Maggie Stoecker

Expression of ezrin correlates with malignant phenotype of lung cancer, and in vitro knockdown of ezrin reverses the aggressive biological behavior of lung cancer cells

Ezrin, one of the ezrin–radixin–moesin proteins, is involved in the formation of cell membrane processes such as lamellipodia and filopodia and acts as a membrane–cytoskeleton linker. Its aberrant expression correlates with development and progression of several human cancers. However, the expression of ezrin and its role in lung cancer are currently unknown. In this study, we performed ezrin small interfering RNA transfection in two lung cancer cell lines and examined the effects on malignant phenotypes in cancer cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, and chamber transwell assays. Ezrin knockdown significantly reduced the proliferation, migration, and invasion of lung cancer cells in vitro. To address the possible mechanisms, we evaluated the expression of adhesion molecules E-cadherin and β-catenin by Western blot and reverse transcriptase-polymerase chain reaction analyses. The results demonstrated that downregulation of ezrin reduced β-catenin and increased E-cadherin at the protein level but had no effects on their mRNA levels, suggesting posttranscriptional regulation of these two adhesion molecules. Immunofluorescence assays revealed that ezrin knockdown restored membranous expression of E-cadherin and decreased cytoplasmic β-catenin in lung cancer cells. In addition, ezrin expression was immunohistochemically evaluated on 135 normal and 183 lung cancer tissues. The expression of ezrin was significantly higher in cancer samples than paired autologous normal lung tissues. In normal bronchial epithelium, ezrin was mainly localized on the apical membrane, while in lung cancers and metastatic foci, ezrin was primarily distributed in cytoplasm. Among lung cancer tissues, expression of ezrin was higher in the invasive front of primary lesions and the highest in lymphatic metastasis. Statistical analysis demonstrated that ezrin expression correlated significantly with lymphatic metastasis and advanced TNM stage. Our data suggest that ezrin may play a crucial role in governing the biological behavior of lung cancer.

Overexpression of CRKL correlates with poor prognosis and cell proliferation in non-small cell lung cancer.

Crk‐Like (CRKL) is an adapter protein that has crucial roles in multiple biological processes, including cell proliferation, adhesion, and migration. Amplification of CRKL gene was found in non‐small cell lung cancer (NSCLC). However, the expression pattern of CRKL protein and its clinical significance in human NSCLC have not been well characterized to date. In this study, expression of CRKL was evaluated in 131 NSCLC tissues by immumohistochemistry. CRKL protein was up‐regulated in the lung carcinomas compared with adjacent normal lung tissue. Overexpression of CRKL was found in 58 of 131 (44.3%) NSCLC samples and correlated with poor tumor differentiation (P = 0.0042), histological type (adenocarcinoma; P = 0.001), advanced p‐TNM stage (P = 0.0004), nodal metastasis (P = 0.0273), high proliferation index (P = 0.0062) and poor overall survival (P = 0.0084). Further univariate and multivariate analysis showed a significant association of CRKL overexpression and worse overall survival in lung cancer patients. In addition, overexpression of CRKL in HBE and H1299 cell lines promoted cell proliferation by facilitating cell cycle progression. Further analysis of cell cycle related molecules showed that CRKL induced cyclin D1, cyclin B1 expression, and increased Rb phosphorylation. In conclusion, this study demonstrated overexpression of CRKL correlated with poor prognosis and lung cancer proliferation by cell cycle regulation.

Histiocytic/dendritic cell transformation of B-cell neoplasms: pathologic evidence of lineage conversion in differentiated hematolymphoid malignancies.

B-cell lymphomas, such as low-grade follicular lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma, can transform to histiocytic/dendritic cell sarcoma (H/DS) in rare cases. The diagnosis of this unconventional neoplastic evolution relies on a combination of immunophenotypic analysis and genotypic studies. A genotype identical to that of the primary B-cell neoplasm in a secondary neoplasm with H/DS immunophenotype supports the lineage conversion to H/DS. Putative mechanisms for this unusual phenomenon include dedifferentiation, common immature progenitor, and transdifferentiation models, the latter of which is suggested by clinical laboratory data at the present time. Elucidation of the molecular mechanisms governing this lineage conversion may facilitate the understanding of carcinogenesis of not only hematopoietic but also nonhematolymphoid neoplasms. The clinical outcome of secondary H/DS is dismal, as observed in sporadic cases, and the optimal treatment remains to be determined.

