Human breast progenitor cell numbers are regulated by WNT and TBX3.

BACKGROUND: Although human breast development is mediated by hormonal and non-hormonal means, the mechanisms that regulate breast progenitor cell activity remain to be clarified. This limited understanding of breast progenitor cells has been due in part to the lack of appropriate model systems to detect and characterize their properties. METHODS: To examine the effects of WNT signaling and TBX3 expression on progenitor activity in the breast, primary human mammary epithelial cells (MEC) were isolated from reduction mammoplasty tissues and transduced with lentivirus to overexpress WNT1 or TBX3 or reduce expression of their cognate receptors using shRNA. Changes in progenitor activity were quantified using characterized assays. We identified WNT family members expressed by cell populations within the epithelium and assessed alterations in expression of WNT family ligands by MECs in response to TBX3 overexpression and treatment with estrogen and progesterone. RESULTS: Growth of MECs on collagen gels resulted in the formation of distinct luminal acinar and basal ductal colonies. Overexpression of TBX3 in MECs resulted in increased ductal colonies, while shTBX3 expression diminished both colony types. Increased WNT1 expression led to enhanced acinar colony formation, shLRP6 decreased both types of colonies. Estrogen stimulated the formation of acinar colonies in control MEC, but not shLRP6 MEC. Formation of ductal colonies was enhanced in response to progesterone. However, while shLRP6 decreased MEC responsiveness to progesterone, shTBX3 expression did not alter this response. CONCLUSIONS: We identified two phenotypically distinguishable lineage-committed progenitor cells that contribute to different structural elements and are regulated via hormonal and non-hormonal mechanisms. WNT signaling regulates both types of progenitor activity. Progesterone favors the expansion of ductal progenitor cells, while estrogen stimulates the expansion of acinar progenitor cells. Paracrine WNT signaling is stimulated by estrogen and progesterone, while autocrine WNT signaling is induced by the embryonic T-box transcription factor TBX3.

Pregnancy-associated breast cancers are driven by differences in adipose stromal cells present during lactation.

INTRODUCTION: The prognosis of breast cancer is strongly influenced by the developmental stage of the breast when the tumor is diagnosed. Pregnancy-associated breast cancers (PABCs), cancers diagnosed during pregnancy, lactation, or in the first postpartum year, are typically found at an advanced stage, are more aggressive and have a poorer prognosis. Although the systemic and microenvironmental changes that occur during post-partum involution have been best recognized for their role in the pathogenesis of PABCs, epidemiological data indicate that PABCs diagnosed during lactation have an overall poorer prognosis than those diagnosed during involution. Thus, the physiologic and/or biological events during lactation may have a significant and unrecognized role in the pathobiology of PABCs. METHODS: Syngeneic in vivo mouse models of PABC were used to examine the effects of system and stromal factors during pregnancy, lactation and involution on mammary tumorigenesis. Mammary adipose stromal cell (ASC) populations were isolated from mammary glands and examined by using a combination of in vitro and in vivo functional assays, gene expression analysis, and molecular and cellular assays. Specific findings were further investigated by immunohistochemistry in mammary glands of mice as well as in functional studies using ASCs from lactating mammary glands. Additional findings were further investigated using human clinical samples, human stromal cells and using in vivo xenograft assays. RESULTS: ASCs present during lactation (ASC-Ls), but not during other mammary developmental stages, promote the growth of carcinoma cells and angiogenesis. ASCs-Ls are distinguished by their elevated expression of cellular retinoic acid binding protein-1 (crabp1), which regulates their ability to retain lipid. Human breast carcinoma-associated fibroblasts (CAFs) exhibit traits of ASC-Ls and express crabp1. Inhibition of crabp1in CAFs or in ASC-Ls abolished their tumor-promoting activity and also restored their ability to accumulate lipid. CONCLUSIONS: These findings imply that (1) PABC is a complex disease, which likely has different etiologies when diagnosed during different stages of pregnancy; (2) both systemic and local factors are important for the pathobiology of PABCs; and (3) the stromal changes during lactation play a distinct and important role in the etiology and pathogenesis of PABCs that differ from those during post-lactational involution.

Correlation between histologic assessment and fluorescence in situ hybridization using MelanoSITE in evaluation of histologically ambiguous melanocytic lesions.

