An Evaluation of a Rural Community-Based Breast Education and Navigation Program: Highlights and Lessons Learned.

Cancer has become the leading cause of death in North Carolina (NC) (North Carolina DHHS, State Center for Health Statistics 2015) and the eastern region of North Carolina (ENC) has experienced greater cancer mortality than the remainder of the state. The Pitt County Breast Wellness Initiative-Education (PCBWI-E) provides culturally tailored breast cancer education and navigation to screening services for uninsured/underinsured women in Pitt and Edgecombe Counties in ENC. PCBWI-E created a network of 23 lay breast health educators, and has educated 735 women on breast health and breast cancer screening guidelines. Navigation services have been provided to 365 women, of which 299 were given breast health assessments, 193 were recommended for a mammogram, and 138 were screened. We have identified five lessons learned to share in the successful implementation of a community-based breast cancer screening intervention: (1) community partnerships are critical for successful community-based cancer screening interventions; (2) assuring access to free or low-cost screening and appropriate follow-up should precede interventions to promote increased use of breast cancer screening; (3) the reduction of system-based barriers is effective in increasing cancer screening; (4) culturally tailored interventions can overcome barriers to screening for diverse racial/ethnic and socioeconomic groups; and (5) multi-component interventions that include multiple community health strategies are effective in increasing screening.

Elevated MicroRNA-33 in Sarcoidosis and a Carbon Nanotube Model of Chronic Granulomatous Disease.

We established a murine model of multiwall carbon nanotube (MWCNT)-induced chronic granulomatous disease, which resembles human sarcoidosis pathology. At 60 days after oropharyngeal MWCNT instillation, bronchoalveolar lavage (BAL) cells from wild-type mice exhibit an M1 phenotype with elevated proinflammatory cytokines and reduced peroxisome proliferator-activated receptor γ (PPARγ)-characteristics also present in human sarcoidosis. Based upon MWCNT-associated PPARγ deficiency, we hypothesized that the PPARγ target gene, ATP-binding cassette (ABC) G1, a lipid transporter with antiinflammatory properties, might also be repressed. Results after MWCNT instillation indicated significantly repressed ABCG1, but, surprisingly, lipid transporter ABCA1 was also repressed, suggesting a possible second pathway. Exploration of potential regulators revealed that microRNA (miR)-33, a lipid transporter regulator, was strikingly elevated (13.9 fold) in BAL cells from MWCNT-instilled mice but not sham control mice. Elevated miR-33 was also detected in murine granulomatous lung tissue. In vitro studies confirmed that lentivirus-miR-33 overexpression repressed both ABCA1 and ABCG1 (but not PPARγ) in cultured murine alveolar macrophages. BAL cells of patients with sarcoidosis also displayed elevated miR-33 together with reduced ABCA1 and ABCG1 messenger RNA and protein compared with healthy control subjects. Moreover, miR-33 was elevated within sarcoidosis granulomatous tissue. The findings suggest that alveolar macrophage miR-33 is up-regulated by proinflammatory cytokines and may perpetuate chronic inflammatory granulomatous disease by repressing antiinflammatory functions of ABCA1 and ABCG1 lipid transporters. The results also suggest two possible pathways for transporter dysregulation in granulomatous disease-one associated with intrinsic PPARγ status and the other with miR-33 up-regulation triggered by environmental challenges, such as MWCNT.

A non-tight junction function of claudin-7-Interaction with integrin signaling in suppressing lung cancer cell proliferation and detachment.