Systemic Mastocytosis With Associated Clonal Hematologic Nonmast Cell Lineage Disease: A Clinicopathologic Review

Systemic mastocytosis (SM) is a heterogeneous disease with 6 subtypes, including systemic mastocytosis with associated clonal hematologic nonmast cell lineage disease (SM-AHNMD). Bone marrow biopsy specimens show multifocal aggregates of mast cells with predominantly spindle-shaped morphology associated with a myeloid or, less frequently, a lymphoproliferative neoplasm defined by World Health Organization criteria. Neoplastic mast cells abnormally express CD2 and/or CD25, which may be detected by flow cytometry or immunohistochemistry. The pathogenesis of SM-AHNMD is not well understood; however, combined KIT tyrosine kinase receptor mutations and additional genetic events in myeloid stem cells may have a pathogenic role. Reactive mast cell hyperplasia, monocytic/histiocytic proliferations, SM without sufficient criteria for a diagnosis of AHNMD, atypical mast cells associated with PDGFRA rearrangements, and other tryptase-positive myeloid proliferations should be excluded. Overall, the prognosis is poor and largely related to the AHNMD. Cytoreductive therapies, splenectomy, allogeneic bone marrow transplant, and tyrosine kinase inhibitors, excluding imatinib, may have potential efficacy in the treatment of these diseases.

Primary mediastinal (thymic) large B cell lymphoma with aberrant expression of CD3 : a case report with review of the literature

We report the first case of primary mediastinal large B cell lymphoma (PMBL) with aberrant expression of CD3. PMBL is a subtype of diffuse large B cell lymphoma (DLBCL) and usually presents with bulky mediastinal lesions. Lineage ambiguity/infidelity is uncommon in DLBCL but has been described in sporadic case reports/series. A literature search identifies 13 additional cases of DLBCL expressing CD3, with the majority displaying cytoplasmic expression. Of the 14 total cases, 6 are pyothorax-associated lymphoma, 4 are conventional DLBCL, 2 are plasmablastic lymphoma, one is primary effusion lymphoma and one is PMBL. Two cases show genotypic ambiguity/infidelity with dual clonal IG and TCR gene rearrangements in addition to ambiguous immunophenotypes. Of the 13 cases tested for EBV status, 11 are positive, suggesting an important role of EBV in promoting lineage ambiguity/infidelity. A low threshold for testing EBV status is advocated in DLBCL with phenotypic ambiguity along with panels of immunohistochemical and molecular studies.

A small cell variant of ALK-positive, CD8-positive anaplastic large cell lymphoma with primary subcutaneous presentation mimicking subcutaneous panniculitis-like T-cell lymphoma.

ALK-positive anaplastic large cell lymphoma (ALK+ ALCL) is an uncommon non-Hodgkins lymphoma of T-cell origin, the majority of which express CD4 and show frequent pan-T-cell antigen loss. While most cases of ALK+ ALCL have the common pattern characterized by anaplastic morphology with hallmark cells, a less common but well-recognized variant with a small cell pattern may pose a diagnostic challenge. We report a case of ALK+ ALCL with small cell morphology and CD8 subset restriction in a 53-year-old male patient who presented primarily with multiple recurrent subcutaneous nodules with histopathologic features simulating a subcutaneous panniculitis-like T-cell lymphoma (SPTCL). The case was initially diagnosed as SPTCL but was reconsidered as ALK+ ALCL when the incidental finding of CD30 positivity on a subsequent biopsy prompted an ALK immunostain, which turned out to be positive in the neoplastic T-cells. The diagnosis of ALK+ ALCL, small cell variant, was then confirmed by detection of an ALK gene rearrangement by FISH analysis. This report highlights a case of ALK+ ALCL with a deceiving clinical and histopathologic presentation, and emphasizes the value of immunohistochemical panel studies and genetic tests in such cases to avoid diagnostic errors.

Primary cutaneous giant cell plasmacytoma in an organ transplant recipient: a rare presentation of a posttransplant lymphoproliferative disorder.

