Prognostic significance of S-phase kinase-associated protein 2 and p27kip1 in patients with diffuse large B-cell lymphoma: effects of rituximab.

BACKGROUND: The F-box protein S-phase kinase-associated protein 2 (Skp2) positively regulates the G1-S transition by promoting degradation of the cyclin-dependent kinase inhibitor p27(kip1) (p27). Recent evidence has indicated an oncogenic role of Skp2 in not only carcinogenesis but also lymphomagenesis. MATERIALS AND METHODS: Clinicopathologic features and immunohistochemical expression of Skp2 and p27 were studied retrospectively in 671 patients treated with cyclophosphamide, vincristine, doxorubicin and prednisolone (CHOP) or cyclophosphamide, vincristine, doxorubicin and prednisolone plus rituximab (R-CHOP). The median follow-up periods were 43.2 months in the CHOP group (n = 425) and 24.0 months in the R-CHOP group (n = 246). RESULTS: High Skp2 or low p27 expression correlated significantly with poor overall survival (OS) and progression-free survival (P < 0.001) in both treatment groups. The prognostic value of Skp2 or p27 expression was independent of the parameters included in the International Prognostic Index by multivariate analysis. Patients with high Skp2 expression in combination with low p27 expression showed the worst survival. CONCLUSIONS: Addition of rituximab to the CHOP regimen did not provide a beneficial outcome to patients with diffuse large B-cell lymphoma with high Skp2 expression and low p27 expression. Skp2 and p27 may be useful prognostic markers in the rituximab era.

Efficient engraftment of primary adult T-cell leukemia cells in newborn NOD/SCID/beta2-microglobulin(null) mice.

Adult T-cell leukemia (ATL) develops via multiple oncogenic steps in human T-cell leukemia virus type I (HTLV-I) carriers. To better understand pathogenesis of ATL, we developed a novel xenogeneic engraftment model in which primary ATL cells are intravenously transplanted into neonatal nonobese diabetic (NOD)/severe-combined immunodeficiency (SCID)/beta2-microglobulin(null) (NOD/SCID/beta2m(null)) mice. Acute-type ATL cells engrafted in the peripheral blood and in the lymph nodes of recipients at a high efficiency. Engrafted ATL cells were dually positive for human CD4 and CD25, and displayed patterns of HTLV-I integration identical to those of donors by Southern blot analysis. These cells infiltrated into recipients' liver, and formed nodular lesions, recapitulating the clinical feature of each patient. In contrast, in smoldering-type ATL cases, multiple clones of ATL cells engrafted efficiently in NOD/SCID/beta2m(null) mice. When smoldering-type ATL cells were retransplanted into secondary NOD/SCID/beta2m(null) recipients, single HTLV-I-infected clones became predominant, suggesting that clones with dominant proliferative activity can be competitively selected in this xenogeneic system. Taken together, the NOD/SCID/beta2m(null) newborn system is useful to understand kinetics, metastasis, and disease progression of ATL in vivo.

Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia.

Gene targeting studies in mice have shown that the lack of Ikaros activity leads to T-cell hyperproliferation and T-cell neoplasia, establishing the Ikaros gene as a tumor suppressor gene in mice. This prompted us to investigate whether mutations in Ikaros play a role in human hematological malignancies. Reverse transcription-PCR was used to determine the relative expression levels of Ikaros isoforms in a panel of human leukemia/lymphoma cell lines and human bone marrow samples from patients with hematological malignancies. Among the cell lines examined, only BV-173, which was derived from a chronic myelogenous leukemia (CML) patient in lymphoid blast crisis, overexpressed the dominant-negative isoform, Ik-6. In 9 of 17 samples of patients in blast crisis of CML, Ikaros activity had been reduced either by drastically reducing mRNA expression (4 of 17) or by overexpressing the dominant-negative isoform Ik-6 (5 of 17). Significantly, expression of Ikaros isoforms seemed normal in chronic phase CML patients and patients with other hematological malignancies. In some cases, overexpression of the dominant-negative Ik-6 protein was confirmed by Western blot analysis, and Southern blot analysis indicated that decreases in Ikaros activity correlated with a mutation in the Ikaros locus. In summary, these findings suggest that a reduction of Ikaros activity may be an important step in the development of blast crisis in CML and provide further evidence that mutations that alter Ikaros expression may contribute to human hematological malignancies.

