Analysis of expression of heat shock protein-90 (HSP90) and the effects of HSP90 inhibitor (17-AAG) in multiple myeloma.

Heat shock protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated an antitumor effect in solid tumors. To test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analysed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55% also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins and akt in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL, MCL-1 and akt. Furthermore, although a low concentration of bortezomib resulted in no cell death, a combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.

Transfection of caspase-3 in the caspase-3-deficient Hodgkin's disease cell line, KMH2, results in enhanced sensitivity to CD95-, TRAIL-, and ARA-C-induced apoptosis.

BACKGROUND: CD95(Fas/apo-1) is a cell surface protein member of the tumor necrosis factor receptor family that serves an important role in the induction of apoptosis in several cell types. Although expression of CD95 has been detected on Hodgkin/Reed Sternberg (HRS) cells in situ, our understanding of the biological significance of this molecule in Hodgkin's disease (HD) is limited. DESIGN: We analyzed both CD95-related apoptotic signaling and its effects on the expression of several factors involved in the regulation of apoptotic mechanisms including: caspase-3, caspase-8, bcl-2, bcl-x, and Bax in HD cell lines (L-428, L-540, HDLM-2, HS-445, and KM-H2). RESULTS: HD cell lines showed similar expression levels of CD95 and all but KM-H2 demonstrated variable increases in apoptosis after CD95 stimulation by the agonistic monoclonal antibody, CH11. There was no significant correlation between CD95 sensitivity and constitutive expression levels of caspase-8, bcl-2, bcl-x, and Bax. Caspase-3 transcript was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) in all cell lines but protein was at low to nearly undetectable levels in KM-H2 cells. Transfection of KM-H2 cells with pro-caspase-3 resulted in a markedly enhanced apoptotic response to CD95 stimulation that was blocked by pretreatment with the caspase-3 inhibitor, DEVD-FMK. In addition, pro-caspase-3-transfected KM-H2 cells showed significantly increased sensitivity to other caspase-3-dependent apoptotic stimuli, including the death-inducing ligand, TRAIL, and the chemotherapeutic agent, Ara-C. CONCLUSION: These data demonstrate that caspase-3 expression plays an important role in CD95-mediated apoptosis in HD cell lines. Furthermore, lack of or decreased expression of caspase-3 in HD cells impairs their apoptotic response not only to CD95 but also to other caspase-3-dependent apoptotic stimuli.

Inhibition of tyrosine kinase activity induces caspase-dependent apoptosis in anaplastic large cell lymphoma with NPM-ALK (p80) fusion protein.

OBJECTIVE: The t(2;5)(p23;q35) translocation creates a fusion gene NPM-ALK (p80) that encodes a product with tyrosine kinase activity believed to play an important role in development of anaplastic large cell lymphoma (ALCL). Our study was aimed to analyze tyrosine kinase activity and phosphotyrosine in ALCLs. We were also interested in determining the effect of tyrosine kinase inhibitors on survival of ALCL. METHODS: Eleven cases of ALCL and three ALCL cell lines with t(2;5)(Karpas-299, SUPM2, SU-DHL-1) and 10 Hodgkin's disease (HD) samples were stained with anti-phosphotyrosine antibody. The tyrosine kinase activity, p80 phosphorylation, and the apoptotic effects of two tyrosine kinase inhibitors, herbimycin A and STI-571, were determined on ALCL cell lines. RESULTS: Herbimycin A had showed both a time- and dose-dependent apoptotic effect on all three cell lines, while STI-571 demonstrated a minimal effect. Following herbimycin A treatment, a decrease in tyrosine kinase activity in the ALCL cell lines and inhibition in NPM-ALK (p80) autophosphorylation was demonstrated by immunoprecipitation and Western blotting. Herbimycin A-induced apoptosis was accompanied by caspase-3 activation. Furthermore, apoptosis induced by herbimycin A was blocked by both z-VAD-FMK and z-DEVD-FMK, suggesting a critical role of caspases. CONCLUSIONS: These findings indicate that tyrosine kinase activity is a common characteristic of ALCLs and necessary for ALCL cell survival. These findings further suggest that therapies targeting tyrosine kinases, including p80, may have clinical utility.

