microRNA-29 negatively regulates EMT regulator N-myc interactor in breast cancer.

BACKGROUND: N-Myc Interactor is an inducible protein whose expression is compromised in advanced stage breast cancer. Downregulation of NMI, a gatekeeper of epithelial phenotype, in breast tumors promotes mesenchymal, invasive and metastatic phenotype of the cancer cells. Thus the mechanisms that regulate expression of NMI are of potential interest for understanding the etiology of breast tumor progression and metastasis. METHOD: Web based prediction algorithms were used to identify miRNAs that potentially target the NMI transcript. Luciferase reporter assays and western blot analysis were used to confirm the ability of miR-29 to target NMI. Quantitive-RT-PCRs were used to examine levels of miR29 and NMI from cell line and patient specimen derived RNA. The functional impact of miR-29 on EMT phenotype was evaluated using transwell migration as well as monitoring 3D matrigel growth morphology. Anti-miRs were used to examine effects of reducing miR-29 levels from cells. Western blots were used to examine changes in GSK3ß phosphorylation status. The impact on molecular attributes of EMT was evaluated using immunocytochemistry, qRT-PCRs as well as Western blot analyses. RESULTS: Invasive, mesenchymal-like breast cancer cell lines showed increased levels of miR-29. Introduction of miR-29 into breast cancer cells (with robust level of NMI) resulted in decreased NMI expression and increased invasion, whereas treatment of cells with high miR-29 and low NMI levels with miR-29 antagonists increased NMI expression and decreased invasion. Assessment of 2D and 3D growth morphologies revealed an EMT promoting effect of miR-29. Analysis of mRNA of NMI and miR-29 from patient derived breast cancer tumors showed a strong, inverse relationship between the expression of NMI and the miR-29. Our studies also revealed that in the absence of NMI, miR-29 expression is upregulated due to unrestricted Wnt/ß-catenin signaling resulting from inactivation of GSK3ß. CONCLUSION: Aberrant miR-29 expression may account for reduced NMI expression in breast tumors and mesenchymal phenotype of cancer cells that promotes invasive growth. Reduction in NMI levels has a feed-forward impact on miR-29 levels.

Loss of tumor suppressor Merlin in advanced breast cancer is due to post-translational regulation.

Unlike malignancies of the nervous system, there have been no mutations identified in Merlin in breast cancer. As such, the role of the tumor suppressor, Merlin, has not been investigated in breast cancer. We assessed Merlin expression in breast cancer tissues by immunohistochemistry and by real-time PCR. The expression of Merlin protein (assessed immunohistochemically) was significantly decreased in breast cancer tissues (although the transcript levels were comparable) simultaneous with increased expression of the tumor-promoting protein, osteopontin (OPN). We further demonstrate that the loss of Merlin in breast cancer is brought about, in part, due to OPN-initiated Akt-mediated phosphorylation of Merlin leading to its proteasomal degradation. Restoring expression of Merlin resulted in reduced malignant attributes of breast cancer, characterized by reduced invasion, migration, motility, and impeded tumor (xenograft) growth in immunocompromised mice. The possibility of developing a model using the relationship between OPN and Merlin was tested with a logistic regression model applied to immunohistochemistry data. This identified consistent loss of immunohistochemical expression of Merlin in breast tumor tissues. Thus, we demonstrate for the first time a role for Merlin in impeding breast malignancy, identify a novel mechanism for the loss of Merlin protein in breast cancer, and have developed a discriminatory model using Merlin and OPN expression in breast tumor tissues.

Functional characterization of the progestagen-associated endometrial protein gene in human melanoma.

Utilizing gene microarray profiling of melanoma samples, we have recently identified a novel gene overexpressed in both thick primary and metastatic melanomas. This gene, progestagen-associated endometrial protein (PAEP), has never before been implicated in the oncogenic processes of melanoma, with its true function in oncogenesis and tumour progression relatively unknown. Overexpression of the PAEP gene in freshly procured thick primary and metastatic melanoma samples (58%) and daughter cell lines (77%) is confirmed by quantitative RT-PCR, immunohistochemistry, Western blotting and mass spectrometric analysis. We suggest that PAEP gene overexpression is involved with melanoma tumour progression as well as an aggressive phenotype. Transfection of melanoma cells with PAEP small interfering RNA (siRNA) reveals a significant decrease in soft agar colony formation and a marked inhibition of both cell migration and cell invasion. Furthermore, we establish stable melanoma transfectants via PAEP lentiviral small hairpin RNA (shRNA), examine their growth characteristics in a murine xenograft model and reveal that tumour growth is significantly inhibited in two separate melanoma cell lines. Our data strongly implicate the PAEP gene as a tumour growth promoter with oncogenic properties and a potential therapeutic target for patients with advanced melanoma.

