APRIL promotes cell-cycle progression in primary multiple myeloma cells: influence of D-type cyclin group and translocation status.

A proliferation-inducing ligand (APRIL) promotes survival and drug resistance in multiple myeloma (MM) cell lines. We studied the effect of APRIL on cell-cycle behavior in primary MM cells and correlated our findings with D-type cyclin expression by immunohistochemistry and/or Western blotting. In MM cases, expressing cyclin D2 APRIL significantly increased the percentage of CD138(+) cells in S + G(2)/M phase (from 8.4% ± 1.9% to 14.3% ± 2.6%, n = 15, P < .01), whereas a lesser effect was seen in cases expressing cyclin D1 (n = 18). Cell-cycle response to APRIL was most marked for cyclin D2-expressing cases with IgH translocations (P < .01) and was accompanied by increased expression of cyclin D2, CDK4, CDK6, and phospho-retinoblastoma protein. Cell-cycle proteins in cyclin D1(+) cells were not modulated by APRIL. Surface expression of B-cell maturation antigen and transmembrane activator and calcium-modulating cyclophilin ligand interactor was not significantly different between cyclin D1(+) and D2(+) MM cells. We observed activation of nuclear factor-κB and PI3-kinase pathways in response to APRIL in both cyclin D1(+) and D2(+) MM cells. In conclusion, APRIL stimulates G(1)/S progression in cyclin D2(+) MM cells bearing IgH translocations but has minimal effect on cyclin D1(+) cells, suggesting MM cells from different cyclin D/translocation classes rely on different mechanisms for cell-cycle re-entry.

A novel role for Wnt/Ca2+ signaling in actin cytoskeleton remodeling and cell motility in prostate cancer.

Wnt signaling is a critical regulatory pathway in development and disease. Very little is known about the mechanisms of Wnt signaling in prostate cancer, a leading cause of death in men. A quantitative analysis of the expression of Wnt5A protein in human tissue arrays, containing 600 prostate tissue cores, showed >50% increase in malignant compared to benign cores (p<0.0001). In a matched pair of prostate cancer and normal cell line, expression of Wnt5A protein was also increased. Calcium waves were induced in prostate cells in response to Wnt5A with a 3 fold increase in Flou-4 intensity. The activity of Ca(2+)/calmodulin dependent protein kinase (CaMKII), a transducer of the non-canonical Wnt/Ca(2+) signaling, increased by 8 fold in cancer cells; no change was observed in beta-catenin expression, known to activate the canonical Wnt/beta-catenin pathway. Mining of publicly available human prostate cancer oligoarray datasets revealed that the expression of numerous genes (e.g., CCND1, CD44) under the control of beta-catenin transcription is down-regulated. Confocal and quantitative electron microscopy showed that specific inhibition of CaMKII in cancer cells causes remodeling of the actin cytoskeleton, irregular wound edges and loose intercellular architecture and a 6 and 8 fold increase in the frequency and length of filopodia, respectively. Conversely, untreated normal prostate cells showed an irregular wound edge and loose intercellular architecture; incubation of normal prostate cells with recombinant Wnt5A protein induced actin remodeling with a regular wound edge and increased wound healing capacity. Live cell imaging showed that a functional consequence of CaMKII inhibition was 80% decrease in wound healing capacity and reduced cell motility in cancer cells. We propose that non-canonical Wnt/Ca(2+) signaling via CaMKII acts as a novel regulator of structural plasticity and cell motility in prostate cancer.

The use of immunoglobulin g4 immunostaining in diagnosing pancreatic and extrapancreatic involvement in autoimmune pancreatitis.

BACKGROUND & AIMS: Autoimmune pancreatitis (AIP) is recognized increasingly as a multisystem disorder. We evaluated the use of immunoglobulin (Ig)G4 immunostaining of pancreatic and extrapancreatic biopsy specimens to make a definitive diagnosis of AIP. METHODS: Seventeen biopsy specimens and 3 gallbladder resections were assessed from 11 patients with clinical and radiologic features of AIP. Biopsy specimens from pancreas, liver, colon, stomach, duodenum, bone marrow, salivary gland, and kidney were analyzed morphologically, immunostained for IgG4-positive plasma cells, and compared with controls. RESULTS: Positive IgG4 immunostaining enabled a definitive diagnosis in 10 of 11 (91%) AIP patients. In both pancreatic and extrapancreatic tissues, high levels of IgG4 immunostaining (>10 IgG4-positive plasma cells/high-power field) were found in 17 of 20 (85%) specimens from AIP patients compared with 1 of 175 (0.6%) specimens from controls (P < .05). Positive extrapancreatic IgG4 immunostaining was found in 8 of 11 (73%) patients, including all those with diagnostic features in the pancreas. Increased tissue IgG4 was found irrespective of serum IgG4 level. CONCLUSIONS: The finding of IgG4 immunostaining within a range of clinically involved tissues supports the hypothesis that AIP is a multisystem disease. Positive IgG4 immunostaining in extrapancreatic tissues may allow a definitive diagnosis of AIP to be made in those with evidence of pancreatic disease, without the necessity of pancreatic biopsy or surgical exploration. Immunostaining of involved tissue for IgG4 may be particularly useful when AIP is suspected clinically but the serum IgG4 level is normal.

