Expression of p16INK4A and p14ARF in hematological malignancies.

The INK4A/ARF locus yields two tumor suppressors, p16INK4A and p14ARF, and is frequently deleted in human tumors. We studied their mRNA expressions in 41 hematopoietic cell lines and in 137 patients with hematological malignancies; we used a quantitative reverse transcription-PCR assay. Normal peripheral bloods, bone marrow and lymph nodes expressed little or undetectable p16INK4A and p14ARF mRNAs, which were readily detected in 12 and 17 of 41 cell lines, respectively. Patients with hematological malignancies frequently lacked p16INK4A expression (60/137) and lost p14ARF expression less frequently (19/137, 13.9%). Almost all patients without p14ARF expression lacked p16INK4A expression, which may correspond to deletions of the INK4A/ARF locus. Undetectable p16INK4A expression with p14ARF expression in 41 patients may correspond to p16INK4A promoter methylation or to normal expression status of the p16INK4A gene. All patients with follicular lymphoma (FL), myeloma or acute myeloid leukemia (AML) expressed p14ARF while nine of 23 patients with diffuse large B cell lymphoma (DLBCL) lost p14ARF expression. Patients with ALL, AML or blast crisis of chronic myelogenous leukemia expressed abundant p16INK4A mRNAs more frequently than patients with other diseases (12/33 vs 6/104, P < 0.01). Patients with FL and high p14ARF expression had a significantly shorter survival time while survival for patients with DLBCL and increased p14ARF expression tended to be longer. These observations indicate that p16INK4A and p14ARF expression is differentially affected among hemato- logical malignancies and that not only inactivation but also increased expression may have clinical significance.

Bone marrow stromal cells produce and respond to activin A: interactions with basic fibroblast growth factor and platelet-derived growth factor.

Activin A, a homodimer of the beta A-chain, regulates hematopoiesis. We recently reported that murine bone marrow (BM) stromal cells, ST2 and MC3T3-G2/PA6, produce activin A [16]. Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF), potent mitogens of BM stromal cells, induced a rapid increase in beta A-chain mRNA levels and activin secretion in these cells. Cycloheximide (CHX) did not inhibit the increases in beta A-chain mRNA levels, suggesting that these growth factors directly stimulate beta A-chain gene expression. Furthermore, activin A stimulated mitogenesis in ST2 cells, by itself and with bFGF and PDGF. Consistent with this observation, we detected mRNAs of activin A receptors in the murine stromal cells. These findings suggest that BM stromal cells, stimulated by bFGF and PDGF, produce activin A, which may stimulate stromal cells themselves in concert with these peptide growth factors.

Growth arrest associated with 12-o-tetradecanoylphorbol-13-acetate-induced hematopoietic differentiation with a defective retinoblastoma tumor suppressor-mediated pathway.

The retinoblastoma tumor suppressor (Rb) gene product plays an essential role in cell-cycle regulation. However, its role in terminal differentiation of hematopoietic cells is speculative. Here we show a model of 12-o-tetradecanoylphorbol-13-acetate (TPA)-induced hematopoietic differentiation and growth arrest with a defective Rb-mediated pathway. TPA treatment arrested the cell cycle of a human hematopoietic cell line, MEG-01s, at the G1-S boundary and induced expression of p21/SDI1/WAF1/CIP1 and p27/KIP1. Both of these proteins were present in cyclin E-associated complexes, the histone H1 and Rb kinase activities of which were then inactivated. However, MEG-01s cells lacked the intact Rb protein and the Rb-mediated pathway was defective. This model raises a question about the role for Rb in terminal differentiation of hematopoietic cells.

Effect of cyclophosphamide on serum cyclosporine levels at the conditioning of hematopoietic stem cell transplantation.

We retrospectively analyzed the factors that affect serum cyclosporine (CsA) concentrations up to day 14 after allogeneic hematopoietic stem cell transplantation (HSCT). In all, 103 transplant recipients who received MTX and CsA for acute GVHD prophylaxis were analyzed. No significant relationships between serum CsA concentrations and gender, age, serum creatinine levels, AST/ALT levels, or antibiotic/fluconazole administration were found by comparing median CsA concentrations or by using longitudinal or regression multivariate analyses. However, the mean of the median serum CsA concentration in patients (n=54) receiving the regimen containing cyclophosphamide (CY) (149.7 ng/ml; 95% confidence interval (CI): 132.1-167.4) was significantly (P<0.0001) lower than that in patients (n=49) receiving the non-CY regimen (217.3 ng/ml; 95% CI: 198.9-235.6). Longitudinal analysis and regression multivariate analysis showed that only administration of CY had a significant effect on the serum CsA concentration. Our results suggest that administration of CY during conditioning can reduce the effects on serum CsA concentrations during the 2 weeks following HSCT. The mechanism of this effect is not clear, but it may be due to the autoinduction of CY.