Posttransplant lymphoproliferative disorder (PTLD) is comprised of a spectrum of lymphoid diseases, ranging from early lesions, such as plasmacytic hyperplasia, to monomorphic neoplasms, including plasmacytoma-like lesions. Although PTLD may involve a variety of organs, primary cutaneous PTLD is rare. We report a unique case of Epstein-Barr virus (EBV)-positive primary cutaneous giant cell plasmacytoma developed 5 years after renal/pancreatic transplant in a 55-year-old male patient. The patient received local radiotherapy without reduction in immunosuppression and responded well. A review of the literature identified additional 49 cases of primary cutaneous B-cell PTLD, including 18 cases of plasmacytoma-like lesions. Primary cutaneous B-cell PTLD usually presents years after transplantation, has male preponderance, tends to occur on extremities, is frequently EBV-associated, and predicts a favorable clinical outcome. Unlike PTLD in general, in which EBV-positive cases usually occur earlier than EBV-negative ones, the longer presentation interval in the cutaneous PTLD seems to be uncorrelated to EBV status. Compared with other subtypes of cutaneous B-cell PTLD, plasmacytoma-like lesions have an increased male preponderance and tendency to present on the extremities. Although the majority of cases have been treated with reduction of immunosuppression, antiviral therapy and/or local radiotherapy, and a few with chemotherapy, the best therapeutic intervention for primary cutaneous B-cell PTLD remains to be further investigated with the analysis of more reported cases and large clinical trials.

Axin gene methylation status correlates with radiosensitivity of lung cancer cells

BackgroundWe previously reported that Axin1 (Axin) is down-regulated in many cases of lung cancer, and X-ray irradiation increased Axin expression and inhibited lung cancer cells. The mechanisms, however, were not clear.MethodsFour lung cancer cell lines were used to detect the methylation status of Axin with or without X-ray treatment. Real-time PCR was used to quantify the expression of Axin, and western blot analysis was applied to measure protein levels of Axin, β-catenin, Cyclin D1, MMP-7, DNMTS, MeCP2 and acetylated histones. Flow cytometric analysis, colony formation assay, transwell assay and xenograft growth experiment were used to study the biological behavior of the cells with hypermethylated or unmethylated Axin gene after X-ray treatment.ResultsHypermethylated Axin gene was detected in 2 of 4 cell lines, and it correlated inversely with Axin expression. X-ray treatment significantly up-regulated Axin expression in H446 and H157 cells, which possess intrinsic hypermethylation of the Axin gene (P<0.01), but did not show up-regulation in LTE and H460 cells, which have unmethylated Axin gene. 2Gy X-ray significantly reduced colony formation (from 71% to 10.5%) in H157 cells, while the reduction was lower in LTE cells (from 71% to 20%). After X-ray irradiation, xenograft growth was significantly decreased in H157 cells (from 1.15 g to 0.28 g) in comparison with LTE cells (from 1.06 g to 0.65 g). Significantly decreased cell invasiveness and increased apoptosis were also observed in H157 cells treated with X-ray irradiation (P<0.01). Down-regulation of DNMTs and MeCP2 and up-regulation of acetylated histones could be detected in lung cancer cells.ConclusionsX-ray-induced inhibition of lung cancer cells may be mediated by enhanced expression of Axin via genomic DNA demethylation and histone acetylation. Lung cancer cells with a different methylation status of the Axin gene showed different radiosensitivity, suggesting that the methylation status of the Axin gene may be one important factor to predict radiosensitivity of the tumor.

Concomitant Waldenstrom macroglobulinemia and IgA plasmablastic myeloma in a patient with untreated IgM paraproteinemia: sequential development of biclonal B-cell neoplasms over a 10-year period in a single individual.