BACKGROUND: Claudins are a family of tight junction (TJ) membrane proteins involved in a broad spectrum of human diseases including cancer. Claudin-7 is a unique TJ membrane protein in that it has a strong basolateral membrane distribution in epithelial cells and in tissues. Therefore, this study aims to investigate the functional significance of this non-TJ localization of claudin-7 in human lung cancer cells. METHODS: Claudin-7 expression was suppressed or deleted by lentivirus shRNA or by targeted-gene deletion. Cell cycle analysis and antibody blocking methods were employed to assay cell proliferation and cell attachment, respectively. Electron microscopy and transepthelial electrical resistance measurement were performed to examine the TJ ultrastructure and barrier function. Co-immunolocalization and co-immunoprecipitation was used to study claudin-7 interaction with integrin ß1. Tumor growth in vivo were analyzed using athymic nude mice. RESULTS: Claudin-7 co-localizes and forms a stable complex with integrin ß1. Both suppressing claudin-7 expression by lentivirus shRNA in human lung cancer cells (KD cells) and deletion of claudin-7 in mouse lungs lead to the reduction in integrin ß1 and phospho-FAK levels. Suppressing claudin-7 expression increases cell growth and cell cycle progression. More significantly, claudin-7 KD cells have severe defects in cell-matrix interactions and adhere poorly to culture plates with a remarkably reduced integrin ß1 expression. When cultured on uncoated glass coverslips, claudin-7 KD cells grow on top of each other and form spheroids while the control cells adhere well and grow as a monolayer. Reintroducing claudin-7 reduces cell proliferation, upregulates integrin ß1 expression and increases cell-matrix adhesion. Integrin ß1 transfection partially rescues the cell attachment defect. When inoculated into nude mice, claudin-7 KD cells produced significantly larger tumors than control cells. CONCLUSION: In this study, we identified a previously unrecognized function of claudin-7 in regulating cell proliferation and maintaining epithelial cell attachment through engaging integrin ß1.

Proteomic identification of tumor- and metastasis-associated galectin-1 in claudin-low breast cancer.

BACKGROUND: Metastasis and mortality remain high among breast cancer patients with the claudin-low subtype because these tumors are aggressive, chemoresistant, and lack targeted therapies. Our objective was to utilize discovery-based proteomics to identify proteins associated with claudin-low primary and metastatic tumors to gain insight into pathways and mechanisms of tumor progression. METHODS: We used nano-LC-MS/MS proteomics to analyze orthotopic and metastatic tumors from the syngeneic murine T11 tumor model, which displays gene expression profiles mirroring human claudin-low tumors. Galectin-1 identity, expression and spatial distribution were investigated by biochemical and immunochemical methods and MALDI/IMS. RNA seq data from mouse and human tumors in our study and publicly available microarray data were analyzed for differential galectin-1 expression across breast cancer subtypes. RESULTS: Galectin-1, an N-acetyllactosamine-binding protein, exhibited the highest sequence coverage and high abundance rank order among nano-LC-MS/MS-identified proteins shared by T11 claudin-low tumors but not normal tissue. Label-free quantitation, Western immunoblot and ELISA confirmed galectin-1 identity and significant differential expression. MALDI/IMS spatial mapping and immunohistochemistry detected galectin-1 in T11 metastatic lung foci. Immunohistochemistry of human claudin-low tumors demonstrated intermediate-to-high intensity galectin-1 staining of tumor and stroma. Gene expression analysis of mouse and human tumors found the highest galectin-1 levels in the claudin-low breast cancer subtype. CONCLUSIONS: Proteomics and genomics reveal high expression of galectin-1 protein and RNA in primary and metastatic claudin-low breast cancer. GENERAL SIGNIFICANCE: This work endorses proteomic approaches in cancer research and supports further investigations of the function and significance of galectin-1 overexpression in claudin-low tumor progression.

Long-term follow-up study of a prospective multicenter sentinel node trial: molecular detection of breast cancer sentinel node metastases.