Herpes simplex virus infection in adult patients after unrelated cord blood transplantation: a single-institute experience in Japan.

Herpes simplex virus (HSV) infection in adult patients who underwent cord blood transplantation (CBT) from unrelated donors was studied. None of nine HSV-seronegative patients developed HSV disease after CBT. Of 28 HSV-seropositive patients, seven (25%) developed HSV disease at a median of 92 days after CBT (range, 52-239 days). The cumulative incidence of HSV disease in HSV-seropositive patients was 27% at 12 months after CBT. The manifestations of HSV disease included gingivostomatitis (three patients), herpes labialis (two patients), localized herpes facialis of the nose (one patient), and disseminated eczema herpeticum (one patient). HSV disease recurred in two patients as gingivostomatitis and disseminated eczema herpeticum. All the patients responded to antiviral therapy. The presence of grade II-IV acute graft-versus-host disease (GVHD) was significantly associated with a higher rate of HSV disease after CBT (51 vs 8%, P=0.015). These results suggest that the recovery of HSV-specific immune responses is delayed in patients who develop grade II-IV acute GVHD after CBT.

Overexpression of the PRAD1 oncogene in a patient with prolymphocytic leukemia with t(11;14)(q13;q32).

Prolymphocytic leukemia (PLL) was diagnosed by morphologic and immunophenotypical studies in a 72-year-old Japanese man. Massive splenomegaly was present but lymphadenopathy was minimal in this case. Chromosomal analysis of peripheral mononuclear cells showed t(11;14)(q13;q32) in all metaphases examined, except for one normal karyotype. Northern blot analysis of RNA prepared from leukemic cells obtained from the patient revealed overexpression of the PRAD1/cyclin D1 proto-oncogene, which has not been described previously in patients with PLL.

Clinical significance of serial measurement of the serum levels of soluble interleukin-2 receptor and soluble CD8 in malignant lymphoma.

We serially measured the serum levels of soluble interleukin-2 receptor (sIL-2R) and soluble CD8 (sCD8) in 36 patients with malignant lymphoma (33 non-Hodgkin's lymphoma cases and three Hodgkin's disease cases). The level of serum sIL-2R was significantly elevated in patients with active disease (18) compared to those in remission (18), and correlated with the clinical stage of the lymphoma. The temporal profile of the sIL-2R level reliably represented the disease status, which was judged clinically, during the course of the disease. In three patients, the tumor bulk paralleled the sIL-2R level. On the other hand, a less significant correlation was found between the serum sCD8 level and disease activity. The serial measurement of sCD8 appeared to be less useful for monitoring the disease activity, although there was a significant correlation between the sCD8 and sIL-2R levels. This study indicates that serial measurement of the serum sIL-2R level may be useful for monitoring the tumor burden in response to treatment and for early detection of disease progression in malignant lymphoma.

[Mantle cell lymphoma with multiple extranodal involvement].

A 79-year-old male was admitted to our hospital because of general fatigue and night sweat. Physical examination showed generalized superficial lymphadenopathy, marked splenomegaly, and tumors in the conjunctiva and the abdomen. Chest X-ray and computed tomography (CT) revealed pleural effusion and intrathoracic lymphadenopathy. Abdominal ultrasonography and CT showed hepatosplenomegaly and intraperitoneal tumors. Upper gastrointestinal fiberscopy revealed multiple polypoid lesions and ulcers in the duodenum and the stomach. Involvement of relatively small-sized lymphocytes with cleaved nuclei was identified in each biopsied specimen from a cervical lymph node, a tumor in the conjunctiva, gastrointestinal polypoid lesions, and the bone marrow. Surface marker analysis of abnormal lymphocytes in the bone marrow revealed that CD5, CD19, and CD20 were strongly positive, but CD23 was weakly positive. Although (11:14)(q13:q32) translocation was not identified by chromosome analysis of bone marrow cells, Northern blot analysis of bone marrow cells revealed overexpression of the PRAD1 oncogene. Diagnosis of mantle cell lymphoma (MCL) was made. Combination chemotherapy by cyclophosphamide and vincristine was not effective, but etoposide perorally given at a dose of 50 mg per day was effective. In MCL, extranodal involvement of a digestive tract and bone marrow is well known. This case suggests that involvement of multiple organs including lacrimal glands and pleura could be characteristic of MCL cells.

