional studies.

Detection of chromosomal instability in paired breast surgery and ductal lavage specimens by interphase fluorescence in situ hybridization.

PURPOSE: Ductal lavage is a new modality for collecting exfoliated breast cells with the goal of detecting early neoplasia. The purpose of our study was to evaluate the correlation between cancer-associated abnormalities in breast lesions and exfoliated breast cells collected by ductal lavage. EXPERIMENTAL DESIGN: We performed histopathologic, cytologic, and molecular cytogenetic analyses on 39 paired cases of surgically excised breast lesions and ductal lavage specimens collected immediately before surgery. RESULTS: Abnormal cytology was detected in 7 of 15 (47%) of the evaluable lavages collected from malignant cases, versus 4 of 19 (21%) of the evaluable lavages harvested from benign cases for a sensitivity and specificity of 47 and 79%, respectively. Interphase fluorescence in situ hybridization analysis of all evaluable lavages revealed numeric changes on chromosomes 1, 8, 11, and/or 17 in 10 of 14 (71%) specimens from malignant cases versus 2 of 18 (11%) from benign cases for a sensitivity and specificity of 71 and 89%, respectively. CONCLUSIONS: Our study demonstrates that cytologic and genetic abnormalities associated with breast cancer progression can be detected in ductal lavage cells collected from women with in situ and invasive breast cancer and suggests that fluorescence in situ hybridization may have superior sensitivity and specificity compared with conventional cytology.