Comparison of umbilical cord tissue-derived mesenchymal stromal cells isolated from cryopreserved material and extracted by explantation and digestion methods utilizing a split manufacturing model.

BACKGROUND AIMS: Umbilical cord (UC) tissue is recognized as an advantageous source of mesenchymal stromal cells (MSCs), whose therapeutic properties are being actively evaluated in pre-clinical and clinical trials. In recognition of its potential value, storage of UC tissue or cells from UC tissue in newborn stem cell banks is now commonplace; however, strategies for isolating UC-derived MSCs (UCMSCs) from UC tissue have not been standardized. The majority of newborn stem cell banks take one of two approaches to cord tissue processing and cryopreservation: enzymatic digestion of the fresh tissue with cryopreservation of the subsequent cell suspension or cryopreservation of the tissue as a composite whole with later, post-thaw isolation of cells by explantation. Evaluation of UCMSCs derived by these two principal preparation and cryopreservation strategies is important to understanding whether the methods currently employed by newborn stem cell banks retain the desirable clinical attributes of UC cells. METHODS: UCMSCs were isolated from 10 UC tissue samples by both explantation and enzymatic digestion methods to allow for comparison of cells from the same donor. Cell isolates from both methods were compared pre- and post-cryopreservation as well as after serial passaging. Cell viability, morphology, growth kinetics, immunophenotype, cytokine secretion and differentiation capacity were evaluated. RESULTS: UCMSCs could be derived from fresh UC tissue by both explantation and digestion methods and from thawed UC tissue by explantation. Initial cell populations isolated by digestion were heterogeneous and took longer to enrich for UCMSCs in culture than populations obtained by explantation. However, once isolated and enriched, UCMSCs obtained by either method showed no significant difference in viability, morphology, rate of proliferation, surface marker expression, levels of cytokine secretion or differentiation capacity. CONCLUSIONS: Derivation of UCMSCs by explantation after thawing UC cryopreserved as a composite tissue may be favorable in terms of initial purity and number of cells achievable by a specific passage. However, we observed no evidence of functional difference between UCMSCs derived by explanation or digestion, suggesting that cells isolated from cryopreserved material obtained by either method maintain their therapeutic properties.

Hypomethylation of LINE-1 in primary tumor has poor prognosis in young breast cancer patients: a retrospective cohort study.

Long interspersed element 1 (LINE-1), a non-coding genomic repeat sequence, methylation status can influence tumor progression. In this study, the clinical significance of LINE-1 methylation status was assessed in primary breast cancer in young versus old breast cancer patients. LINE-1 methylation index (MI) was assessed by absolute quantitative assessment of methylated alleles (AQAMA) PCR assay. Initially, LINE-1 MI was assessed in a preliminary study of 235 tissues representing different stages of ductal breast cancer development. Next, an independent cohort of 379 primary ductal breast cancer patients (median follow-up 18.9 years) was studied. LINE-1 hypomethylation was shown to occur in DCIS and invasive breast cancer. In primary breast cancer it was associated with pathological tumor stage (p = 0.026), lymph node metastasis (p = 0.022), and higher age at diagnosis (>55, p < 0.001). In multivariate analysis, LINE-1 hypomethylation was associated with decreased OS (HR 2.19, 95 % CI 1.17-4.09, log-rank p = 0.014), DFS (HR 2.05, 95 % CI 1.14-3.67, log-rank p = 0.016) and increased DR (HR 2.83, 95 % CI 1.53-5.21, log-rank p = 0.001) in younger (≤55 years), but not older patients (>55 years). LINE-1 analysis of primary breast cancer demonstrated cancer-related age-dependent hypomethylation. In patients ≤55 years, LINE-1 hypomethylation portends a high-risk of DR.

Prognostic value of basal phenotype in HER2-overexpressing breast cancer.

BACKGROUND: Primary breast cancers that overexpress human epidermal growth factor receptor 2 have variable biological features and clinical outcomes. A subgroup of HER2-overexpressing tumors that express basal-like immunohistochemical markers-the so-called basal-HER2+ subtype--is associated with poor prognosis. We investigated the clinical relevance of this basal-HER2+ subtype within HER2-overexpressing breast tumors. METHODS: Database review identified consecutive patients with HER2-overexpressing breast cancer. Archival tumor specimens from these patients were immunostained for estrogen receptor (ER), HER2, and basal cytokeratin (CK) expression, then subtyped as luminal-HER2+ (ER positive and basal CK negative), HER2+ (ER negative and basal CK negative), and basal-HER2+ (ER negative and basal CK positive). Subtypes were correlated with clinicopathologic features and overall survival. RESULTS: Immunohistochemical assessment of 131 HER2-overexpressing breast tumors identified 79 (60%) luminal-HER2+ tumors, 40 (31%) HER2+ tumors, and 12 (9%) basal-HER2+ tumors. There was no difference in the use of adjuvant trastuzumab and chemotherapy among patients with these subtypes. Five-year overall survival was 65% for patients with basal-HER2+ tumors versus 94% (P = 0.0035) and 96% (P = 0.0031) for patients with luminal-HER2+ and HER2+ tumors, respectively. The basal-HER2+ subtype was associated with the worst prognosis after adjusting for age, tumor size, lymph node status, and adjuvant treatment (hazard ratio 5.06, 95% confidence interval 1.1-23.2, P = 0.037). CONCLUSIONS: The basal-HER2+ subtype highlights the heterogeneous biology of HER2-overexpressing breast cancer. The basal-HER2+ subtype is independently associated with poor survival and may provide insight into breast cancer cell response to anti-HER2 therapy.