Elevated levels of Ser/Thr protein phosphatase 5 (PP5) in human breast cancer.

Ser/Thr protein phosphatase 5 (PP5) regulates several signaling-cascades that suppress growth and/or facilitate apoptosis in response to genomic stress. The expression of PP5 is responsive to hypoxia inducible factor-1 (HIF-1) and estrogen, which have both been linked to the progression of human breast cancer. Still, it is not clear if PP5 plays a role in the development of human cancer. Here, immunostaining of breast cancer tissue-microarrays (TMAs) revealed a positive correlation between PP5 over-expression and ductal carcinoma in situ (DCIS; P value 0.0028), invasive ductal carcinoma (IDC; P value 0.012) and IDC with metastases at the time of diagnosis (P value 0.0001). In a mouse xenograft model, the constitutive over-expression of PP5 was associated with an increase in the rate of tumor growth. In a MCF-7 cell culture model over-expression correlated with both an increase in the rate of proliferation and protection from cell death induced by oxidative stress, UVC-irradiation, adriamycin, and vinblastine. PP5 over-expression had no apparent effect on the sensitivity of MCF-7 cells to taxol or rapamycin. Western analysis of extracts from cells over-expressing PP5 revealed a decrease in the phosphorylation of known substrates for PP5. Together, these studies indicate that elevated levels of PP5 protein occur in human breast cancer and suggest that PP5 over-expression may aid tumor progression.

Renal medullary carcinoma: ultrastructural studies may benefit diagnosis.

Renal medullary carcinoma is a recently described highly aggressive malignancy that in most instances exhibits a constellation of clinical and light microscopic features sufficiently distinctive to enable a quick and confident diagnosis. Presented are three examples where, because of unusual elements in the clinical presentation, electron microscopic examination proved beneficial in establishing the diagnosis.

Cytologic diagnosis of alveolar soft part sarcoma of the lower extremity by fine needle aspiration and correlation with core biopsy: a case report.

BACKGROUND: Alveolar soft part sarcoma is a rare soft tissue tumor of uncertain origin that is generally slow growing but unmistakably malignant due to its propensity for metastasis to lung, bone and brain early in the course of disease. Fine needle aspiration biopsy (FNAB) of these tumors and recognition of the characteristic cytologic features precludes more invasive diagnostic measures and facilitates appropriate treatment. CASE: A 54-year-old African-American man presented to our institution with a 2-week history of left leg pain. Imaging studies revealed a left leg soft tissue mass just below the popliteal fossa and multiple bilateral lung lesions suggestive of metastatic neoplasm. FNAB of the left lower extremity mass yielded uniform clusters of cells and sigle cells with large nuclei and single prominent nucleoli. Histologically, the biopsy showed nests of large polygonal cells with abundant eosinophilic cytoplasm, round regular nuclei and prominent nucleoli. A periodic acid-Schiff (PAS) stain highlighted intracytoplasmic rhomboidal crystals, a feature diagnostic of alveolar soft part sarcoma. CONCLUSION: Alveolar soft part sarcoma may be diagnosed by its unique morphologic characteristics and should be considered in the differential diagnosis of all cytologically sampled soft tissue lesions.

Solid-pseudopapillary tumor: a report of three cases in adult males diagnosed utilizing three different modalities.

Solid-pseudopapillary tumor of the pancreas is a rare neoplasm of uncertain origin with low malignant potential and often indolent behavior occurring predominantly in adolescent and young women. Here we report the cases of three adult males with pancreatic masses, one with metastasis, diagnosed as solid-pseudopapillary tumor by cytology, electron microscopy (EM), and routine histology. This neoplasm is uncommon in both males and adults and uncommonly metastasizes. The cases reported emphasize the utility of different diagnostic modalities, and here we review the diagnostic features by cytology, EM, and routine histology to correctly characterize this neoplasm. It is extremely important to correctly diagnose this indolent neoplasm due to the excellent prognosis with surgical resection.

Employer Expectations for Newly Trained Pathologists: Report of a Survey From the Graduate Medical Education Committee of the College of American Pathologists.

