Melanoma is the cancer with the highest increase in incidence, and transformation of radial growth to vertical growth (i.e., noninvasive to invasive) melanoma is required for invasive disease and metastasis. We have previously shown that p42/p44 MAP kinase is activated in radial growth melanoma, suggesting that further signaling events are required for vertical growth melanoma. The molecular events that accompany this transformation are not well understood. Akt, a signaling molecule downstream of PI3K, was introduced into the radial growth WM35 melanoma in order to test whether Akt overexpression is sufficient to accomplish this transformation. Overexpression of Akt led to upregulation of VEGF, increased production of superoxide ROS, and the switch to a more pronounced glycolytic metabolism. Subcutaneous implantation of WM35 cells overexpressing Akt led to rapidly growing tumors in vivo, while vector control cells did not form tumors. We demonstrated that Akt was associated with malignant transformation of melanoma through at least 2 mechanisms. First, Akt may stabilize cells with extensive mitochondrial DNA mutation, which can generate ROS. Second, Akt can induce expression of the ROS-generating enzyme NOX4. Akt thus serves as a molecular switch that increases angiogenesis and the generation of superoxide, fostering more aggressive tumor behavior. Targeting Akt and ROS may be of therapeutic importance in treatment of advanced melanoma.

Background: Immune hemolytic anemia is a well-known complication after allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant hemolytic anemia results in increased red blood cell transfusions and medical sequelae including iron overload. Case Report: We present a case report of immune hemolytic anemia that occurred after allogeneic HSCT from an ABO major-mismatched, HLA-matched unrelated donor. The patient had high anti-donor A type antibodies that were unresponsive to treatment with steroids and rituximab, resulting in persistent transfusion dependence. A detailed time course of anti-A titers, plasma cell content of the marrow, and B-cell content of the blood is presented. Treatment with bortezomib, a protease inhibitor, eliminated residual host-type plasma cells secreting anti-A and restored normal donor-derived erythropoiesis. Conclusion: This report, and a review of literature for treatment of immune hemolytic anemia after allogeneic HSCT, supports the utility of bortezomib as plasma cell-targeted therapy in this setting.

Background: Lymphoid enhancer binding factor 1 (LEF-1) has recently been reported as a potential immunohistochemical (IHC) marker for basal cell adenoma (BCA) and other salivary gland tumors, which may contribute to an increased accuracy in differentiating basaloid salivary gland neoplasms. We evaluated the utility of LEF-1 in fine needle aspiration (FNA) and resection specimens to distinguish pleomorphic adenoma (PA), BCA, basal cell adenocarcinoma (BCAC), and adenoid cystic carcinoma (ACC) as well as in non-neoplastic salivary gland (NNSG). Methods: Cases including 66 PA (35 FNA, 31 resections), 12 BCA (5 FNA, 7 resections), 42 ACC (11 FNA, 31 resections), 1 BCAC FNA, and 10 NNSG (5 FNA, 5 resections) were obtained and stained for LEF-1. Results: On cell block (CB), 51% of PA and 60% of BCA were LEF-1 positive while 91% of ACC were LEF-1 negative. Among resections, there was a higher percentage of LEF-1 positive PA (84%) and BCA (86%), and a higher percentage of LEF-1 negative ACC (97%). LEF-1 staining had a low to moderate sensitivity for detecting benign basaloid neoplasms on FNA CB and resection specimens (52.5% and 84%, respectively), but a higher specificity (92% and 97% respectively), and positive predictive value (95% and 97% respectively). Conclusion: When comparing benign (PA and BCA) and the most common malignant basaloid salivary gland tumor (ACC), positive LEF-1 favors a benign neoplasm. Additional studies with LEF-1, specifically including other rare basaloid salivary gland neoplasms are needed to further clarify the role of LEF-1 in diagnosing these lesions on FNA.

High levels of HER2 expression identify those patients who might benefit from treatments that target HER2. Among women with metastatic breast cancer, the predictive markers may be different from the primary tumor. We compared predictive markers: Estrogen Receptor (ER), Progesterone Receptor (PR) and HER2 of primary breast carcinomas with those of lymph node (LN) and blood spread metastases (BM). ER, PR and HER2 status were compared between the primary breast tumor and the LN metastasis and blood spread metastasis. ER, PR and HER2 were performed on primary tumor core biopsies and available FNA cell blocks and on metastatic lesions using FDA approved antibodies and HercepTest (Dako). ER and PR were positive when >/=10%. Her2 was positive (amplified/expressed) when 3+ >30% by immunostain or >2.2 by FISH. Sixty four metastatic breast cancer patients were included in this analysis. Forty-eight patients had LN metastases (35 [73 %] diagnosed by FNA) and twenty seven patients had BM (16 [60 %] diagnosed by FNA). P value was determined comparing primary breast with BM and LN for ER, PR and HER2. ER p values when compared for primary breast with BM and LN were 0.45 and 0.57 respectively, and for PR were 0.31 and 0.06 and for HER2 were 0.45 and 0.07. All three predictive markers are similar in the primary and two metastatic sites (lymph node, bloodspread). Only in primary versus lymph node metastases is there a tendency for PR and HER2 (P values 0.06, 0.07) to be different. For HER2, the majority of lymph node metastases are in cell blocks (FNA), fixed in ethanol rather than formalin, which may have caused false positive HER2 expression.