BACKGROUND: This prospective multicenter sentinel lymph node (SLN) trial investigated whether molecular analysis would improve the detection of SLN metastases and their prognostic value. We report mammaglobin quantitative real-time polymerase chain reaction (qRT-PCR) results and clinical outcome for 547 patients (mean follow-up 7 years). METHODS: Breast cancer patients (excluding stage IV disease or palpable nodes) were enrolled from 1996 to 2005 at 16 institutional review board-approved sites. Alternate 2-mm serial sections of each SLN were examined by hematoxylin and eosin staining with or without immunohistochemistry at multiple levels or blinded and assayed by Taqman qRT-PCR according to previously established thresholds. RESULTS: Mammaglobin remains a highly specific (99%), sensitive (97% primary tumor; 82% N1 SLN) marker for breast cancer. Mammaglobin SLN expression was associated with other prognostic factors, was detected in most patients with distant recurrence (48 of 79; 61%), and was associated with decreased recurrence-free survival (log rank P < 0.0001). Molecular analysis upstaged 13% (52 of 394) node-negative (N0) patients who exhibited a significantly lower distant recurrence-free survival compared to node-negative, PCR-negative patients (80 vs. 91%; P < 0.04). N0 patients with PCR-positive SLN were 3.4 times more likely to experience relapse than PCR-negative patients (odds ratio 3.4; 95% confidence interval 1.6-7.1; P = 0.001). However, molecular staging failed to predict most of the N0 patient recurrences (25 of 34) and was not a statistically significant independent predictor of distant recurrence. CONCLUSIONS: To our knowledge, these data are the first to prospectively compare PCR detection of SLN metastases with long-term outcome in breast cancer patients. Molecular staging of SLN detected clinically significant disease missed by standard pathology. Further refinement and optimization of molecular staging is indicated to improve clinical utility.

Race May Not Impact Endocrine Therapy-Related Changes in Breast Density.

BACKGROUND: Reduction in breast density may be a biomarker of endocrine therapy (ET) efficacy. Our objective was to assess the impact of race on ET-related changes in volumetric breast density (VBD). METHODS: This retrospective cohort study assessed longitudinal changes in VBD measures in women with estrogen receptor-positive invasive breast cancer treated with ET. VBD, the ratio of fibroglandular volume (FGV) to breast volume (BV), was measured using Volpara software. Changes in measurements were evaluated using a multivariable linear mixed effects model. RESULTS: Compared with white women (n = 191), black women (n = 107) had higher rates of obesity [mean ± SD body mass index (BMI) 34.5 ± 9.1 kg/m2 vs. 30.6 ± 7.0 kg/m2, P < 0.001] and premenopausal status (32.7% vs. 16.7%, P = 0.002). Age- and BMI-adjusted baseline FGV, BV, and VBD were similar between groups. Modeled longitudinal changes were also similar: During a follow-up of 30.7 ± 15.0 months (mean ± SD), FGV decreased over time in premenopausal women (slope = -0.323 cm3; SE = 0.093; P = 0.001), BV increased overall (slope = 2.475 cm3; SE = 0.483; P < 0.0001), and VBD decreased (premenopausal slope = -0.063%, SE = 0.011; postmenopausal slope = -0.016%, SE = 0.004; P < 0.0001). Race was not significantly associated with these longitudinal changes, nor did race modify the effect of time on these changes. Higher BMI was associated with lower baseline VBD (P < 0.0001). Among premenopausal women, VBD declined more steeply for women with lower BMI (time × BMI, P = 0.0098). CONCLUSIONS: Race does not appear to impact ET-related longitudinal changes in VBD. IMPACT: Racial disparities in estrogen receptor-positive breast cancer recurrence and mortality may not be explained by differential declines in breast density due to ET.

Therapeutic Effect of Y-27632 on Tumorigenesis and Cisplatin-Induced Peripheral Sensory Loss through RhoA-NF-κB.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect of cancer therapy that frequently requires a reduction or cessation of treatments and negatively impacts the patient's quality of life. There is currently no effective means to prevent or treat CIPN. In this study, we developed and applied CIPN in an immunocompetent, syngeneic murine Lewis Lung Carcinoma (LLCab) model that enabled the elucidation of both tumor and host responses to cisplatin and treatments of Y-27632, a selective inhibitor of Rho kinase/p160ROCK. Y-27632 not only preserved cisplatin's efficacy toward tumor suppression but also the combination treatment inhibited tumor cell proliferation and increased cellular apoptosis. By alleviating the cisplatin-induced loss of epidermal nerve fibers (ENFs), Y-27632 protected tumor-bearing mice from cisplatin-induced reduction of touch sensation. Furthermore, quantitative proteomic analysis revealed the striking cisplatin-induced dysregulation in cellular stress (inflammation, mitochondrial deficiency, DNA repair, etc.)-associated proteins. Y-27632 was able to reverse the changes of these proteins that are associated with Rho GTPase and NF-κB signaling network, and also decreased cisplatin-induced NF-κB hyperactivation in both footpad tissues and tumor. Therefore, Y-27632 is an effective adjuvant in tumor suppression and peripheral neuroprotection. These studies highlight the potential of targeting the RhoA-NF-κB axis as a combination therapy to treat CIPN. IMPLICATIONS: This study, for the first time, demonstrated the dual antineoplastic and neuroprotective effects of Rho kinase/p160ROCK inhibition in a syngeneic immunocompetent tumor-bearing mouse model, opening the door for further clinical adjuvant development of RhoA-NF-κB axis to improve chemotherapeutic outcomes.