CONTEXT: -Multiple sources have identified challenges that training programs face in preparing graduates for the "real world" practice of pathology, and many training programs have sought to decrease the gap between skills acquired during training and those required in practice. However, there exists the possibility that some of the difficulty experienced by newly trained pathologists and employers might arise from differences between employer expectations of new hires and what applicants expect from their first job. OBJECTIVE: -To define the constellation of skills and attributes employers prioritize when hiring newly trained pathologists. DESIGN: -A survey of fellows of the College of American Pathologists in practice for 5 or more years in the United States was administered and the results were analyzed. RESULTS: -A total of 630 pathologists who were responsible for hiring a new-in-practice pathologist completed the survey. Regardless of practice setting, certain skills and attributes were rated critically important in new hires, including ethics/integrity, work ethic, and professionalism. Seventy-one percent reported having some difficulty hiring entry-level pathologists and cited inadequate training/experience during residency, and applicants having unrealistic expectations regarding work load/hours as the most common reasons. CONCLUSIONS: -Prospective employers not only expect well-developed diagnostic skills in their job applicants, but also require evidence of a strong work ethic and outstanding professionalism. Successful applicants must display willingness to assume responsibilities and flexibility regarding existing and new responsibilities. A secondary but important finding of this survey was that most jobs are garnered through word-of-mouth recommendations; therefore, it is crucial for pathologists-in-training to hone their networking skills.

Loss of Merlin induces metabolomic adaptation that engages dependence on Hedgehog signaling.

The tumor suppressor protein Merlin is proteasomally degraded in breast cancer. We undertook an untargeted metabolomics approach to discern the global metabolomics profile impacted by Merlin in breast cancer cells. We discerned specific changes in glutathione metabolites that uncovered novel facets of Merlin in impacting the cancer cell metabolome. Concordantly, Merlin loss increased oxidative stress causing aberrant activation of Hedgehog signaling. Abrogation of GLI-mediated transcription activity compromised the aggressive phenotype of Merlin-deficient cells indicating a clear dependence of cells on Hedgehog signaling. In breast tumor tissues, GLI1 expression enhanced tissue identification and discriminatory power of Merlin, cumulatively presenting a powerful substantiation of the relationship between these two proteins. We have uncovered, for the first time, details of the tumor cell metabolomic portrait modulated by Merlin, leading to activation of Hedgehog signaling. Importantly, inhibition of Hedgehog signaling offers an avenue to target the vulnerability of tumor cells with loss of Merlin.

Dual CD117 expression in gastrointestinal stromal tumor (GIST) and paraganglioma of Carney triad: a case report.

Carney triad is a rare syndrome, with only 20 complete cases reported. We report a 36-year-old white woman with complete Carney triad, including metastatic gastric stromal tumor (GIST), pulmonary chondroma, and nonfunctioning extra-adrenal paraganglioma. Immunohistochemistry was positive for CD34 and CD117 (c-kit) in the GIST, and positive for chromogranin and CD117 in the paraganglioma. Ultrastructural studies demonstrated skeinoid fibers in the GIST. To our knowledge, this is the 21st complete Carney triad case reported and the first report of dual expression CD117 in both GIST and paraganglioma, a finding with intriguing pathogenetic implications related to the organization of the autonomic nervous system.

Assessment and Management of Professionalism Issues in Pathology Residency Training: Results From Surveys and a Workshop by the Graduate Medical Education Committee of the College of American Pathologists.

Professionalism issues are common in residency training and can be very difficult to recognize and manage. Almost one-third of the milestones for pathology recently instituted by the Accreditation Council for Graduate Medical Education encompass aspects of professionalism. Program directors are often unsure of how and when to remediate residents for unprofessional behavior. We used a case-based educational approach in a workshop setting to assist program directors in the management of unprofessional behavior in residents. Eight case scenarios highlighting various aspects of unprofessional behavior by pathology residents were developed and presented in an open workshop forum at the annual pathology program director's meeting. Prior to the workshop, 2 surveys were conducted: (1) to collect data on program directors' experience with identifying, assessing, and managing unprofessional behavior in their residents and (2) to get feedback from workshop registrants on how they would manage each of the 8 case scenarios. A wide range of unprofessional behaviors have been observed by pathology program directors. Although there is occasionally general agreement on how to manage specific behaviors, there remains wide variation in how to manage many of the presented unprofessional behaviors. Remediation for unprofessional behavior in pathology residents remains a difficult and challenging process. Additional education and research in this area are warranted.

A kidney epithelial cell line from a Bolivian squirrel monkey.