CONTEXT: The 4-probe, multicolor, fluorescence in situ hybridization (FISH) panel targeting chromosomes 6 and 11 has shown promising sensitivity and specificity in distinguishing between benign nevi and malignant melanoma. Only a few studies have assessed the potential utility of FISH in classification of histologically ambiguous melanocytic lesions. In the United States, this assay is exclusively licensed to NeoGenomics Laboratories (Irvine, California), which provides the technical component and has developed an innovative service (MelanoSITE) allowing pathologists to interpret FISH results using a dedicated Web portal. Thus far, use of MelanoSITE as a diagnostic adjunct in the diagnosis of melanocytic lesions has not, to our knowledge, been reported in the literature. OBJECTIVE: To analyze 1.5 years of experience with the MelanoSITE melanoma FISH assay in the evaluation of histologically ambiguous lesions in the context of second opinion and routine dermatopathology practice. DESIGN: A prospective histologic/FISH correlation study of 140 cases. RESULTS: Twenty-seven percent of abnormal FISH results were false-positive results because of tetraploidy. After correcting for known false-positive results, all lesions considered atypical nevi showed normal FISH signals. Abnormal FISH signals were reported in 30% of lesions considered histologically borderline and in 48% of lesions in which a diagnosis of melanoma was favored. CONCLUSIONS: Four-probe, multicolor FISH results for melanoma correlate with the microscopic assessments of histologically ambiguous lesions. Pathologists using MelanoSITE must be aware of the high rate of false-positive results from tetraploidy.

Molecular grading of tumors of the upper urothelial tract using FGFR3 mutation status identifies patients with favorable prognosis.

BACKGROUND: Mutations in FGFR3 have been shown to occur in tumors of the upper urothelial tract and may be indicative of a good prognosis. In bladder tumors, the combination of FGFR3 mutation status and Ki-67 level has been used to define a tumor's molecular grade and predict survival. Pathological evaluation of upper urothelial tumors is currently the best predictor of prognosis, but suffers from variability in pathological assessments. This study investigated the association with prognosis of FGFR3 mutations alone and in combination with Ki-67 in this patient population. METHODS: Genomic DNA was isolated from tumor samples of 80 patients with upper urothelial cancer. The presence of mutation in FGFR3 was evaluated using real-time polymerase chain reaction. Ki-67 protein expression was determined by immunohistochemistry. Kaplan-Meier survival analysis evaluated the relationship of FGFR3 mutations and Ki-67 levels with survival. RESULTS: FGFR3 mutations were identified in 40% of tumors and were predominantly associated with noninvasive tumors. Overall survival was higher in patients with FGFR3 mutant tumors (P = 0.02) and in molecular grade 1 tumors as determined by FGFR3 and Ki-67 (P = 0.02). CONCLUSION: In this study, we confirm the occurrence of FGFR3 mutations in tumors of the upper urothelial tract and its association with a good prognosis. Both FGFR3 and molecular grading are predictors of overall survival. Molecular grading can help to assess the prognosis of patients with upper urinary tract cancer and may represent a new tool for managing this population of patients.

Subcutaneous thrombotic vasculopathy syndrome: an ominous condition reminiscent of calciphylaxis: calciphylaxis sine calcifications?

Ischemic skin necrosis can be a cause of severe morbidity and mortality. It can be due to a number of systemic conditions such as (1) thrombotic vasculopathy syndromes, (2) calciphylaxis, (3) septic or cholesterol emboli, and (4) cutaneous vasculitis. We present 3 patients with a clinicopathological syndrome consisting of ischemic skin necrosis associated with histological pattern of subcutaneous thrombotic vasculopathy-extensive microvascular thrombosis confined to small vessels and capillaries of the subcutaneous tissue. All 3 patients were obese and had severe pre-existing medical conditions. Skin biopsies showed intravascular thrombosis involving small arterioles and capillaries of the subcutaneous tissue. Distribution of vascular involvement by thrombotic process was similar to that observed in calciphylaxis, but calcifications were not observed. Two patients died within 3 months of diagnosis. One patient died 2 years after the presentation. Review of 15 biopsies of calciphylaxis revealed areas of subcutaneous thrombotic vasculopathy in 11 cases (73%). Our study shows that subcutaneous thrombotic vasculopathy syndrome is a potentially lethal condition showing overlapping features between thrombotic vasculopathy syndromes and calciphylaxis. Clinicopathological analysis suggests that it may be a rare variant of calciphylaxis sine calcifications or an early prodromal stage of calciphylaxis. This conclusion is in keeping with increasing appreciation of importance of thrombosis and vascular injury in calciphylaxis.