Demonstration of reversed flow in segmental branches of the portal vein with hand-held color Doppler ultrasonography after hematopoietic stem cell transplantation.

Hepatic veno-occlusive disease (VOD) is a severe complication of hematopoietic stem cell transplantation (SCT). When monitored with hand-held color Doppler ultrasonography during day -7 to +35 around SCT, reversed blood flow in the segmental branches of the portal vein was detected in nine of 56 patients who had undergone SCT. Three of nine patients had clinical evidence of VOD, but six patients did not fulfill the criteria for diagnosis of VOD initially. Two patients progressed to clinical VOD at a later date and the reversed portal flow disappeared with or without treatment for VOD in the other four patients. Monitoring for reversed portal flow with color Doppler ultrasonography may be a useful tool for the early diagnosis of VOD, and may improve prognosis by allowing early initiation of treatment.

Aggressive natural killer-cell leukemia revisited: large granular lymphocyte leukemia of cytotoxic NK cells.

Aggressive natural killer-cell leukemia (ANKL) is a rare form of large granular lymphocyte leukemia, which is characterized by a systemic proliferation of NK cells. The clinical features of 22 ANKL cases were analyzed. Hepatomegaly (64%), splenomegaly (55%) and lymphadenopathy (41%) were also frequently observed. Leukemic cells were identified as CD1-, CD2+, surface CD3-, CD4-, CD5-, CD7+, CD8+/-, CD10-, CD11b+/-, CD13-, CD16+, CD19-, CD20-, CD25-, CD33(-), CD34-, CD38+, CD56+, CD122+, HLA-DR+ and TCR-. Two of the 16 cases examined for CD57 were positive and three of the seven cases examined for cytoplasmic CD3. Epstein-Barr virus was detected in the tumor cells of 11 of the 13 cases examined. No common cytogenetic abnormalities were identified and 6q anomaly was detected in only one. Three of 13 patients treated with chemotherapy containing anthracycline/anthraquinone attained complete remission, in contrast to none of the eight who were treated with regimens without anthracycline. Although the overall prognosis was poor with a median survival of 58 days, those who attained remission showed better prognosis (P=0.005). These findings suggest that ANKL is an entity of mature cytotoxic NK-cell neoplasms with distinct phenotype and disease presentations. Intensive treatment for ANKL may result in a better prognosis.

Drug-induced in vitro tolerance to allogeneic antigens. I. Establishment of a tolerance induction system in a fully allogeneic murine combination.

C3H/HeSlc (H-2k) spleen cells were cultured with mitomycin C (MMC)-treated C57BL/6CrSlc (H-2b) spleen cells for 2 days and incubated with 5-fluorouracil (5-FU) for a further 9 hr. Thereafter, those C3H/He spleen cells were recultured with the same allogeneic cells for 5 days. Cell-mediated cytotoxicity (CMC) and mixed lymphocyte reaction (MLR) were profoundly suppressed, antigen-specifically, in such C3H/He spleen cells. In contrast, interleukin 2(IL-2) production was not impaired in the restimulating mixed lymphocyte culture (MLC) with C57BL/6. Moreover, an adequate amount of exogenous IL-2 added to the restimulating MLC did not lead to a restoration of the depressed CMC. Suppressor cell activity in the CMC assay was not detected in the C3H/He spleen cells exposed to such a tolerance induction. These results suggest that the unresponsiveness to alloantigens in CMC and MLR was induced through a clonal deletion mechanism, and there may exist a 5-FU-resistant--thus less-proliferative--cell population that can produce IL-2 even after the tolerance induction.

Drug-induced in vitro tolerance to allogeneic antigens. II. Further analysis of in vitro-tolerized spleen cells in a fully allogeneic murine combination.