Constitutive expression of NF-kappa B is a characteristic feature of mycosis fungoides: implications for apoptosis resistance and pathogenesis.

The NF-kappa B family of transcription factors is an important regulator of genes expressed during inflammatory responses, immunoglobulin (Ig) class switching, cellular differentiation, and apoptosis. Recently, members of the NF-kappaB family, including p65(Rel A), have been implicated in promoting survival of various hematopoeitic neoplasms, including T cell malignancies such as adult T cell leukemia-lymphoma. We investigated the expression of active NF-kappa B p65(Rel A) in cases of mycosis fungoides (MF) and the effect of chemical inhibitors of NF-kappa B on apoptosis in cutaneous T cell lymphoma (CTCL) cell lines. Paraffin-embedded tissues from 23 cutaneous lesions and a single lymph node biopsy from patients diagnosed with MF were evaluated for p65(Rel A) expression by using a monoclonal mouse antibody that detects the activated form of p65(Rel A). Apoptosis after treatment with the NF-kappa B inhibitors gliotoxin, MG132, BAY 11-7082, and BAY 11-7085 was quantitatively measured in the CTCL cell lines HuT-78 and HH by propidium iodide (PI)/cell cycle analysis for detection of a hypodiploid (sub-G(0)) population and by determination of increased Annexin V/7-amino-actinomycin D (7-AAD) expression. Nuclear extracts from CTCL cells before and after chemical inhibition were analyzed for NF-kappa B nuclear DNA-binding activity by electrophoretic mobility shift assay (EMSA) with quantitative densitometry. Nuclear expression of p65(Rel A) before and after treatment with the various inhibitory compounds was measured by immunofluorescence staining in each CTCL cell line. Neoplastic T lymphocytes from 22 of 24 cases of MF showed strong nuclear and cytoplasmic expression of active p65(Rel A). Compared with untreated control cells, a marked increase in apoptosis, a significant decrease in NF-kappa B DNA-binding activity, and a marked decrease in nuclear p65(Rel A) expression were seen in cells from both CTCL cell lines after chemical NF-kappa B inhibition. These data show that the active form of NF-kappa B p65(Rel A) is commonly expressed in neoplastic T lymphocytes in patients with MF. In CTCL cell lines, the significant decrease in nuclear NF-kappa B expression and the marked increase in spontaneous apoptosis caused by chemical NF-kappa B inhibition suggest a critical role for NF-kappa B in the pathogenesis and tumor cell maintenance of CTCLs. HUM PATHOL 31:1482-1490.

Monoclonal IgH gene rearrangement in microdissected nodules from nodular sclerosis Hodgkin disease.

Recently, single-cell PCR studies have demonstrated that Hodgkin and Reed-Sternberg (HRS) cells are clonally related in many cases of Hodgkin disease. To investigate the lineage and clonality of neoplastic cells in local environments in nodular sclerosis Hodgkin disease (NSHD), we microdissected multiple distinct nodules from patients with NSHD and analyzed them for IgH gene rearrangement by PCR. These results were correlated with immunophenotype, Epstein-Barr-encoded RNA (EBER) expression, and clinical outcome. Forty individual nodules from 10 patients with NSHD (11 specimens) were microdissected from formalin-fixed paraffin-embedded tissue. DNA extracts were analyzed for IgH gene rearrangement by using PCR with FRIIIa and JHa primers. Cases were immunophenotyped in paraffin sections with antibodies to CD20(L26), CD79a(HM57), CD45RO(A6), CD15 (Leu-M1), and CD30(Ber-H2). Infection of HRS cells by Epstein-Barr virus was evaluated by using EBER in situ hybridization (EBER-ISH). DNA extracts from 12 of 40 microdissected nodules from 8 of 10 patients demonstrated a monoclonal pattern by IgH-PCR. Three patients demonstrated 2 individual nodules with different monoclonal patterns. One patient demonstrated 2 nodules with bands that appeared similar in size but were found to be different from one another upon further testing. All 28 remaining nodules demonstrated a polyclonal pattern. Six of 10 patients were positive for the Epstein-Barr virus genome by EBER-ISH. No correlation was found between IgH monoclonality, immunophenotypic features, Epstein-Barr virus infection, or clinical outcome. It was concluded that a subset of NSHD cases contain detectable monoclonality within individual nodules by IgH-PCR, suggesting that HRS cells are clonally related within local microenvironments.