Squirrel monkeys are the most commonly used New World primates in biomedical research, but in vitro studies are restricted by the limited number of cell lines available from this species. We report here the development and characterization of a continuous, kidney epithelial cell line (SQMK-FP cells) derived from a newborn squirrel monkey. Karyotype was consistent with Bolivian squirrel monkey (submetacentric chromosome pair 15 and acrocentric chromosome pair 16). All cells examined were hyperdiploid with chromosome numbers ranging from 52 to 57. Ultrastructural analysis of SQMK-FP cells revealed the presence of cell junctions with radiating filaments, indicating desmosomes and numerous surface projections containing longitudinally oriented filaments typical of tubular epithelium. Biochemically, SQMK-FP cells exhibit glucocorticoid resistance typical of the squirrel monkey. Glucocorticoid receptor (GR) binding is low in SQMK-FP cells because of high expression of the FK506-binding immunophilin FKBP51 that inhibits GR binding. SQMK-FP cells constitute a tubular epithelial cell line that has biochemical properties characteristic of squirrel monkeys and represents an alternate cell model to B-lymphoblast SML cells to study the biology of the squirrel monkey in vitro.

Chondrocyte viability in refrigerated osteochondral allografts used for transplantation within the knee.

PURPOSE: To evaluate cell viability and matrix characteristics of refrigerated osteochondral allografts implanted up to 44 days after harvest. METHODS: Sixteen refrigerated allografts underwent histologic and ultrastructural examination and fluorescence excitation analysis prior to implantation. The average size of the graft implanted was 6.2 cm(2) (+/-3.4 cm(2)). Refrigerated allografts averaged 30 days (range, 17 to 44 days) from donor expiration to implantation. Nine specimens underwent cell viability testing. The percent viability of refrigerated allografts prior to implantation averaged 67%. RESULTS: No significant correlations were noted between histologic score, electron microscopy score, matrix staining percent (MSP) score, and viability. When time to implantation was assessed, an inverse correlation was noted with MSP score (r =.539) (P < 0.05), indicating less matrix staining in grafts refrigerated longer after harvest. CONCLUSION: The current data indicate that refrigerated osteochondral allografts can be maintained for up to 44 days with average chondrocyte viability of 67%.

Halicephalobus gingivalis: a rare cause of fatal meningoencephalomyelitis in humans.

The genus Halicephalobus consists of eight species of free-living nematodes. Only one species (H. gingivalis) has been reported to infect vertebrates. Human infection is extremely rare, and only four cases have been reported in the literature. These nematodes seem to exhibit neurotropism, but their life cycle, mode of infection, and risk factors are poorly understood. Neurohelminthiases are not commonly recognized in the United States and when they do occur, pose great diagnostic challenges because of lack of appropriate non-invasive screening and/or confirmatory tests. We report a challenging case of meningoencephalomyelitis caused by a Halicephalobus sp., in which the patient had a rapidly deteriorating clinical course. The case did not raise any clinical suspicion of neurohelminthiases, although increased eosinophils were present in the cerebrospinal fluid. This case presents an opportunity to highlight the importance of considering parasitic infection in meningoencephalitis or meningoencephalomyelitis presenting atypically.

Hedgehog signaling in tumor cells facilitates osteoblast-enhanced osteolytic metastases.

The remodeling process in bone yields numerous cytokines and chemokines that mediate crosstalk between osteoblasts and osteoclasts and also serve to attract and support metastatic tumor cells. The metastatic tumor cells disturb the equilibrium in bone that manifests as skeletal complications. The Hedgehog (Hh) pathway plays an important role in skeletogenesis. We hypothesized that the Hh pathway mediates an interaction between tumor cells and osteoblasts and influences osteoblast differentiation in response to tumor cells. We have determined that breast tumor cells have an activated Hh pathway characterized by upregulation of the ligand, IHH and transcription factor GLI1. Breast cancer cells interact with osteoblasts and cause an enhanced differentiation of pre-osteoblasts to osteoblasts that express increased levels of the osteoclastogenesis factors, RANKL and PTHrP. There is sustained expression of osteoclast-promoting factors, RANKL and PTHrP, even after the osteoblast differentiation ceases and apoptosis sets in. Moreover, tumor cells that are deficient in Hh signaling are compromised in their ability to induce osteoblast differentiation and consequently are inefficient in causing osteolysis. The stimulation of osteoblast differentiation sets the stage for osteoclast differentiation and overall promotes osteolysis. Thus, in the process of developing newer therapeutic strategies against breast cancer metastasis to bone it would worthwhile to keep in mind the role of the Hh pathway in osteoblast differentiation in an otherwise predominant osteolytic phenomenon.