Waldenstrom macroglobulinemia and plasma cell myeloma are both mature B-cell neoplasms primarily involving bone marrow and frequently demonstrating paraproteinemia. Plasma cell myeloma has been described in association with other indolent B-cell lymphomas, particularly chronic lymphocytic leukemia, but concomitant Waldenstrom macroglobulinemia and plasma cell myeloma has not been previously described. We report the first case of sequential development of Waldenstrom macroglobulinemia and plasma cell myeloma over a 10-year period in a 73-year-old man with untreated IgM paraproteinemia. The bone marrow biopsy demonstrated dual populations of lymphoid cells: small mature lymphocytes and immature plasma cells. Although both Waldenstrom macroglobulinemia and plasma cell myeloma were restricted to κ light chain, plasma cell myeloma expressed IgA, whereas the plasmacytic component in Waldenstrom macroglobulinemia produced IgM. The biclonal nature of these 2 B-cell neoplasms was further supported by immunofixation electrophoresis and cytogenetic studies. Although the clinical outcome of plasma cell myeloma when concomitant with other indolent B-cell neoplasms is unfavorable, our patient seemed to respond to high-dose bortezomib plus lenalidomide.

“Cannibalistic” phagocytosis in acute megakaryoblastic leukemia (AML M7) with t(10;17)(p15;q22)

Phagocytic activity is occasionally observed in the malignant cells of hematopoietic neoplasms, such as blasts in acute myeloid leukemia (AML). In the vast majority of cases of leukemic cell phagocytosis, the target or interiorized elements are bystander mature hematopoietic components or their precursors, including erythrocytes, granulocytes, and platelets [1– 10]. Occasionally, bystander lymphocytes are interiorized [2]. The terms hemophagocytosis and/or erythrophagocytosis, therefore, have been used to describe such phagocytic activity in leukemic blasts. This deviation of leukemic cells’ behavior has been reported to be associated with certain cytogenetic abnormalities in AML [1–8]. Here, we describe a unique case of acute megakaryoblastic leukemia (AML M7) with t(10;17)(p15;q22) and prominent cytophagocytic activity. Interestingly, in addition to the usual hemophagocytic activity of leukemic blasts, the leukemic blasts themselves become the phagocytosed target in this case. We designate this blast versus blast phagocytic activity ‘‘cannibalistic’’ phagocytosis or ‘‘cannibalism’’. The patient is a 53-year-old man with a history of chronic renal failure secondary to primary hypertension. He was on hemodialysis since his renal transplant failed 10 years previously. The patient recently developed chronic fatigue, and was found to have pancytopenia. His complete blood cell count showed the following: white blood cells 4.86 10/mL, hemoglobin 8.4 g/dL, and platelets 236 10/mL. The peripheral blood smear showed many circulating blasts (27% of total leukocytes). Subsequent bone marrow examination revealed markedly increased blasts in the aspirate smear. The blasts are medium to large in size, have high nuclear/cytoplasmic ratios, oval to indented nuclear contours, fine chromatin, 1–2 prominent nucleoli, and scant basophilic cytoplasm. Some blasts contain cytoplasmic vacuoles, while others have cytoplasmic blebs [Figure 1 (A– D)]. A few blasts display peri-nuclear clearing with fine azurophilic granules, but Auer rods are not observed. Gigantic blasts with multinucleation are occasionally noted. The blasts comprise approximately 80% of all marrow nucleated cells. Cytochemical analysis shows that blasts are negative for myeloperoxidase and a-naphthyl esterase reactivity. Maturation to mature neutrophils is minimal to essentially absent by morphology. Interestingly, leukemic cell ‘‘cannibalism’’ is frequently observed. The leukemic cell ‘‘cannibalism’’ is characterized by one typically larger leukemic blast (outer cell or cannibal cell) with a crescentic nucleus engulfing another typically smaller blast (interiorized or endocytosed cell) with a round to oval nucleus rimmed by a halo of phagocytic vacuoles [Figure 1(A–D), arrows]. Also seen is the occasional multi-layer cell ‘‘cannibalism’’ simulating an onion-skin appearance [Figure 1(C,D), arrows]. In contrast, erythrophagocytosis by leukemic blasts, though present, is less frequently noted [Figure 1(B), arrow head]. Of note, significant hemophagocytosis or phagocytosis of leukemic blasts by mature histiocytes/macrophages is not evident on the aspirate smear. The bone marrow core biopsy shows approximately 90% cellularity with replacement of marrow spaces by blasts [Figure 1(E)]. Immunoperoxidase analysis reveals the blasts express megakaryocytic antigen markers factor VIII-related antigen [Figure 1(F1)]