Multiparameter immunohistochemical analysis of the cell cycle proteins cyclin D1, Ki-67, p21WAF1, p27KIP1, and p53 in mantle cell lymphoma.

BACKGROUND: Mantle cell lymphoma (MCL) is characterized by overexpression of cyclin D1, a G1 cyclin that participates in the control of cell cycle progression at the G1 to S phase transition. In addition to cyclin D1, other cell cycle regulatory molecules may be involved in the proliferation and progression of MCL. Mutation of p53, deletion of p16(INK4a), and loss of p21(WAF1) expression have been reported in some cases of blastoid MCL. OBJECTIVE: We sought to examine levels of expression of these proteins in typical and blastoid MCL and to determine whether differences were present between these subtypes of lymphomas. DESIGN: A retrospective series of typical and blastoid MCLs was evaluated for expression of the cell cycle-related proteins cyclin D1, p21(WAF1), p27(KIP1), Ki-67, and p53, as well as mitotic index. Paraffin-embedded archival tissues from 24 MCL specimens (17 typical, 7 blastoid) were immunostained with antibodies to p21(WAF1), p27(KIP1), p53, Ki-67, and cyclin D1. The percentage of positive cells for each specimen was estimated by counting 1500 cells under oil immersion microscopy. Levels of antigen expression were compared for the typical and blastoid MCLs. The mitotic index was estimated using twenty 100x oil immersion fields (OIFs) for each specimen. RESULTS: Cyclin D1 expression was seen in 22/24 specimens (92%). Blastoid MCLs were characterized by a significantly higher mean mitotic index (>20 mitoses/20 OIFs) and Ki-67 index (>45%) when compared with typical MCLs (P <.001 and P <.008, respectively; Fisher's exact test). High expression of p27(KIP1) (>25% staining) was seen more frequently in typical MCLs than in the blastoid variants (P =.03; Fisher's exact test). No significant differences were found between typical and blastoid MCLs for the expression of p21(WAF1) or p53. CONCLUSIONS: A significantly higher mitotic index and Ki-67 index were found in blastoid MCLs as compared with typical MCLs. Low p27(KIP1) expression was associated with the blastoid MCL variant. These findings confirm the high proliferative nature of blastoid MCL and suggest a role for p27(KIP1) in the negative regulation of the cell cycle in MCL.

Metastatic melanoma of the vulva identified by peritoneal fluid cytology.

Malignant melanoma of the vulva is an uncommon disease, with a significant portion of cases demonstrating metastasis to inguinal lymph nodes with potential distal spread. Identification of such metastases often requires fine-needle aspiration or biopsy. The cytologic diagnosis of metastatic vulvar melanoma from peritoneal effusions has not been previously described. We present the case of a 54-yr-old woman who underwent en bloc radical vulvectomy with bilateral inguinal lymphadenectomy for melanoma of the right labium minora. No evidence of metastatic disease was identified, and all surgical margins were free of tumor. Despite chemotherapy, the patient returned approximately 2 yr later with abdominal pain and distention. Computed tomography revealed marked ascites and three hepatic lesions. Cytologic examination of the ascites revealed recurrent, metastatic melanoma. Although very rare, metastatic melanoma of the vulva may present as a malignant effusion. In such an event, the diagnosis may be rendered by exfoliative cytology.

Cytologic diagnosis of peripheral T-cell lymphoma manifesting as ascites. A case report.