PURPOSE: We wished to determine whether virally- induced endothelial tumors are rejected by CD4 and CD8 lymphocytes, and whether there are differences in requirements for costimulation in the rejection of these tumors by lymphocyte subsets. EXPERIMENTAL DESIGN: We have developed a model of endothelial tumorigenesis through the sequential introduction of SV40 large T antigen and oncogenic H-ras into endothelial cells. These cells (SVR cells) form highly aggressive angiosarcomas in immunocompromised mice, but do not grow in syngeneic C57BL/6 mice. Using both acute blockade with systemic administration of antibodies and mice genetically deficient in the costimulatory molecules CD28, CD40, and CD40L, we have delineated the requirements of costimulation required to reject this virally-induced endothelial tumor. RESULTS: Control of SVR angiosarcoma is mediated through T lymphocytes, and both CD4 and CD8 lymphocytes are capable of controlling SVR angiosarcoma growth in vivo. Mice genetically deficient in CD28, CD40, and CD40L were able to reject SVR tumors, but depletion of these mice of CD8, but not CD4 cells led to rapid tumor growth. This data suggests that CD4 mediated rejection has a greater dependence of costimulation than CD8 mediated rejection. Surprisingly, acute depletion of costimulatory molecules in immunocompetent C57BL/6 mice led to rapid tumor growth. CONCLUSIONS: Significant differences exist in the immune status of mice acutely depleted of costimulatory molecules versus genetically deficient mice. Our results suggest that acute depletion is more immunosuppressive than genetic depletion. Humans who undergo costimulatory blockade may require periodic surveillance for virally-induced tumors.

Parathyroid hormone (PTH) stimulates osteoblasts to produce the proinflammatory cytokine interleukin-6 (IL-6), causing bone resorption. In patients with primary hyperparathyroidism, elevated serum levels of IL-6 normalize after resection of parathyroid tumours. Because IL-6 is also expressed in normal parathyroids and in other endocrine cells (adrenal and islet), we hypothesized that parathyroid tumours might contribute directly to the elevated serum IL-6 levels in patients with hyperparathyroidism. Immunohistochemistry identified IL-6, PTH, and chromogranin-A (an endocrine and neuroendocrine tumour marker) in normal, adenomatous and hyperplastic parathyroids. Using immunofluorescence and confocal microscopy, IL-6 co-localized with PTH and with chromogranin-A in parathyroid cells. All cultured parathyroid tumours secreted IL-6 at levels markedly higher than optimally stimulated peripheral blood mononuclear cells. Supernates from cultured parathyroids stimulated proliferation of an IL-6-dependent cell line, and anti-IL-6 MoAb abolished this stimulatory effect. IL-6 mRNA was documented in cultured parathyroid tumours, cultured normal parathyroids, fresh operative parathyroid tumours and fresh operative normal specimens. In conclusion, these data show that parathyroid tumours and normal parathyroids contain, produce and secrete IL-6. Our findings present a novel pathway by which human parathyroids may contribute markedly to IL-6 production and elevation of serum IL-6 levels in patients with hyperparathyroidism. The physiological relevance of IL-6 production by human parathyroids remains to be determined, but IL-6 secretion by parathyroid tumours may contribute to bone loss and to other multi-system complaints observed in these patients.

Fine needle aspiration (FNA) is a rapid tool for detection of breast carcinomas. Evaluation of estrogen and progesterone receptors (ER, PR) and HER2 expression by immunohistochemistry (IHC) are routinely performed in breast carcinomas. Formalin fixation of tissue for a minimum of 6 hours, and for HER2 not more than 48 hours is the current recommended practice. In this retrospective study, we compared ER, PR and HER2 expression in breast carcinomas using archival ethanol-fixed FNA cell block with formalin fixed resection tissue block preparations. 34 archival breast carcinoma FNA cell blocks of primary origin with subsequent resection tissue blocks were identified retrospectively. All 34 cases were diagnosed as invasive ductal carcinoma. Cases with neoadjuvant or adjuvant chemotherapy were excluded. Cell blocks were initially fixed in 50% ethanol (4–12 hrs), followed by formalin fixation (minimum 6 hrs). Tissue blocks were formalin-fixed within 4–8 hrs for 6–48 hrs. ER, PR, and HER2 IHC results on the cell blocks and tissue blocks were compared. Alcohol fixed cell block samples for detection of ER and PR by IHC show good agreement with tissue block samples and are therefore a reliable method (weighted Kappa of 0.773 and 0.785, respectively) to triage patients for hormonal treatment. However, HER2 results show only moderate agreement with a weighted kappa of 0.571. The increase in discrepant results may be due to ethanol fixation which results in false positive increased HER2 expression. These results demonstrate the importance of adherence to the College of American Pathologists/ASCO guidelines for HER2 IHC.