Basal-like breast cancer defined by FOXC1 expression offers superior prognostic value: a retrospective immunohistochemical study.

BACKGROUND: Basal-like breast cancer (BLBC) has a poor prognosis and is often identified by the triple-negative phenotype (TNP) and/or basal cytokeratins (CKs). Overexpression of mRNA for forkhead box C1 (FOXC1) transcription factor was recently identified as a pivotal prognostic biomarker of BLBC. We investigated the prognostic value of FOXC1 protein expression in invasive breast cancer and compared its prognostic significance to that of TNP and basal CKs. METHODS: Archived TNP specimens of primary invasive ductal breast cancer from 759 patients were examined by immunohistochemical staining for FOXC1, CK5/6, and CK14; prognostic significance was assessed using multivariate analyses. In addition, the impact of adding FOXC1 versus basal CKs to TNP-based BLBC assessment was assessed. RESULTS: FOXC1 protein expression was a significant predictor of overall survival on univariate (hazard ratio [HR] 3.364 95% confidence interval [CI] 1.758-6.438, P = 0.0002) and multivariate (HR 3.389 95% CI 1.928-7.645, P = 0.0001) analyses, despite its correlation with younger age (P = 0.0003). Interestingly, nodal status was not significant on multivariate analysis when FOXC1 expression status was included in the analysis. BLBC defined by TNP plus FOXC1 demonstrated superior prognostic relevance compared to BLBC defined by TNP or TNP plus basal CKs. CONCLUSIONS: Immunohistochemical detection of FOXC1 expression in TNP invasive breast cancer is an independent prognostic indicator that is superior to conventional immunohistochemical surrogates of BLBC. Prospective validation is warranted to further define the diagnostic, prognostic, and predictive utility of FOXC1 in breast cancer management and clinical trial design.

Excessive complement activation is associated with placental injury in patients with antiphospholipid antibodies.

OBJECTIVE: Studies that use a murine model of antiphospholipid syndrome have demonstrated a critical role for complement activation that leads to fetal and placental injury in the presence of antiphospholipid antibodies (APAs). We examined the placentas of patients with APAs to demonstrate a similar association with tissue injury in humans. STUDY DESIGN: Immunohistochemical analyses with the use of antibodies to the complement products C4d, C3b, and C5b-9 were performed on paraffin-embedded tissue sections of placentas from 47 patients with APAs and 23 normal control patients. RESULTS: We found evidence of increased complement deposition in the trophoblast cytoplasm (C4d and C3b), trophoblastic cell and basement membrane (C4d), and extravillous trophoblasts (C4d) of patients with APAs, compared with control patients. We report a correlation between placental pathologic features and complement deposition (C4d) in the trophoblastic cytoplasm, cell membrane, and basement membrane. CONCLUSION: These findings are consistent with murine studies that implicate complement as a critical factor in the fetal tissue injury observed in antiphospholipid syndrome.

Cross-platform comparison of independent datasets identifies an immune signature associated with improved survival in metastatic melanoma.

Platform and study differences in prognostic signatures from metastatic melanoma (MM) gene expression reports often hinder consensus arrival. We performed survival/outcome-based pairwise comparisons of three independent MM gene expression profiles using the threshold-free algorithm rank-rank hypergeometric overlap analysis (RRHO). We found statistically significant overlap for genes overexpressed in favorable outcome (FO) groups, but no overlap for poor outcome (PO) groups. This "favorable outcome signature" (FOS) of 228 genes coinciding on all three overlapping gene lists showed immune function predominated in FO MM. Surprisingly, specific cell signature-enrichment analysis showed B cell-associated genes enriched in FO MM, along with T cell-associated genes. Higher levels of B and T cells (p<0.05) and their relative proximity (p<0.05) were detected in FO-to-PO tumor comparisons from an independent MM patients cohort. Finally, expression of FOS in two independent Stage III MM tumor datasets correctly predicted clinical outcome in 12/14 and 44/70 patients using a weighted gene voting classifier (area under the curve values 0.96 and 0.75, respectively). This RRHO-based, cross-study analysis emphasizes the RRHO approach power, confirms T cells relevance for prolonged MM survival, supports a favorable role for B cells in anti-melanoma immunity, and suggests B cells potential as means of intervention in melanoma treatment.

Personalizing breast cancer staging by the inclusion of ER, PR, and HER2.