C3H/HeSlc (C3H, H-2k) spleen cells were made tolerant in vitro to C57BL/6CrSlc (B6, H-2b) at the cell-mediated cytotoxicity (CMC) level by in vitro stimulation for 48 hr with mitomycin C (MMC)-treated B6 spleen cells, and treatment with 5 micrograms/ml of 5-fluorouracil for a further 9 hr. These cells were given intraperitoneally to neonate (C3HxB6) F1 mice to examine whether these tolerized spleen cells would cause lethal graft-versus-host disease (GVHD). Despite the lack of CMC, the tolerized C3H spleen cells caused lethal GVHD in most of the neonate F1 mice. Evaluating from various immune parameters, it was evident that T cell populations responsible for IL-2 production, cytostasis, and delayed footpad reaction (DFR) were retained intact after in vitro tolerance induction, probably because of their less-proliferative characteristics in response to fully allogeneic antigen stimulation, and were considered to be responsible for lethal GVHD. Contribution of natural killer (NK) cells to lethal GVHD was not ruled out.

B-lymphoid/myeloid stem cell origin in Ph-positive acute leukemia with myeloid markers.

We report two cases of Philadelphia chromosome (Ph)-positive acute leukemia with definite myeloid markers. Ph was the sole chromosomal abnormality at presentation, and neither eosinophilia, basophilia, thrombocytosis nor hepatosplenomegaly was present. In both cases, Ph+ myeloblasts showed positive stain for myeloperoxidase and naphthol ASD chloroacetate esterase, which fulfilled the FAB criteria of acute myelogenous leukemia (AML). Ph+ myeloblasts co-expressed myeloid and B-lymphoid antigens (CD10, CD13, CD19 and CD33). In case 1, myeloblasts rearranged M-BCR, and the expression of M-BCR/ABL chimeric RNA was demonstrated by using the reverse transcription polymerase chain reaction (RT-PCR). They also clonally rearranged IGH. Ph clone disappeared on cytogenetic analysis in remission, and granulocytes in remission did not have rearranged M-BCR. In case 2, morphocytochemically distinct myeloid and lymphoid blast populations were seen. Myeloblasts and lymphoblasts were enriched > 96% as CD19-/CD33+ and CD19+/CD33- populations, respectively. Both of them possessed the identical rearrangement of IGH and M-BCR, indicating a common leukemic progenitor cell origin. Furthermore, m-BCR/ABL was detected in addition to M-BCR/ABL on RT-PCR. Accordingly, both cases were diagnosed as de novo Ph+ acute leukemia rather than as chronic myelogenous leukemia in blastic crisis. Their mixed B-lymphoid/myeloid characteristics strongly suggest that so-called 'Ph+ AML' is derived from Ph+ myeloid/B-lymphoid stem cells.

A randomized phase II trial of low-dose aclarubicin vs very low-dose cytosine arabinoside for treatment of myelodysplastic syndromes.

We performed a randomized phase II trial comparing low-dose aclarubicin (LC-ACR) with very low-dose cytosine arabinoside (VLD-AC) in 39 consecutive untreated patients with myelodysplastic syndromes (MDS), including refractory anemia (RA), RA with excess of blasts (RAEB) and RAEB in transformation (RAEB-t). Nineteen patients received the VLD-AC therapy; 2 good responses (GR) and 2 partial responses (PR) were obtained in 11 patients with RAEB and RAEB-t, while 2 PR were obtained in 8 RA patients. Eighteen patients received the LD-ACR therapy; 2 GR and 4 PR were obtained in 11 RAEB/RAEB-t patients while 2 PR in 7 RA patients. There was no significant difference in the therapeutic effects and survival between these two groups of patients. These observations suggest that the LD-ACR therapy is effective in some patients with MDS and can be used as an alternative to the low-dose Ara-C therapy.

Expression of sex-hormone receptors and clinicopathological findings in hepatocellular-carcinoma.