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

The Short-Term Effect of Weight Loss Surgery on Volumetric Breast Density and Fibroglandular Volume.

PURPOSE: Obesity and breast density are both associated with an increased risk of breast cancer and are potentially modifiable. Weight loss surgery (WLS) causes a significant reduction in the amount of body fat and a decrease in breast cancer risk. The effect of WLS on breast density and its components has not been documented. Here, we analyze the impact of WLS on volumetric breast density (VBD) and on each of its components (fibroglandular volume and breast volume) by using three-dimensional methods. MATERIALS AND METHODS: Fibroglandular volume, breast volume, and their ratio, the VBD, were calculated from mammograms before and after WLS by using Volpara™ automated software. RESULTS: For the 80 women included, average body mass index decreased from 46.0 ± 7.22 to 33.7 ± 7.06 kg/m2. Mammograms were performed on average 11.6 ± 9.4 months before and 10.1 ± 7 months after WLS. There was a significant reduction in average breast volume (39.4 % decrease) and average fibroglandular volume (15.5 % decrease), and thus, the average VBD increased from 5.15 to 7.87 % (p < 1 × 10-9) after WLS. When stratified by menopausal status and diabetic status, VBD increased significantly in all groups but only perimenopausal and postmenopausal women and non-diabetics experienced a significant reduction in fibroglandular volume. CONCLUSIONS: Breast volume and fibroglandular volume decreased, and VBD increased following WLS, with the most significant change observed in postmenopausal women and non-diabetics. Further studies are warranted to determine how physical and biological alterations in breast density components after WLS may impact breast cancer risk.

Identification and characterization of optimal gene expression markers for detection of breast cancer metastasis.

Sentinel lymph node (SLN) status is highly predictive of overall axillary lymph node involvement in breast cancer. Historically, SLN-positive patients have undergone axillary lymph node dissection in a second surgery. Intraoperative SLN analysis could reduce the cost and complications of a second surgery; however, existing histopathological methods lack standardization and exhibit poor sensitivity. Rapid molecular methods may lead to improved intraoperative diagnosis of SLN metastasis. In this study, we used a genome-wide gene expression analysis of breast and other tissues to identify seven putative markers for detecting breast cancer metastasis. We assessed the utility of these markers for identifying clinically actionable metastases in lymph nodes through reverse transcriptase-polymerase chain reaction analysis of SLNs from 254 breast cancer patients. Polymerase chain reaction signals were compared to pathology on a per-patient basis. The optimal two-gene combination, mammaglobin and cytokeratin 19, detected clinically actionable metastasis in breast SLNs with 90% sensitivity and 94% specificity. Application of stringent criteria for identifying presumptive hematoxylin- and eosin-positive samples increased sensitivity and specificity to 91 and 97%, respectively. This study represents the first comprehensive demonstration of the utility of gene expression markers for detecting clinically actionable breast metastases. An intraoperative molecular assay using these markers has the potential to significantly reduce second surgeries for patients undergoing SLN dissection.

Characterization of Lewis lung clonal variants in a model of syngeneic pulmonary murine metastases.