Impact of sex incompatibility on the outcome of single-unit cord blood transplantation for adult patients with hematological malignancies.

Donor-recipient sex incompatibility has been associated with transplant outcomes in allogeneic hematopoietic SCT. Such outcomes might be because mHA encoded by Y chromosome genes could be immunological targets for allogeneic T cells and B cells to induce GVHD, GVL effect and graft failure. However, its effect on the outcome of cord blood transplantation (CBT) is yet to be clarified. We retrospectively analyzed 191 adult patients who received single-unit CBT after myeloablative conditioning for malignant disease in our institute. In multivariate analysis, male recipients with female donors had a higher incidence of extensive chronic GVHD (hazard ratio (HR) 2.97, P=0.02), and female recipients with male donors had a lower incidence of platelet engraftment (HR 0.56, P=0.02) compared with female recipients with female donors as the reference. Nevertheless, there was no increase in mortality following sex-incompatible CBT. These data suggested that donor-recipient sex compatibility does not have a significant impact on survival after myeloablative CBT for hematological malignancies.

Tumor necrosis factor and interleukin-1 induce activin A gene expression in a human bone marrow stromal cell line.

Activin A, a homodimer of the beta A chain, regulates hematopoiesis. In a human bone marrow-derived stromal cell line, KM-102, phorbol myristate acetate, tumor necrosis factor-alpha and interleukin-1 beta induced great increases in beta A chain mRNA levels and production of activin A activities. The phorbol ester-induced beta A chain gene expression was inhibited by cycloheximide and down regulation of protein kinase C, whereas the cytokine-induced expression was little affected by these treatments. These results indicate that the inflammatory cytokines directly stimulate beta A chain gene expression via protein kinase C-independent pathways.

Detection of cyclin D1 (bcl-1, PRAD1) overexpression by a simple competitive reverse transcription-polymerase chain reaction assay in t(11;14)(q13;q32)-bearing B-cell malignancies and/or mantle cell lymphoma.

In mantle cell lymphoma, the t(11;14)(q13;q32) and its molecular counterpart, bcl-1 rearrangement, are consistent features and lead to cyclin D1 (bcl-1, PRAD1) proto-oncogene overexpression. In order to detect cyclin D1 overexpression, we developed a simple assay involving a reverse transcription followed by competitive polymerase chain reaction (PCR). A single upstream primer was derived from a homologous region between cyclin D1 and the other D-type cyclins, cyclins D2 and D3, while three downstream primers were specific to their respective D-type cyclins. Because the upstream primer was shared in PCR amplification of the three sequences, each PCR product served as a competitor and the quantification of the target was made by comparison of the intensity of the three products. With this assay we analyzed 45 hematopoietic cell lines and 40 clinical specimens. Cyclin D1 was rarely expressed in lymphoid cell lines except in t(11;14)(q13;q32)-bearing B-cell malignancies and/or mantle cell lymphoma, which expressed cyclin D1 predominantly. In myeloid cell lines, the levels of cyclin D1 expression varied and never exceeded the sum of cyclin D2 and D3 levels. Cyclin D3 was ubiquitously expressed while cyclins D1 and D2 were differentially used. The observations suggest that human cyclin D3 may play a fundamental role in hematopoiesis and that cyclins D1 and D2 may have different lineage- or differentiation-dependent functions. With this assay, small aliquots of clinical specimens such as 100 microL peripheral blood were enough to detect cyclin D1 overexpression without a well-controlled standard. The technique was validated as highly comparable with Northern analysis. This rapid and reliable detection of cyclin D1 overexpression may have practical clinical utility in the analysis and management of B-cell malignancies.

Oncogenic collaboration of the cyclin D1 (PRAD1, bcl-1) gene with a mutated p53 and an activated ras oncogene in neoplastic transformation.

Cyclin D1 is one of the key regulators in G1 progression in the cell cycle and is also a candidate oncogene (termed PRAD1 or bcl-1) in several types of human tumors. We report a collaboration of the cyclin D1 gene with ras and a mutated form of p53 (p53-mt) in neoplastic transformation. Transfection of cyclin D1 alone or in combination with ras or with p53-mt was not sufficient for focus formation of rat embryonic fibroblasts. However, focus formation induced by co-transfection of ras and p53-mt was enhanced in the presence of the cyclin D1-expression plasmid. Co-transfection of ras- and p53-mt-transformants with the cyclin D1-expression plasmid resulted in reduced serum dependency in vitro. Furthermore, the transformants expressing exogenous cyclin D1 grew faster than those without the cyclin D1 plasmid when injected into nude mice. These observations strengthen the significance of cyclin D1 overexpression through gene rearrangement or gene amplification observed in human tumors as a step in multistep oncogenesis; deregulated expression of cyclin D1 may reduce the requirement for growth factors and may stimulate in vivo growth.