Angioimmunoblastic T-cell lymphoma: histological progression associates with EBV and HHV6B viral load.

The clinical and histological presentations of angioimmunoblastic T-cell lymphoma (AITL) often mimic an infectious process. Epstein-Barr virus (EBV) and human herpes virus (HHV6) are known to be associated with AITL, but whether these viral infections play a role in its pathogenesis is unclear. It also remains to be investigated whether there might be other viruses associated with AITL. We first screened 26 well-characterised cases of AITL for herpesvirus by polymerase chain reaction (PCR) with universal primers and found evidence of only EBV and HHV6B infection. Subsequent PCR using virus-specific primers demonstrated EBV and HHV6B infection in 40/49 biopsies (36/42 cases) and 21/49 biopsies (19/42 cases) of AITL respectively with both viral infections found in 17/49 specimens (15/42 cases). Importantly, simultaneous infection with both viruses was found only in specimens showing histological pattern II (n = 2) or III (n = 15). Interestingly, among specimens containing both viruses, there was a tendency towards an inverse correlation between the EBV and HHV6B viral load as shown by quantitative PCR. In specimens positive only for EBV, the viral load was significantly higher in specimens with histological pattern III than those with pattern II. High EBV load was also significantly associated with B-cell monoclonality. Double EBV encoded small RNA (EBER) in situ hybridisation and immunohistochemistry indicated that EBV-infected B cells had a late postgerminal centre immunophenotype. Our results demonstrate an association between EBV and HHV6B infection and the histological progression of AITL, suggesting that these viruses may play a role in the pathogenesis of this lymphoma.

DNA replication licensing in peripheral B-cell lymphoma.

Peripheral B-cell lymphomas representing 90% of lymphoid neoplasms are divided into low- and high-growth fraction lymphomas. Here we investigate regulation of DNA replication licensing during B-cell lymphomagenesis. Combined analysis of origin licensing factors Mcm2 and geminin with the proliferation marker Ki67 in SLL/CLL, MCL, DLBCL and Burkitt lymphoma reveals for the first time the precise cell cycle state of these entities. Given that tight Mcm2 downregulation defines the quiescent state (G0) and that both high- and low-growth fraction lymphomas express Mcm2, the data demonstrate that neoplastic lymphocytes of SLL/CLL and MCL reside in an "in-cycle" G1 state and not in G0 as previously thought. Absence of the S/G2/M phase marker geminin in SLL/CLL and MCL further indicates failure of cell cycle progression in these tumours. In contrast, the high-growth fraction lymphomas DLBCL and Burkitt lymphoma exhibit differential expression of geminin, with the geminin/Ki67 ratio increasing for more aggressive neoplasms in keeping with a shortened G1 phase and thus representing an important discriminator for differential diagnosis. These data provide new insights into abrogation of cell cycle control during B cell lymphomagenesis and suggest that combined analysis of origin licensing factors may contribute to improved treatment decisions and prognosis in haematopoietic malignancies.

Anaplastic lymphoma kinase (ALK 1) staining and molecular analysis in inflammatory myofibroblastic tumours of the bladder: a preliminary clinicopathological study of nine cases and review of the literature.

