RIP kinase is involved in arsenic-induced apoptosis in multiple myeloma cells.

These studies explore the molecular effect of arsenicals on MM cells. Freshly isolated cells derived from patients with advanced, chemo-refractory myeloma as well as human myeloma cell lines, ARP-1, RPMI-8226 and H929 were exposed to the organic arsenical melarsoprol and to the inorganic compound AT. Both agents potently induced apoptosis in myeloma cells. Exposure to 1-5 microM AT or melarsoprol for 6 hours suppressed NF-kappa B DNA binding and enhanced of c-Jun kinase (JNK) activity. Arsenic also activated caspase-3 resulting in the cleavage of poly (ADP-ribose) polymerase (PARP) and Fas/TNF alpha related receptor interacting protein (RIP). In contrast to reported observations in acute promyelocytic leukemia, myeloma cell apoptosis was not associated with either the downregulation of Bcl-2 protein or with alterations in the expression of other Bcl-2 family members, Bax, Bak, Bag, and Bcl-xl. This study first shows that arsenic induces apoptotic signaling in MM through the cleavage of TNF alpha related receptor interacting protein (RIP). RIP is a key downstream protein in FasL/ TNF alpha /TRAIL induced apoptosis and a major antiapoptotic adaptor of pathways through NF-kappa B and JNK. RIP has not been previously characterized in myeloma. This study supports the hypothesis that arsenicals share common mediators (RIP, NF-kappa B, PARP, caspase-3) with death receptor induced apoptosis. These studies provide an important insight into the molecular mechanism of AT induced apoptosis and can be used in the development of adjuvant therapy for MM, presently an incurable disease.

Multiple myeloma disrupts the TRANCE/ osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression.

Bone destruction, caused by aberrant production and activation of osteoclasts, is a prominent feature of multiple myeloma. We demonstrate that myeloma stimulates osteoclastogenesis by triggering a coordinated increase in the tumor necrosis factor-related activation-induced cytokine (TRANCE) and decrease in its decoy receptor, osteoprotegerin (OPG). Immunohistochemistry and in situ hybridization studies of bone marrow specimens indicate that in vivo, deregulation of the TRANCE-OPG cytokine axis occurs in myeloma, but not in the limited plasma cell disorder monoclonal gammopathy of unknown significance or in nonmyeloma hematologic malignancies. In coculture, myeloma cell lines stimulate expression of TRANCE and inhibit expression of OPG by stromal cells. Osteoclastogenesis, the functional consequence of increased TRANCE expression, is counteracted by addition of a recombinant TRANCE inhibitor, RANK-Fc, to marrow/myeloma cocultures. Myeloma-stroma interaction also has been postulated to support progression of the malignant clone. In the SCID-hu murine model of human myeloma, administration of RANK-Fc both prevents myeloma-induced bone destruction and interferes with myeloma progression. Our data identify TRANCE and OPG as key cytokines whose deregulation promotes bone destruction and supports myeloma growth.

Polymerase chain reaction detection of Kaposi's sarcoma-associated herpesvirus-optimized protocols and their application to myeloma.

Since its discovery in 1994, KSHV (also called human herpesvirus-8 or HHV8) has been implicated in a variety of disorders. Although the association of KSHV with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease has been well established, its presence in some other diseases, such as multiple myeloma, remains controversial. Because most KSHV studies are based on polymerase chain reaction (PCR) analysis, the conflicting data may be attributable to variations in the methods, primer sets, and target sequences selected. To establish an efficient and reliable PCR approach for KSHV detection we designed eight sets of primers to six regions (ORFK1, ORFK2, ORFK9, ORK26, ORF72, and ORF74) of the KSHV genome using appropriate database and software. The detection sensitivity of these primers was carefully assessed and their reliability was strictly validated in a series of positive (15 KS and PEL samples) and negative (16 lymphoid tissues) controls. We found that primer sets to the ORFK9 region showed the highest sensitivity, whereas primer sets to ORFK1 and ORF74 showed the lowest sensitivity. Primer sets to ORFK9, ORF26 and ORF72 regions detected all of the positive cases, whereas other primer sets showed varying detection rates or nonspecific bands. All 16 negative controls were negative with all primer sets. However, six of 16 negative controls became positive when we used nested PCR targeting ORF26. Therefore, multiple target KSHV sequences increase the detection efficiency, while nested PCR protocols are likely to introduce false positivity. Using ORFK9, ORF26 and ORF72 primer sets, we screened bone marrow biopsies from 18 cases of multiple myeloma, and failed to detect any KSHV sequences. This finding supports the conclusion that KSHV is not associated with multiple myeloma. Indeed, our results further confirm that although KSHV is universally present in Kaposi's sarcoma and primary effusion lymphoma, it is not ubiquitious.