Production, safety and antitumor efficacy of recombinant Oncofetal Antigen/immature laminin receptor protein.

We describe here for the first time an efficient high yield production method for clinical grade recombinant human Oncofetal Antigen/immature laminin receptor protein (OFA/iLRP). We also demonstrate significant antitumor activity for this protein when administered in liposomal delivery form in a murine model of syngeneic fibrosarcoma. OFA/iLRP is a therapeutically very promising universal tumor antigen that is expressed in all mammalian solid tumors tested so far. We have cloned the human OFA/iLRP cDNA in a bacterial expression plasmid which incorporates a 6x HIS-tag. Large scale cultures of the plasmid transformed Escherichia coli were performed and the crude HIS-tagged OFA/iLRP was isolated as inclusion bodies and solubilized in guanidine chloride. The protein was then purified by successive passage through three column chromatography steps of immobilized metal affinity, anion exchange, and gel filtration. The resulting protein was 94% pure and practically devoid of endotoxin and host cell protein. The purified OFA/iLRP was tested in mice for safety and efficacy in tumor rejection with satisfactory results. This protein will be used for loading onto autologous dendritic cells in an FDA approved phase I/II human cancer vaccine trial in OFA/iLRP-positive breast cancer patients.

Metaplastic breast carcinoma with osseous differentiation: a case report.

Metaplastic breast carcinoma is a rare entity with the distinguishing feature of having epithelial and mesenchymal tissue types incorporated within one tumor. This is a case report of a patient found to have a rare metaplastic breast carcinoma with prominent osseous differentiation. Radiologic and pathologic correlation is provided.

From Renaissance art to contemporary electron microscopy: DeGroft's rediscovery of Titian's "lost" portrait of Federico II Gonzaga, Duke of Mantua, of 1539-40.

At the Ultrapath X meeting in Florence, the regular session opened with a presentation of Aaron DeGroft's engrossing story of investigating the authenticity of a portrait of Federico II Gonzaga, Duke of Mantua. In the early 1900s, this work had been deemed to be an authentic production by Titian, a great artist of the Italian Renaissance. A respected art historian, however, discovered a conflict of dates that led to the conclusion that this work was not authentic. In a process sometimes analogous to the practice of surgical pathology, Dr. DeGroft pursued a review of the original materials that refutes this seeming contradiction of dates. Dr. DeGroft also undertook an extensive art historical examination and scientific analysis, including the use of electron microscopy, to persuasively conclude that this portrait is authentic. Further, his work provided a bridge from the conference setting in Florence, rich in Renaissance art, to the contemporary update on ultrastructural pathology provided by the conference.

Fibromatosis: the breast cancer imitator.

Fibromatosis of the breast is a rare benign tumor that should be included in the differential diagnosis for breast cancer. It is usually indistinguishable from malignancy on ultrasound, mammography, physical examination, and on gross evaluation. Distinction is easily made by histologic findings. This benign tumor does not metastasize, but is locally aggressive and tends to recur postoperatively, which accounts for considerable morbidity. We present two cases and a discussion from the perspective of the radiologist, the surgeon, and the pathologist.

Inspissation of pancreatic zymogen material in cystic fibrosis.

Cystic fibrosis is characterized by the elaboration of abnormal, thick, tenacious mucus resulting in obstructive disease in sites such as the lung and pancreas. In the pancreas, acinar plugs of mucus have been reported as the earliest recognizable morphologic lesion in cystic fibrosis. Since mucus is not normally elaborated within the pancreatic lobular tissue, the mechanism of accumulation of mucus in acini is enigmatic. To investigate this phenomenon, well-preserved autopsy pancreatic tissue was studied ultrastructurally. This study demonstrated very prominent mucous metaplasia in these diseased organs. Acinar plugs, though, developed before mucous metaplasia. Subsequent histochemical study was performed, which demonstrated that the early acinar plugs exhibited the same staining properties as zymogen granules and were distinct from the staining pattern of mucus in pancreatic tissue of cystic fibrosis patients. These findings, then, indicate that zymogen material, not mucus, becomes inspissated in the acini of the pancreas in early cystic fibrosis, and that subsequent mucous metaplasia occurs as the obstruction and exocrine atrophy progress.