Inflammatory myofibroblastic tumours (IMFT) may arise at any anatomical site, including lung, soft tissues, retroperitoneum and bladder. Although morphologically similar, these lesions encompass a spectrum of entities with differing aetiology, ranging from reactive/regenerative proliferations to low-grade neoplasms with a risk of local recurrence, but no significant metastatic potential. Vesical IMFT usually presents as a polypoid mass with a pale firm cut surface and can be of considerable size, mimicking a malignant tumour clinically and radiologically. Its good outcome, however, warrants conservative surgical excision, emphasising the importance of identification and distinction from malignant tumours of the bladder that may require more radical surgery and/or adjuvant therapy. We conducted a preliminary retrospective, comparative immunocytochemical study of 20 bladder tumours, including nine IMFTs, five spindle cell (sarcomatoid) carcinomas, two rhabdomyosarcomas, two leiomyosarcomas and two neurofibromas. The results confirmed IMFT positivity for smooth muscle actin, desmin and cytokeratin in 78-89% cases, resulting in potential confusion with sarcomatoid carcinoma or leiomyosarcoma. In contrast, cytoplasmic anaplastic lymphoma kinase (ALK 1) staining was present in eight IMFT (89%), but was not seen in any other lesion examined. The ALK 1 staining was confirmed by fluorescence in situ hybridisation, with translocation of the ALK gene present in 15-60% tumour cells in four of six IMFT examined, but not in four cases of sarcomatoid carcinoma or three of leiomyosarcoma. In conclusion, ALK 1 staining may be of value in the distinction of vesical IMFT from morphologically similar entities, and often reflects ALK gene translocations in these lesions.

CD10 expression in extranodal dissemination of angioimmunoblastic T-cell lymphoma.

Angioimmunoblastic T-cell lymphoma (AITL) is a systemic disease that often has evidence of extranodal involvement at presentation. In a recent study of lymph nodes in AITL, we showed that the neoplastic T cells in most cases can be identified by aberrant expression of CD10. The aim of this study was to investigate whether CD10 expression by the neoplastic T cells is maintained in extranodal sites. Ten cases of AITL with histologic and immunophenotypic evidence of extranodal dissemination were studied. Seven cases of peripheral T-cell lymphoma unspecified (PTLu), that included biopsies of involved extranodal sites, two cases of enteropathy type T-cell lymphoma (ETTL), and one case of extranodal NK/T lymphoma, nasal type were selected as controls. Diagnostic lymph node biopsies and biopsies of extranodal sites were reviewed. PCR for T-cell clonality and single layer immunostaining for CD3, CD20, CD10, and CD21 and double layer immunostaining for CD20/CD10 were performed. All 10 cases of AITL had characteristic histologic features and molecular evidence of the disease in lymph node biopsies. In these cases, aberrant CD10 expression was maintained in the lung, cecum, tonsil, nasopharynx, and one of six involved bone marrow trephines. In these extranodal biopsies, the distribution of CD10-positive tumor cells correlated with that of the follicular dendritic cell meshwork (FDC). The five bone marrow trephines that lacked aberrant CD10 expression were devoid of morphologic and immunohistochemical evidence of FDC. In these five cases, there was evidence of aberrant CD10 expression in other involved sites that had FDC. The neoplastic cells in PTLu, ETTL, and extranodal NK/T lymphoma, nasal type were CD10 negative. Our data show that aberrant CD10 expression is a useful phenotypic marker for diagnosis of AITL in most involved extranodal sites, except bone marrow, and suggest a possible role of FDC in the pathogenesis of AITL.

Productive HIV-2 infection in the brain is restricted to macrophages/microglia.

OBJECTIVES: HIV-2 can use a broader range of co-receptors than HIV-1 in vitro, and is less dependent on CD4 for infection. The aim of this study was to detect productive HIV-2 infection in the brain and investigate whether HIV-2 has an expanded tropism for brain cells in vivo, in comparison with HIV-1, which productively infects macrophages/microglia. DESIGN: Brain samples taken at autopsy from eight patients who died from AIDS, six HIV-2 and two HIV-1/HIV-2 dually seropositive, with HIV encephalitis (HIVE), collected in Abidjan, Côte d'Ivoire in 1991, were examined for the presence and localization of productive HIV-2 infection. METHODS: Using immunohistochemistry, the presence of HIV-2 p26 in formalin-fixed, wax-embedded brain tissue sections was investigated. Double-staining with glial fibrillary acidic protein (GFAP), CD45- and CD68-specific antibodies was performed to identify infected cell types. RESULTS: HIV-2 p26 was detected in brain tissue from four of the HIV-2 cases and one of the dually infected individuals. The productively infected cells were either microglia or infiltrating macrophages. CONCLUSIONS: The productively infected cells in the brains of HIV-2 infected individuals are macrophages/microglia. No evidence was found for productive infection of astrocytes, neurons or oligodendrocytes. Thus, the broader in vitro cell tropism, promiscuous coreceptor usage and relative independence of CD4 by HIV-2 compared to HIV-1 does not broaden its range of target cells in the brain.