Bilateral optic neuropathy with IgGkappa multiple myeloma improved after myeloablative chemotherapy.

A 49-year-old woman with immunoglobulin GK multiple myeloma developed progressive visual loss with bilateral upper and lower central arcuate scotomas. Funduscopic and electrophysiologic studies indicated bilateral optic neuropathy. The immunoglobulin G fraction of the patient's serum reacted with retinal ganglionic cells in bovine retina. The visual abnormalities remitted after myeloablative chemotherapy and disappearance of the paraprotein.

Hepatic outflow obstruction and liver failure due to leukemic cell infiltration in chronic lymphocytic leukemia.

We report a patient with advanced progressive CLL who presented with liver failure and hepatic venus outflow obstruction (HVOD) due to lymphocytic leukemic infiltrates. Initiation of antileukemic therapy resulted in a rapid and prompt resolution of these life-threatening complications. This is apparently the first report of a CLL patient with HVOD and liver failure, attributable to liver infiltration by leukemic cells.

Multicolor spectral karyotyping identifies new recurring breakpoints and translocations in multiple myeloma.

Karyotypic information on multiple myeloma (MM) is less extensive than that on other myeloid or lymphoid malignancies due to low mitotic activity of plasma cells. An add(14)(q32) marker chromosome has been reported to be the most frequent recurring abnormality in clonally abnormal cases; in approximately one third of the latter cases, this marker has been identified as a der(14)t(11;14)(q13;q32) chromosome. To map chromosomal breakpoints, characterize the add(14)(q32) marker chromosomes, and to identify other recurring translocations in MM, we used spectral karyotyping (SKY) to analyze a panel of nine bone marrow (BM) biopsy samples from eight patients and 10 tumor cell lines derived from MM patients. SKY involves hybridization of 24 fluorescently labeled chromosome painting probes to metaphase spreads in such a manner that simultaneous visualization of each of the chromosomes in a different color is accomplished. By this method, it was possible to define all chromosomal rearrangements and identify all of the clonal marker chromosomes in tumor cells. By detailed mapping of breakpoints of rearrangement, it was also possible to identify several novel recurring sites of breakage that map to the chromosomal bands 3q27, 17q24-25, and 20q11. The partner chromosomes in translocations that generated the add (14)(q32) marker chromosomes were identified in all cases in which they were detected by G-banding (one biopsy and six cell lines). In addition, two new translocations involving band 14q32, ie, t(12;14)(q24;q32) and t(14;20)(q32;q11) have also been identified. These studies demonstrate the power of SKY in resolving the full spectrum of chromosome abnormalities in tumors.

Characterization of nonrandom chromosomal gains and losses in multiple myeloma by comparative genomic hybridization.

Clonal chromosomal changes in multiple myeloma (MM) and related disorders are not well defined, mainly due to the low in vivo and in vitro mitotic index of plasma cells. This difficulty can be overcome by using comparative genomic hybridization (CGH), a DNA-based technique that gives information about chromosomal copy number changes in tumors. We have performed CGH on 25 cases of MM, 4 cases of monoclonal gammopathy of uncertain significance, and 1 case of Waldenstrom's macroglobulinemia. G-banding analysis of the same group of patients demonstrated clonal chromosomal changes in only 13 (43%), whereas by CGH, the number of cases with clonal chromosomal gains and losses increased to 21 (70%). The most common recurrent changes detected by CGH were gain of chromosome 19 or 19p and complete or partial deletions of chromosome 13. +19, an anomaly that has so far not been detected as primary or recurrent change by G-banding analysis of these tumors, was noted in 2 cases as a unique change. Other recurrent changes included gains of 9q, 11q, 12q, 15q, 17q, and 22q and losses of 6q and 16q. We have been able to narrow the commonly deleted regions on 6q and 13q to bands 6q21 and 13q14-21. Gain of 11q and deletion of 13q, which have previously been associated with poor outcome, can thus be detected by CGH, allowing the use of this technique for prognostic evaluation of patients, without relying on the success of conventional cytogenetic analysis.

Hexamethylene bisacetamide induces programmed cell death (apoptosis) and down-regulates BCL-2 expression in human myeloma cells.