BACKGROUND: Patients with malignant lymphoma seldom present with effusions without a known history of malignancy. Because of this, initial diagnosis of malignant lymphoma by effusion cytology is uncommon, with few reported cases. CASE: A 75-year-old male presented with fatigue, decreased appetite and progressively increasing abdominal girth over five weeks. Cytologic examination of ascitic fluid obtained by paracentesis revealed non-Hodgkin's lymphoma with a T-cell phenotype, confirmed by immunophenotypic and molecular studies. Approximately one week later, histologic examination of liver and bone marrow revealed involvement by lymphoma, demonstrating immunophenotypic and molecular profiles identical to those obtained from neoplastic lymphocytes recovered from the ascites fluid. CONCLUSION: This case demonstrates a rare presentation of peripheral T-cell lymphoma, clinically manifesting as ascites. In cases such as ours, where the effusion consists predominantly of small to intermediate-sized lymphocytes, distinguishing lymphoma from reactive lymphocytosis may be difficult. This case not only demonstrates the value of effusion cytology for lymphoma diagnosis but also illustrates how the use of various immunophenotypic and molecular techniques may assist the pathologist in properly diagnosing these difficult cases.

Immunohistochemical analysis of mycosis fungoides on paraffin-embedded tissue sections.

Mycosis fungoides (MF) is typically characterized by dermal and epidermal infiltration of T lymphocytes with a helper/inducer phenotype. Immunophenotypic analysis of such cases was traditionally performed by flow cytometry or immunohistochemistry on cryostat sections. With the advent of new monoclonal antibodies developed against T-cell antigens, including CD3, CD4, CD5, and CD8, it is now possible to immunophenotype T-cell subpopulations in paraffin-embedded tissues. To investigate the potential use of these antibodies for the evaluation of cutaneous lesions, 35 specimens (34 skin and 1 lymph node) from 29 patients with MF were retrospectively reviewed and immunophenotyped in paraffin sections with antibodies to CD3 (T-cell CD3), CD4 (NCL-CD4-1F6), CD5 (NCL-CD5-4C7), CD8 (CD8/144B), and CD20 (L26). Epidermal and dermal distribution of T and B cells were analyzed, and we assessed the ratios of CD4+ to CD8+ T cells. All of our 35 cases demonstrated a predominant CD3+ T-cell population. In 32 cases, the neoplastic cells expressed CD3, CD4, and CD5 consistent with a T-helper/inducer phenotype. In three cutaneous cases, the neoplastic CD4+ T cells showed minimal or absent expression of CD5, indicating an aberrant phenotype. In the majority of cases, minimal CD8+ T cells were present in the background, but in four cases, the CD4:CD8 ratios were 2:1 or less. Thirty-two cutaneous cases demonstrated epidermotropism exclusively by CD4+ T cells; one case showed both CD4+ and CD8+ T cells. In 17 cutaneous cases, scattered dermal CD20+ B cells were found individually or in small clusters within the background surrounding the neoplastic infiltrates. We concluded, therefore, that the immunophenotypic analysis of T-cell subpopulations using monoclonal antibodies of CD3, CD4, CD5, and CD8 was useful for histologic evaluation and confirmation of MF lesions in paraffin-embedded tissue. These antibodies might also provide an effective method of immunophenotyping other neoplastic and non-neoplastic T-cell populations in paraffin-embedded tissues.

Expression of the tumor necrosis factor receptor-associated factors (TRAFs) 1 and 2 is a characteristic feature of Hodgkin and Reed-Sternberg cells.