In the normal rodent breast, the pineal hormone melatonin controls the development of ductal and alveolar tissue. Melatonin counteracts tumor occurrence and tumor cell progression in vivo and in vitro in animal and human breast cancer cell cultures. It acts predominantly through its melatonin MT1 receptor. Our aim was to investigate the presence or absence of the MT1 melatonin receptor in the aggressive triple negative group of human breast carcinoma (TNBC) and its possible relationship to the course of the disease. A total of 167 patients with a ER-, PR-, Her-2/neu- phenotype in which tissue for receptor studies was available were examined. The MT1 receptor immunostain was evaluated semiquantitatively as staining intensity (0, 1, 2, 3), percentage of stained cells and the weighted index (WI) (staining intensity times percentage of stained cells). A score of WI < 60 was regarded as "negative". There was a striking difference in incidence of MT1 positivity and staining intensity between carcinomas in African American (AA) and Caucasian (C) women. The AA showed a higher incidence of MT1 negative tumors (41/84 = 48.8 % in AA, 6/51 = 11.8 % in C) and a lower average WI. MT1 positivity in TNBC was associated with a lower stage and a smaller tumor size at time of diagnosis. In multivariable survival analysis, MT1 negative TNBC in all cases regardless of race showed a significantly higher hazard ratio for disease progression, shorter progression free survival, and disease-related death, and shorter OS. This was especially pronounced in the AA group but did not reach statistical significance in the smaller group of C alone. These results suggest that melatonin or a melatonin receptor agonist may be useful biologic additions in the treatment of some forms of TNBC, especially in AA who generally show a more aggressive course of their disease.

P62 and ubiquitin are small regulatory proteins demonstrated to have implications in the prognosis and survival of various malignancies including: hepatocellular, breast, ovarian, and some gastrointestinal carcinomas. Several trials studied the link of their activity to the extrinsic apoptosis pathway and showed that their autophagy modification has a critical stand point in tumorigenesis. These findings explain their vital role in controlling the process of cell death and survival. It has been shown recently that p62 and ubiquitin overexpression in different types of cancers, such as triple negative breast and ovarian cancers, have directly correlated with incidence of distant metastases. We aim to evaluate p62/ubiquitin expression in gastrointestinal carcinomas of gastric, colonic, and pancreatic origin, and correlate with annotated clinicopathologic data. In gastric carcinoma (61), positive p62 nuclear expression was noted in 57% and cytoplasmic in 61%, while positive ubiquitin was nuclear expressed in 68.8%, and cytoplasmic in 29.5%. In colon carcinoma (45), positive p62 nuclear expression was noted in 29% and cytoplasmic in 71%, while positive ubiquitin was nuclear in 58% and cytoplasmic in 44%. In pancreatic cancer (18), positive p62 nuclear expression was noted in 78% and cytoplasmic in 56%, while positive ubiquitin was nuclear in 83% and cytoplasmic in 72%. Normal gastric (6), colon (4), and pancreatic (4) tissues were negative for both P62 and ubiquitin (nuclear and cytoplasmic staining <20%). Ubiquitin high expression was associated with more lymph node metastases in colon (4.14 vs 1.70, P = 0.04), and pancreatic adenocarcinomas (3.07 vs 0.33, P = 0.03). Also, ubiquitin high expression was associated with worse pancreatic adenocarcinoma overall survival (1.37 vs 2.26 mos, P = 0.04). In addition, gastric cancer patients with high p62 expression tend to have more poorly differentiated grade when compared to those with low expression (21 vs 17, P = 0.04) but less lymph node metastases (2.77 vs 5.73, P = 0.01). P62 and ubiquitin expression did not correlate with other clinicopathologic parameters in gastric, colon or pancreatic denocarcinomas. The results suggest that p62 and ubiquitin are highly expressed in gastric, colonic, and pancreatic carcinomas. High ubiquitin expression was noted to have an impact on number of lymph node metastases in patients with colon and pancreatic adenocarcinomas, but on overall survival only in patients with pancreatic adenocarcinoma. Also, P62 high expression is correlated with poor differentiation, but less lymph node metastases, in gastric carcinoma.