Lung cancer is the leading cause of cancer-related mortality world-wide. Since the majority of cancer deaths result from metastatic complications, understanding cellular alterations contributing to organ specific metastases is a continuing cancer research goal. Desirable models involve easy, efficient methodologies for development of pulmonary metastases utilizing genetically related syngeneic tumor cell lines varying in clonogenic frequency and growth rate for comparative studies. This work focused on development and characterization of primary and metastatic Lewis lung subclones (LLCC3, LLC1, LLCab) in a histocompatible C57B1/6 model. Surgical resection of primary tumors utilizing these cell lines resulted in reliable development of pulmonary metastases (> 90% of injected mice), while tail-vein injection proved sporadic (20% of injected mice). The preliminary analysis of selected cell-surface molecules indicates potential genetic differences that may underlie phenotypic variations. The combination of subcutaneous resection methodology and variant cell lines results in robust metastatic lung cancer for testing potential therapeutic interventions.

Suppression of Lewis lung tumor development in alpha 1,3 galactosyltransferase knock-out mice.

BACKGROUND: Humans lack the gene alpha 1,3 galactosyltransferase (GalT) and instead produce abundant cytolytic antibodies against cells bearing the antigen [gal alpha1,3 gal] (alphaGal). We have previously studied humoral anti-alphaGal responses in GalT knock-out (GalT KO) mice and shown that murine anti-alphaGal IgM, like human anti-alphaGal IgM, causes extensive complement-mediated cytolysis of GalT+ murine Lewis Lung carcinoma cells (LLCa) in vitro. Here we test the hypothesis that anti-alphaGal immune responses can inhibit the in vivo development of GalT+ tumors. MATERIALS AND METHODS: GalT KO mice orally immunized to produce anti-alphaGal antibodies (n =52) and naïve non-immunized KO mice (n=37) were challenged s.c. with 10(5) LLCa tumor cells. Anti-alphaGal antibody titers were measured before and after LLCa challenge. RESULTS: Anti-alphaGal IgM titers present at challenge correlated with protection from tumor development (p<0.04). Seventy-five percent of mice with titers > or = 1:1280 remained tumor-free versus 43% of naïve mice. Tumor onset was delayed in mice with circulating anti-alphaGal IgM versus naïve animals (p=0.02). LLCa challenge itself induced and augmented anti-alphaGal IgM and post-challenge titers correlated highly with protection from tumor development (p<0.001). No mice with post-challenge anti-alphaGal IgM titers > or = 1:1280 developed tumors, compared to 83% of mice lacking antibody. Inhibition studies showed that 30% of post-challenge IgM recognized LLCa antigens distinct from alphaGal. Anti-alphaGal IgG was low or undetectable both pre- and post challenge and did not affect tumor formation. CONCLUSION: The finding that anti-alphaGal IgM suppresses GaIT+ tumor development in vivo supports the premise that immunotherapy using GalT expression can utilize human anti-alphaGal responses and induce significant anti-tumor effects.

Selective preoperative magnetic resonance imaging in women with breast cancer: no reduction in the reoperation rate.

HYPOTHESIS: The use of preoperative magnetic resonance (MR) imaging may have an effect on the reoperation rate in women with operable breast cancer. DESIGN: Retrospective cohort study. SETTING: University medical center. PATIENTS: Women with operable breast cancer treated by a single surgeon between January 1, 2006, and December 31, 2010. INTERVENTION: Selective preoperative MR imaging based on breast density and histologic findings. MAIN OUTCOME MEASURES: Reoperation rate and pathologically avoidable mastectomy at initial operation. RESULTS: Of 313 patients in the study, 120 underwent preoperative MR imaging. Patients undergoing MR imaging were younger (mean age, 53.6 vs 59.5 years; P < .001), were more often of non-Hispanic white race/ethnicity (61.7% vs 52.3%, P < .05), and more likely had heterogeneously dense or very dense breasts (68.4% vs 22.3%, P < .001). The incidence of lobular carcinoma (8.3% in the MR imaging group vs 5.2% in the no MR imaging group, P = .27) and the type of surgery performed (mastectomy vs partial mastectomy, P = .67) were similar in both groups. The mean pathological size of the index tumor in the MR imaging group was larger than that in the no MR imaging group (2.02 vs 1.72 cm, P = .009), but the extent of disease was comparable (75.8% in the MR imaging group vs 82.9% in the no MR imaging group had pathologically localized disease, P = .26). The reoperation rate was similar between the 2 groups (19.1% in the MR imaging group vs 17.6% in the no MR imaging group, P = .91) even when stratified by breast density (P = .76), pT2 tumor size (P = .35), or lobular carcinoma histologic findings (P = .26). Pathologically avoidable mastectomy (multifocal or multicentric MR imaging and unifocal histopathological findings) was observed in 12 of 47 patients (25.5%) with preoperative MR imaging who underwent mastectomy. CONCLUSION: The selective use of preoperative MR imaging to decrease reoperation in women with breast cancer is not supported by these data. In a considerable number of patients, MR imaging overestimates the extent of disease.