A comprehensive analysis of a web-based dermatopathology second opinion consultation practice.

CONTEXT: Sharing cases and seeking second opinion consultations is an important part of everyday pathology practice. Internet-based communications and upcoming digital slide technologies have the potential to decrease barriers and open access to the best expertise. We recently developed a dedicated Web-based process for communication with outside practices seeking second opinion consultations. The software allowed us to collect data about the current needs and use of a second opinion consultation practice, a topic that has not been addressed in research studies thus far. OBJECTIVE: To analyze the needs for and performance of a Web-based second opinion consultation practice in dermatopathology. DESIGN: We performed a retrospective analysis on paper and digital records. RESULTS: The average turn-around time from the time of biopsy to the time the report was issued was 7-days. Eighty-two percent of cases were reported the same day they were received. Biopsies of melanocytic lesions, inflammatory dermatoses, and squamous lesions comprised 82% of consultations. Among the remaining cases, soft tissue tumors, adnexal neoplasms, alopecia, and nonmelanoma nonsquamous lesions were the most common diagnoses. In 69% of cases, the outside report contained information about the diagnosis favored by the submitting pathologists. In 5%, there was a significant change in the diagnosis. CONCLUSIONS: Web-based communication facilitates rapid turn-around time and reduces costs and barriers to second opinion consultation.

The alpha6beta4 integrin maintains the survival of human breast carcinoma cells in vivo.

The alpha6beta4 integrin has been widely implicated in carcinoma function in vitro; however, in vivo data are scarce. To determine the importance of alpha6beta4 in tumor progression, a SUM-159 breast carcinoma cell line that is essentially devoid of alpha6beta4 expression was generated using an RNA interference strategy. Loss of alpha6beta4 expression inhibits colony formation in soft agar assays, suggesting a vital role for alpha6beta4 in survival signaling and anchorage-independent growth. Orthotopic injection of the beta4-deficient cell line into the mammary fat pad of immunocompromised mice yielded significantly fewer and smaller tumors than the control cell line, revealing a role for the alpha6beta4 integrin in tumor formation. Under conditions that mimicked the in vivo environment, decreased expression of the alpha6beta4 integrin led to enhanced apoptosis as determined by the percentage of Annexin V-FITC+, PI- cells and the presence of caspase-3 cleavage products. Recombinant vascular endothelial growth factor (VEGF) significantly inhibited the cell death observed in the beta4-deficient cell line, demonstrating the importance of VEGF expression in this survival pathway. Furthermore, loss of alpha6beta4 expression leads to enhanced apoptosis and reduced expression of VEGF in breast carcinoma cells in vivo. Importantly, the specificity of alpha6beta4 in both the in vitro and in vivo assays showed that reexpression of the beta4 subunit into the beta4-deficient cell line could rescue the functional phenotype. Taken together, these data implicate the alpha6beta4 integrin in tumor formation by regulating tumor cell survival in a VEGF-dependent manner.

Genetics analysis of mouse mutations Abnormal feet and tail and rough coat, which cause developmental abnormalities and alopecia.

Mutations in the mouse Brachyury (T) gene are characterized by a dominant reduction of tail length and recessive lethality. Two quantitative trait loci, Brachyury-modifier 1 and 2 (Brm1 and Brm2) are defined by alleles that enhance the short-tail Brachyury phenotype. Here we report on a genetic analysis of a visible dominant mutation Abnormal feet and tail (Aft) located in the vicinity of Brm1. Affected animals display kinky tails and syndactyly in the hindlimbs, both likely resulting from a defect in apoptosis. We observed an unusual genetic incompatibility between Aft and certain genetic backgrounds. We show that Aft and T are likely to interact genetically, since some double heterozygotes are tailless. In addition to the tail and hindlimb phenotypes, Aft-bearing mutants display characteristic late-onset skin lesions. We therefore tested for allelism between Aft and a closely linked recessive mutation rough coat (rc) and found that these two mutations are likely nonallelic. Our results provide a valuable resource for the study of mammalian skin development and contribute to the genetic analysis of Brachyury function.