Tumor necrosis factor receptor-associated factors (TRAFs) are a recently established group of proteins involved in the intracellular signal transduction of several members of the tumor necrosis factor receptor (TNFR) superfamily. Recently, specific members of the TRAF family have been implicated in promoting cell survival as well as activation of the transcription factor NF-kappaB. We investigated the constitutive expression of TRAF1 and TRAF2 in Hodgkin and Reed-Sternberg (HRS) cells from archived paraffin-embedded tissues obtained from 21 patients diagnosed with classical Hodgkin's disease (HD). In a selective portion of cases, examination of HRS cells for Epstein-Barr virus (EBV)-encoded RNA was performed by in situ hybridization, and the results were compared with the magnitude of TRAF1 and TRAF2 staining. We also determined the TRAF profile in the classical HD cell lines L428, KMH2, and HS445 by Western blotting using a series of antibodies that specifically recognize the six individual TRAF family proteins (TRAF1-TRAF6). Moderate to high constitutive expression of TRAF1 and TRAF2 were found in 19 of 21 and 20 of 21 cases of classical HD, respectively. Of the remaining cases, one case showed weak expression of TRAF1, and another case showed weak expression of both proteins. No relationship was found between the staining intensity of the TRAF proteins and EBV expression in HRS cells. Strong constitutive expression of TRAF1 was also identified in the HD cell line L428, compared with the relatively weak expression observed in KMH2 and HS445. All three HD cell lines showed strong expression of TRAF2 protein and moderate, comparatively equal expression of TRAF4 and TRAF6. In contrast, TRAF3 was not expressed in the HD cell lines. Although KMH2 showed weak expression, the remaining HD cell lines also lacked TRAF5 protein. These data demonstrate that constitutive expression of TRAF1 and TRAF2 is a characteristic feature of HRS cells from both patient and cell line specimens. Furthermore, with the exception of TRAF1 expression, HRS cells from the three HD cell lines showed similar TRAF protein expression patterns. Overall, these findings demonstrate the expression of several TRAF proteins in HD. Significantly, the altered regulation of selective TRAF proteins may reflect HRS cell response to stimulation from the microenvironment and potentially contribute both to apoptosis resistance and cell maintenance of HRS cells.

Characterization of the interleukin-1beta-converting enzyme/ced-3-family protease, caspase-3/CPP32, in Hodgkin's disease: lack of caspase-3 expression in nodular lymphocyte predominance Hodgkin's disease.

Apoptosis (programmed cell death) serves an important role in the normal morphogenesis, immunoregulation, and homeostatic mechanisms in both normal and neoplastic cells. Caspase-3/CPP32, a member of the ICE/Ced-3-family of cysteine proteases, is an important downstream mediator of several complex proteolytic cascades that result in apoptosis in both hematopoietic and nonhematopoietic cells. Previous studies have demonstrated that caspase-3 is commonly expressed in classical Hodgkin's disease (CHD); however, the biological significance of its expression in Hodgkin's disease is unknown. In this report, the expression of caspase-3 in nodular lymphocyte predominance Hodgkin's disease (NLPHD) was evaluated by immunohistochemistry; in addition, we investigated the role of caspase-3 in CD95 (Fas)-mediated apoptosis in three CHD cell lines. Formalin-fixed, paraffin-embedded tissue sections from 11 cases of NLPHD were immunostained for caspase-3 using a polyclonal rabbit antibody that detects both the 32-kd zymogen and the 20-kd active subunit of the caspase-3 protease. Only 1/11 cases of NLPHD demonstrated caspase-3 immunopositivity in lymphocytic/histiocytic cells. Caspase-3 expression was also evaluated in three CHD cell lines, HS445, L428, and KMH2. Whereas caspase-3 expression was detected in HS445 and L428 cell lines, no expression was found in KMH2 cells by immunohistochemical staining. Treatment of HS445 and L428 cell lines for 72 hours with agonistic CD95 monoclonal antibody induced marked apoptosis that was significantly inhibited by pretreatment with the caspase-3 inhibitor, DEVD-FMK, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and flow cytometric analysis of 7-amino-actinomycin D staining. In addition, a significant increase in caspase-3 activity as determined by an enzyme colorimetric assay was detected in HS445 and L428 cells after 48 hours of CD95 stimulation. In marked contrast, treatment of caspase-3-deficient KMH2 cells with anti-CD95 mAb did not demonstrate an increase in caspase-3 activity or induce apoptosis. These data demonstrate caspase-3 is important for CD95-mediated apoptosis in CHD cell lines. In addition, the majority of NLPHD cases examined in this study failed to express detectable levels of caspase-3, suggesting these tumor cells may be resistant to apoptotic stimuli dependent on caspase-3 activity. Furthermore, these data suggest the differential expression of caspase-3 noted between NLPHD and CHD may provide additional evidence that each is a unique disease entity.