Multiple myeloma (MM) is a B cell malignancy characterized by the expansion of monoclonal Ig-secreting plasma cells with low proliferative activity. It is postulated that inhibition of physiologic cell death is an underlying factor in the pathophysiology of MM. The development of chemoresistance is a common feature in patients with MM. In the present studies, hexamethylene bisacetamide (HMBA), a hybrid polar compound that is a potent inducer of terminal differentiation of various transformed cells, is shown to inhibit the growth of several human myeloma cell lines (ARP-1, U266, and RPMI 8226), including doxorubicin-resistant RPMI 8226 variants that overexpress the multidrug-resistance gene, MDR-1, and its product, p-glycoprotein. In addition to growth arrest and suppression of clonogenicity, HMBA induces apoptosis both in freshly isolated human myeloma cells and in cell lines, as determined by morphologic alterations, cell cycle distribution and endonucleosomal DNA fragmentation. Further, HMBA decreases BCL-2 protein expression in myeloma cells within 12-48 hr. Overexpression of BCL-2 protein in ARP-1 cells confers resistance to HMBA-induced apoptosis. Taken together, these data suggest that HMBA is a potent inducer of apoptosis in human myeloma cells, which may act through suppressing the anti-apoptotic function of the bcl-2 gene. HMBA, and related hybrid polar compounds, may prove useful in the management of this presently incurable disease.

Technetium-99m-MIBI versus fluorine-18-FDG in diffuse multiple myeloma.

Experience of scintigraphic detection of bone lesion and active bone marrow involvement of multiple myeloma, especially with sestamibi and FDG-PET scans is in evolution. We report a case of intense sestamibi uptake in bone marrow correlating with the extent of the disease, while FDG-PET scans showed activity only in areas of active disease progression associated with pain. Technetium-99m-sestamibi appears to indicate the extent of the disease, while [18F]FDG-PET scans show sites of active tumor proliferation and may be useful in directing local therapy such as radiation.

Proteinaceous (angiocentric sclerosing) lymphadenopathy: a polyclonal systemic, nonamyloid deposition disorder.

Proteinaceous lymphadenopathy with hypergammaglobulinemia (PLWH) is an exceedingly rare disease of unknown etiology. Described primarily as a pathologic entity, relatively little is known about its clinical manifestations or its response to therapy. The disease is often referred to and treated as an unusual form of plasma cell dyscrasia or light chain deposition disease. We have recently encountered a young patient with PLWH who presented with generalized lymphadenopathy, marked liver function abnormalities, hypocomplementemia, cryoglobulinemia, decreased T4/T8 ratio, and ophthalmopathy. Contrary to the notion that PLWH is a clonal disorder, we found no evidence of clonality in this patient. The most characteristic finding in this and in another patient, previously seen at our institution, was marked angiocentric hyaline sclerosis of the small and mid-sized blood vessels of involved lymph nodes and organs. Based on these findings, we propose the term angiocentric sclerosing lymphadenopathy, which more accurately defines this clinicopathologic entity that appears to be distinct from light chain deposition disease and other plasma cell dyscrasias.

Hypercalcaemia and increased serum interleukin-6 levels induced by all-trans retinoic acid in patients with multiple myeloma.

All-trans retinoic acid (ATRA) inhibits human myeloma cell growth in vitro, presumably through the down-regulation of interleukin 6 receptors (IL-6R). Based on these and other studies, we initiated a phase II clinical trial using ATRA in patients with advanced refractory multiple myeloma (MM). We report that three out of six treated patients developed severe hypercalcaemia following administration of ATRA, which was accompanied by a significant rise in serum IL-6 levels. Normal calcium levels were restored after the discontinuation of the drug and the administration of standard anti-hypercalcaemic care. We suspect that down-regulation of IL-6R resulted in increased serum IL-6 levels, leading to advanced bone resorption and hypercalcaemia. We conclude that the use of ATRA in patients with advanced MM is not warranted.

Protein kinase C mediates basic fibroblast growth factor protection of endothelial cells against radiation-induced apoptosis.