American Society of Clinical Oncology-recommended surveillance and physician specialty among long-term breast cancer survivors.

BACKGROUND: It is unclear whether it is appropriate to transfer the follow-up care of breast cancer (BrCa) survivors from cancer specialists to primary care physicians (PCPs). This contemporary study compared physician specialty and documented the long-term surveillance of survivors who underwent surgery at an American academic center. METHODS: Women in this institutional review board-approved study underwent breast surgery between 1996 and 2006. Data were collected for 270 patients with stage I to III BrCa (mean follow-up, 6 years). Charts were reviewed based on American Society of Clinical Oncology (ASCO) guidelines for recommended surveillance frequency and care. RESULTS: The majority of patients (90%; n = 242) were followed by specialists with 10% (n = 28) followed by PCPs. Patients with advanced disease and a greater risk of disease recurrence more often received specialist care. Patients followed by specialists were more often seen at ASCO-recommended intervals (eg, 89% vs 69% of patients followed by a PCP at follow-up Year 6; P < .01); however, many patients were followed inconsistently. Breast disease was often not the focus of PCP visits or mentioned in clinic notes (18% patients). Women seen by specialists were more likely to have documented clinical examinations of the breast (93% vs 44% at Year 6), axilla (94% vs 52%), or annual mammograms (74% vs 48%; P = .001-.02). CONCLUSIONS: Consistent compliance with surveillance guidelines and chart documentation needs improvement among all providers; however, specialists more consistently met ASCO guidelines. If transfer of care to a PCP occurs, it should be formalized and include follow-up recommendations and defined physician responsibilities. Providers and patients should be educated regarding surveillance care and current guidelines incorporated into standard clinical practice.

Prognostic implications of isolated tumor cells and micrometastases in sentinel nodes of patients with invasive breast cancer: 10-year analysis of patients enrolled in the prospective East Carolina University/Anne Arundel Medical Center Sentinel Node Multicenter Study.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is a more sensitive and accurate nodal staging procedure than axillary lymph node dissection (ALND). Because of increased pathologic evaluation in the sentinel node era, more nodal micrometastases (MIC) (> 0.2 mm to 2 mm) and isolated tumor cells (ITC; < or = 0.2 mm) have been identified. We present the 10-year analysis of our prospective SLN study, focusing on regional axillary node status and distant metastases in patients with nodal ITC and MIC. STUDY DESIGN: From 1996 to 2005, breast cancer patients were enrolled in an Institutional Review Board-approved, multicenter study. SLNs were examined at multiple levels by hematoxylin and eosin; most (85%) hematoxylin and eosin-negative SLNs were also examined by cytokeratin immunohistochemistry. Data from 1,259 patients with invasive breast cancer and in whom an SLN was found were reviewed for this analysis. RESULTS: Of the 1,259 patients, 893 (71%) had negative SLNs, 25 (2%) had ITCs, 57 (5%) had MIC, and 284 (23%) had positive SLNs. None of the 13 patients with ITCs who underwent an ALND had additional positive nodes, compared with 27% (11 of 41) of patients with MIC. At a mean followup of 4.9 years, the distant recurrence rates for SLN-negative, ITC, MIC, and SLN-positive groups were 6%, 8%, 14%, and 21%, respectively. The presence of MIC in the SLN was associated with a significantly shorter disease-free interval than was SLN negativity (p < 0.02 by Cox regression model). CONCLUSIONS: This prospective breast cancer study found that sentinel node MIC, but not ITCs, were associated with additional positive nodes and with distant recurrence. These data suggest that ALND may be unnecessary in patients with ITCs. But ALND and more aggressive adjuvant therapy should be considered in patients with SLN micrometastases.