Expression of p21(Cip1/Waf1/Sdi1) and p27(Kip1) cyclin-dependent kinase inhibitors during human hematopoiesis.

Expression of p21 and p27 cyclin-dependent kinase inhibitors is associated with induced differentiation and cell-cycle arrest in some hematopoietic cell lines. However, it is not clear how these inhibitors are expressed during normal hematopoiesis. We examined various human hematopoietic colonies derived from cord blood CD34(+) cells, bone marrow, and peripheral blood cells using a quantitative reverse transcription-polymerase chain reaction assay, immunochemistry, and/or Western blot analysis. p21 mRNA was expressed increasingly over time in all of the colonies examined (granulocytes, macrophages, megakaryocytes, and erythroblasts), whereas p27 mRNA levels remained low, except for erythroid bursts. Erythroid bursts expressed both p21 and p27 mRNAs with differentiation but expressed neither protein, whereas both proteins were expressed in megakaryocytes and peripheral blood monocytes. In bone marrow, p21 was immunostained almost exclusively in a subset of megakaryocytes and p27 protein was present in megakaryocytes, plasma cells, and endothelial cells. In megakaryocytes, reciprocal expression of p27 to Ki-67 was evident and an inverse relationship between p21 and Ki-67 positivities was also present, albeit less obvious. These observations suggest that a complex lineage-specific regulation is involved in p21 and p27 expression and that these inhibitors are involved in cell-cycle exit in megakaryocytes.

Cyclin D1 overexpression detected by a simple competitive reverse transcription-polymerase chain reaction assay for lymphoid malignancies.

t(11;14)(q13;q32) observed in B-cell malignancies is associated with cyclin D1 (bcl-1, PRAD1, CCND1) overexpression. We devised a simple competitive reverse transcription-polymerase chain reaction (RT-PCR) assay for rapid detection of cyclin D1 overexpression. Sharing a single upstream primer derived from a homologous sequence in cyclins D1, D2 and D3, each PCR product serves as a competitor and cyclin D1 overexpression is determined by comparing the intensities of the three amplified products. We analyzed cyclin D1 in clinical specimens from 104 patients with lymphoid malignancies. Cyclin D1 overexpression was evident in 13 of 104 (7/72 non-Hodgkin's lymphomas, 0/6 adult T-cell lymphoma/leukemias, 0/4 Hodgkin's diseases, 0/11 acute lymphoblastic leukemias, 3/4 multiple myelomas, 1/2 Waldenström's macroglobulinemias, 1/2 prolymphocytic leukemias and 1/3 chronic lymphocytic leukemias). Among 72 patients for whom cytogenetic studies had been done, all 7 patients with t(11;14) were positive. The relative expression levels of D-type cyclins altered dramatically in the presence of t(11;14). Thus, this RT-PCR assay can identify tumors with cyclin D1 overexpression. Cyclin D1 overexpression was frequent in extranodal specimens (11 out of 32 vs. 2 of 72 lymph nodes) and was restricted to specific types of lymphoid malignancies, as observed using other methods. This reliable assay should be suitable to provide clinical guidance for the diagnosis and management of lymphoid malignancies, especially in the case of extranodal involvement.

Overexpression of the PRAD1 oncogene in a patient with multiple myeloma and t(11;14)(q13;q32).

The t(11;14)(q13;q32) chromosomal translocation is associated with several B-cell lymphoproliferative disorders and is thought to result in upregulation of expression of PRAD1/cyclin D1 proto-oncogene. A patient with multiple myeloma of IgG kappa-type with t(11;14)(q13;q32) is now shown to overexpress PRAD1. The clinical stage of the disease was advanced (IIIA), with a myeloma cell count of 94.6% in the bone marrow. Chromosomal analysis of bone marrow cells showed t(11;14)(q13;q32) in five of 20 metaphases as well as other karyotypic features. Northern blot analysis of RNA prepared from myeloma cells revealed overexpression of PRAD1. Multiple myeloma with t(11;14)(q13;q32) has been associated with an aggressive clinical course. Although neither myeloma cells in the peripheral blood nor extramedullary lesions were apparent in the present patient, the myeloma was refractory to several chemotherapeutic regimens from the beginning. Detection of PRAD1 expression may offer an easier alternative to cytogenetic analysis in myeloma and is a potentially useful indicator of a poor prognosis.