IMPORTANCE: Nonanatomic factors, such as histologic grade and biomarkers, can guide breast cancer management but are not included in the current TNM staging system. OBJECTIVE: To use as an example the triple-negative phenotype (TNP) defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) to examine whether such inclusion improves the prognostic accuracy of TNM staging for breast cancer. DESIGN, SETTING, AND PARTICIPANTS: Women diagnosed with primary invasive ductal breast cancer from January 1, 1991, through December 31, 2008, were identified from a prospective institutional database. Excluded were patients who received neoadjuvant therapy, those whose staging information was incomplete, or those whose tumor lacked ER, PR, and HER2 data. Breast cancers were categorized by TNM stage and by the presence or absence of TNP. MAIN OUTCOMES AND MEASURES: Overall survival at 5 years. RESULTS: Database review identified 1842 consecutive eligible patients with breast cancer. When patients were stratified by TNM stage, overall survival curves for those with TNP breast cancer matched those for patients whose non-TNP breast cancer was 1 TNM stage higher. Multivariable analysis showed that TNP status was a powerful prognostic variable, and the likelihood ratio test revealed that the prognostic accuracy of the TNM staging system that incorporated TNP was superior to the current TNM staging system (P< .001). A TNM staging system that incorporated TNP reduced early-stage compression by 15%. CONCLUSIONS AND RELEVANCE: The internationally recognized and easily reproducible examination of ER, PR, and HER2 status exemplifies how nonanatomic factors can improve the prognostic accuracy of breast cancer staging.

Microbial dysbiosis is associated with human breast cancer.

Breast cancer affects one in eight women in their lifetime. Though diet, age and genetic predisposition are established risk factors, the majority of breast cancers have unknown etiology. The human microbiota refers to the collection of microbes inhabiting the human body. Imbalance in microbial communities, or microbial dysbiosis, has been implicated in various human diseases including obesity, diabetes, and colon cancer. Therefore, we investigated the potential role of microbiota in breast cancer by next-generation sequencing using breast tumor tissue and paired normal adjacent tissue from the same patient. In a qualitative survey of the breast microbiota DNA, we found that the bacterium Methylobacterium radiotolerans is relatively enriched in tumor tissue, while the bacterium Sphingomonas yanoikuyae is relatively enriched in paired normal tissue. The relative abundances of these two bacterial species were inversely correlated in paired normal breast tissue but not in tumor tissue, indicating that dysbiosis is associated with breast cancer. Furthermore, the total bacterial DNA load was reduced in tumor versus paired normal and healthy breast tissue as determined by quantitative PCR. Interestingly, bacterial DNA load correlated inversely with advanced disease, a finding that could have broad implications in diagnosis and staging of breast cancer. Lastly, we observed lower basal levels of antibacterial response gene expression in tumor versus healthy breast tissue. Taken together, these data indicate that microbial DNA is present in the breast and that bacteria or their components may influence the local immune microenvironment. Our findings suggest a previously unrecognized link between dysbiosis and breast cancer which has potential diagnostic and therapeutic implications.

Term stillbirth caused by oral Fusobacterium nucleatum.

BACKGROUND: Intrauterine infection is a recognized cause of adverse pregnancy outcome, but the source of infection is often undetermined. We report a case of stillbirth caused by Fusobacterium nucleatum that originated in the mother's mouth. CASE: A woman with pregnancy-associated gingivitis experienced an upper respiratory tract infection at term, followed by stillbirth a few days later. F. nucleatum was isolated from the placenta and the fetus. Examination of different microbial floras from the mother identified the same clone in her subgingival plaque but not in the supragingival plaque, vagina, or rectum. CONCLUSION: F. nucleatum may have translocated from the mother's mouth to the uterus when the immune system was weakened during the respiratory infection. This case sheds light on patient management for those with pregnancy-associated gingivitis.

Immunohistochemical expression of endometrial L-selectin ligand is higher in donor egg recipients with embryonic implantation.

OBJECTIVE: To correlate L-selectin ligand (LSL) expression in human endometrium with embryonic implantation. DESIGN: Retrospective cohort analysis. SETTING: University-based fertility center. PATIENT(S): Donor egg recipients (DERs) who underwent programmed hormonal replacement for ET with prior mock cycle luteal phase endometrial biopsy. INTERVENTION(S): Immunohistochemical expression of LSL using MECA-79 antibody was examined. Slides were scored with a new scoring system, the IHC-Level (range 0-4) as follows: strength of staining-absent (0), weak (1), or strong (2); plus distribution of staining-absent (0), <50% of tissue (1), and >50% (2). Cellular apex and cytoplasm were scored independently in both the endometrial glandular and surface epithelium. MAIN OUTCOME MEASURE(S): Endometrial LSL expression in pregnant versus nonpregnant patients. RESULT(S): MECA-79 IHC-Level of the apex of surface epithelium was significantly higher for pregnant versus nonpregnant DERs (3.8 vs. 3.4). When controlling for embryo morphology, there continues to be a significant difference in apex score on surface epithelium (3.8 vs. 3.3, respectively). The new scoring system results correlated with an established scoring system, the HSCORE. CONCLUSION(S): We demonstrate significantly higher expression of LSL at the apex of human endometrial surface epithelium obtained from DERs with embryonic implantation. Furthermore, we present the IHC-Level, a method of evaluating immunohistochemistry that may be applied to other markers of endometrial receptivity.