Basic fibroblast growth factor (bFGF) was found to protect bovine aortic endothelial cells against the lethal effects of ionizing radiation by inhibiting the programmed cell death (apoptosis) induced in these cells by radiation exposure. The involvement of the bFGF receptor tyrosine kinase in this function was demonstrated by abrogation of the radioprotective effect of bFGF by a specific inhibitor of the bFGF receptor tyrosine kinase, the tyrphostin AG213. The downstream signaling after stimulation of the bFGF receptor tyrosine kinase in bovine aortic endothelial cells involved translocation of the alpha isotype of cytoplasmic protein kinase C (PKC) into the membrane and its activation within 30 s after bFGF stimulation. The involvement of PKC in the radioprotective effect conferred by bFGF was suggested by the demonstration that nonspecific PKC activation by short-term exposure (30 min) to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA; 30 ng/ml) mimicked the radioprotective effect of bFGF. Furthermore, treatment of the cells with the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (20 microM) abrogated the radioprotective effect of bFGF, as was observed after the depletion of cellular PKC by overnight preincubation with high-dose TPA (200 nM). Agarose gel electrophoresis of DNA extracted from irradiated bovine aortic endothelial cells showed that both TPA (30 ng/ml; 30 min) and bFGF (1 ng/ml) inhibited the apoptotic degradation of DNA induced in these cells by radiation exposure (500 cGy). Both the bFGF- and the TPA-mediated inhibition of apoptosis could be reversed by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (20 microM). These data demonstrate the involvement of PKC in the inhibition of radiation-induced apoptosis by bFGF and the rescue of endothelial cells from this mode of radiation-induced cell death.

Induction of differentiation of myeloid leukemic cells by busulphan: in vivo and in vitro observations.

Treatment of a 74 year old patient with chronic myelogenous leukemia (CML) with busulphan resulted in an abrupt and pronounced decrease of the white blood cell (WBC) count with restoration of normal peripheral blood cell morphology and regression of splenomegaly. The Philadelphia positive (Ph+) clone was however still detectable. The alterations in the WBC count and morphology were not preceded by marrow hypoplasia but correlated closely with a marked decrease in the serum levels of Transcobalamin I (TC I), a vitamin B12-binding protein derived from immature myeloid precursors and a reciprocal rise in serum TC III, a vitamin B12-binding protein originating from terminally differentiated mature granulocytes. Studies on the HL-60 cell line showed that busulphan is capable of inducing leukemic cells to differentiate into granulocyte-like cells. These observations, taken together, suggest that in addition to its potent myelosuppressive effects, busulphan may induce apparent clinical remissions in some CML patients by promoting terminal cell differentiation.

Excessive hepatitis-B surface antigen production after corticosteroids and the development of immunoblastic lymphoma.

The development of B-cell immunoblastic lymphoma in a 51 year old patient with hepatitis B virus related immune complex nephritis is described. The lymphoma was diagnosed eleven months after the administration of high dose corticosteroids which resulted in a striking increase in the HBsAg serum titer up to 1:1,000,000. This unusual association raises a number of interesting questions regarding the link between immune complex nephritis and immunoblastic lymphoma. The significance of corticosteroid therapy leading to excessive HBsAg secretion and the role played by this intense antigenic stimulation in the pathogenesis of lymphoma in this particular patient are also discussed.

Biology and treatment of multiple myeloma.

The uniformly fatal plasma cell malignancy, multiple myeloma (MM), currently represents 10-15% of hematologic neoplasms in the USA and has been steadily increasing in incidence for several decades. Therapeutic alternatives have lagged significantly behind insights into the biology and pathogenesis of this entity. Traditionally felt to be a neoplasm of fully differentiated plasma cells, evidence has been mounting that the self renewing population consist of cells derived from a much earlier compartment; perhaps prior to B-cell lineage commitment or even at the level of an earlier 'stem cell'. Bcl-2 protein overexpression has been almost uniformly seen in both clinical myeloma specimens as well as in myeloma cell lines. The failure to consistently identify the t(14;18) translocation, normally found in follicular lymphomas and characteristically associated with overexpression of bcl-2, implies a unique mechanism in MM. A number of cytokines, including TNF alpha, IL-1 and IL-6 have been found to play a central role not only in the biology of the malignant clone but also in the bony and other systemic manifestations of this disease. Since both IL-6 and bcl-2 protein have been shown to prevent programmed cell death, this may be the unifying event in MM. Standard therapy for MM has been an alkylating agent and corticosteroid. Combination chemotherapy provides more prompt palliation but no clear survival advantage. In advanced stages, adriamycin may offer some survival advantage. High dose chemotherapy with or without stem cell support offers a potentially curative therapeutic approach. New interventions directed at the complex cytokine networks pertinent to the pathogenesis of MM are an exciting new area of investigation. Identification of new prognostic parameters as well as new active agents remains the central theme in clinical myeloma research.