Characterization of NF-kappaB expression in Hodgkin's disease: inhibition of constitutively expressed NF-kappaB results in spontaneous caspase-independent apoptosis in Hodgkin and Reed-Sternberg cells.

Although the neoplastic cells of classical Hodgkin's disease (CHD) demonstrate high levels of constitutively active nuclear NF-kappaB, the precise physiologic and clinical significance of NF-kappaB expression is currently undefined. Expression of active NF-kappaB p65(Rel A) was evaluated in patient samples of CHD and nodular lymphocyte predominance Hodgkin's disease. The action of the chemical NF-kappaB inhibitors gliotoxin and MG132 and the effect of NF-kappaB inhibition utilizing an adenovirus vector carrying a dominant-negative IkappaBalpha mutant (Ad5IkappaB) were then demonstrated in CHD cell lines (L428, KMH2, and HS445). Hodgkin and Reed-Sternberg (HRS) cells from all patient and cell line specimens showed strong immunopositivity for active p65(Rel A). Expression was also seen in lymphocytic/histiocytic cells from all cases of nodular lymphocyte predominance Hodgkin's disease. After chemical NF-kappaB inhibition, p65(Rel A) was significantly reduced in nuclear extracts from cultured HRS cells as revealed by electrophoretic mobility shift assays. Furthermore, chemical NF-kappaB inhibition resulted in time- and concentration-dependent apoptosis in HRS cells. With the exception of MG132-induced apoptosis in HS445, apoptosis by chemical NF-kappaB inhibition was not significantly altered by preincubation with various caspase inhibitors (z-DQMD-FMK, z-DEVD-FMK, z-VAD-FMK, z-VEID-FMK, and z-IETD-FMK). Regardless of the chemical inhibitor used, no significant change in caspase-3 functional activity was found in CHD cell lines. HRS cells infected with Ad5IkappaB also showed a marked increase in spontaneous apoptosis compared with wild type adenovirus-infected and control cells. Overall, the inhibition of active NF-kappaB in HRS cells resulting in spontaneous caspase-independent apoptosis demonstrates a critical role for NF-kappaB in HRS cell survival and resistance to apoptosis.

Comparison of two PF4/heparin ELISA assays for the laboratory diagnosis of heparin-induced thrombocytopenia.

Serum samples from 105 patients with suspected heparin-induced thrombocytopenia (HIT) were evaluated using the 14C-serotonin release assay (SRA), considered the "gold standard" for the diagnosis of HIT, and two enzyme-linked immunosorbent assays (ELISA) that measure anti-platelet factor (PF) 4/heparin antibodies to determine the performance characteristics of the newly available ELISA assays. Relative to the SRA, the sensitivity and specificity of the Asserachrom HPIA assay were 73% and 77%, respectively, in this population of patients. The sensitivity and specificity of the GTI-HAT assay were 60% and 93%, respectively. In serum negative by SRA, GTI-HAT and HPIA detected antibodies in 9% and 25%, respectively. Antibodies were detected by HPIA in 18% of the sera negative by both SRA and GTI-HAT. In a second study, samples evaluated from patients (n = 10) treated for established thrombosis with a low-molecular-weight heparin and who had no decrease in platelet counts, showed a weak antibody titer in 50% of the patients after 12 days of therapy by GTI-HAT, whereas the HPIA identified a strong antibody titer in 75% of the patients after 4 days. These data suggest that the currently available ELISA methods for the detection of anti-PF4/heparin antibodies offer a limited sensitivity and specificity in comparison to SRA. The ELISA assays on their own are thus of limited value for the laboratory diagnosis of HIT. SRA-positive sera do not always have positive antibody titers, and antibodies can be present in SRA-negative sera. Furthermore, these data show that ELISA methods differ in their relative sensitivities and specificities for the detection of anti-PF4/heparin antibodies.