Clinical and anti-HLA antibody profile of nine renal transplant recipients with failed grafts: donor-specific and non-donor-specific antibody development.

This study applied the single antigen microsphere technology to the retrospective analysis of sequential post-transplant serum samples in the context of the patient's clinical course. Detailed information on nine of the study patients was presented as representative of the larger cohort and illustrative of different patterns of anti-HLA antibody development and different clinical scenarios that culminated in graft failure. Our major observations are summarized as follows: 1. These data confirm the high sensitivity of the single antigen bead method: In some patients, DSA and NDSA that were undetected by standard methods were found pre-transplant and in sequential post-transplant samples. 2. The precise role that anti-HLA antibody plays in a particular rejection are complicated in cases in which humoral rejection is not diagnosed in the biopsy: The possible involvement of ADCC and mechanisms involving an indirect role for antibody in the rejection process should be carefully investigated. 3. Although anti-HLA antibodies are associated with graft rejection, the time interval between detection and rejection can vary dramatically between patients. Both DSA and NDSA can be adsorbed by the graft and erratically detected in the circulation, in some cases remaining undetected until nephrectomy. 4. Anti-HLA antibody strengths often fluctuate widely over a patient's clinical course, with de novo DSA generally of greater strength than de novo NDSA. 5. In addition to DSA, we have observed the consistent induction of diverse, cross-reactive NDSA. This occurs not only during the post-transplant course but also after graft failure, when immunosuppression is tapered prior to nephrectomy. Our data support further studies to evaluate the value of prospective monitoring of anti-HLA antibodies to better understand the place of anti-HLA antibodies in acute rejection. This may improve our ability to reverse some acute rejection episodes. Since acute rejection has been considered a predictor of late graft loss via chronic allograft nephropathy, understanding and modifying the antibody response is critical to extending the longevity of transplanted organs. Finally, since the strong sensitization to NDSA will seriously hamper the ability to identify a compatible donor for a future transplant, these data reinforce the importance of minimizing HLA mismatches between the donor and the recipient.

Induction of cytolytic anti-Gal antibodies in alpha-1,3-galactosyltransferase gene knockout mice by oral inoculation with Escherichia coli O86:B7 bacteria.

Naturally occurring antibodies against [Gal alpha-1,3-Gal] structures (anti-Gal antibodies) are the primary effectors of human hyperacute rejection (HAR) of nonhuman tissue. Unlike most mammals, humans lack a functional alpha-1,3-galactosyltransferase (GalT) gene and produce abundant anti-Gal antibodies, putatively in response to GalT(+) enteric bacteria. GalT knockout (KO) mice have been generated as a small animal model of HAR but inconsistently express anti-Gal antibodies. We hypothesized that enteric exposure of GalT KO mice to live GalT(+) bacteria would produce cytolytic anti-Gal antibodies. Naive mice lacking anti-Gal antibodies were orally immunized with 10(10) live GalT(+) Escherichia coli O86:B7 bacteria and assayed for anti-Gal antibody titer, isotype, and cytolytic activity. Fecal samples were tested for E. coli O86:B7 prior to and after inoculation. In two separate experiments, 77 to 100% (n = 31) of mice developed serum anti-Gal immunoglobulin G (IgG; titer, 1:5 to 1:80) and/or anti-Gal IgM antibodies (titer, 1:5 to 1:1,280) 14 days postinoculation. Induced anti-Gal antibodies caused complement-mediated cytolysis of GalT(+) target cells, with extensive cytolysis observed consistently at serum IgM titers of >/=1:320. Absorption with synthetic [Gal alpha-1,3-Gal] inhibited both antibody binding and cytolysis. E. coli O86:B7 was recovered from stool samples from 83 to 94% of inoculated mice but not from naive mice, thus confirming enteric exposure. These findings demonstrate that oral inoculation with E. coli O86:B7 is a novel and effective method to induce cytolytic anti-Gal antibodies in GalT KO mice and support the premise that enteric exposure to GalT(+) bacteria induces anti-Gal antibodies in humans. These studies also suggest a role for GalT KO mice in elucidating anti-Gal responses in microbial immunity.