We studied the expresssion of estrogen (ER), progesterone (PgR), and androgen receptors (AR) in hepatocellular carcinoma (HCC) and found expression in (7/36), 3.2% (1/31), 16.7% (4/24) of cases, respectively. The expression of ER did not correlate with histological grade, tumor size, or stage of disease. On the other hand, all the patients with AR-positive tumors had stage IV disease, and only 25% of the patients with AR-negative tumors had stage IV disease. The survival was not influenced by the expression pattern of ER, however, the survival of the patients with AR-positive tumors tended to be worse than that of patients with AR-negative tumors. We suggest that the AR expression correlates better with poor outcome in HCC than the ER expression.

Thymus-dependent increase in number of T cells in parathymic lymph nodes induced by the biscoclaurine alkaloid, cepharanthine.

In mice treated with cepharanthine (Cepha), a biscoclaurine alkaloid, the number of T cells was increased in the parathymic lymph nodes (PtLNs) which are considered to be the specialized lymph nodes in local differentiation of T cells. Such PtLN cells exhibited augmented proliferative responses to T cell mitogens and exogenous interleukin 2 (IL 2) and showed a great ability to produce IL2, which suggests an increase in mature T cells in the PtLN. However, such increases in the number of T cells and in the mitogen response were not observed in adult thymectomized mice. A low level of plasma prostaglandin E2 (PGE2) was observed in Cepha-treated mice. Replenishment of the PGE2 in such mice with exogenous PGE2 prevented the increase in T cells in the PtLN. These results suggest that the migration of mature T cells from the thymus to PtLN is increased by Cepha and that Cepha is able to regulate their traffic by a prostaglandin-mediated system.

Autoreactive antibodies following autologous peripheral blood stem cell transplantation.

A 35-year-old woman diagnosed with multiple myeloma (IgG, lambda type, stage IIIA) received an autologous peripheral blood stem cell transplant (PBSCT). She was euthyroid without autoreactive antibodies prior to the transplant. The patient complained of malaise, weight loss and low grade fever 1 month after transplant, despite rapid haematopoietic recovery. Thyroid function tests on day 34 revealed hyperthyroidism associated with anti-thyroid peroxidase antibody. Antinuclear antibody was also detected, and platelet-associated immunoglobulin was increased. These findings disappeared spontaneously by day 62 without treatment. Autoimmune diseases may occur transiently after autologous PBSCT.

Myasthenia gravis after allogeneic bone marrow transplantation.

A 37-year-old Japanese man with chronic myeloid leukemia (CML) developed myasthenia gravis 29 months after bone marrow transplantation (BMT) from an HLA one locus-mismatched brother. Proximal muscle weakness and bilateral ptosis occurred along with the exacerbation of chronic GVHD shortly after sudden cessation of cyclosporine (CYA) and prednisolone. The diagnosis of myasthenia gravis was made based on clinical symptoms and elevation of an anti-acetylcholine receptor antibody titer and all symptoms related to myasthenia gravis promptly diminished with the start of treatment for chronic GVHD. In most previously reported cases, the underlying disease was aplastic anemia (6 of 7 cases) and donors were of the opposite sex (6 of 7 cases). The haplotypes HLA B7 (3 of 5 cases), B35 (3 of 5 cases), and DR2 (3 of 3 cases) were common. All cases suffered from chronic GVHD. The present case had only chronic GVHD and HLA B7 as a background for myasthenia gravis after BMT. The abrupt cessation of immunosuppressive therapy may also be related to the development of myasthenia gravis after BMT.

Lamivudine therapy for a hepatitis B surface antigen (HBsAg)-positive leukemia patient receiving myeloablative chemotherapy and autologous stem cell transplantation.

Hepatitis B virus (HBV) can be a cause of fatal liver failure after chemotherapy for viral carrier patients and limits the indication of myeloablative therapy for them. We describe an HBsAg-positive leukemia patient who successfully underwent autologous PBSC transplant. After chemotherapeutic treatment his serum HBV DNA level rose in association with hepatitis. To prevent progression to fulminant hepatitis, we administered lamivudine, a viral reverse transcriptase inhibitor, during the transplantation procedure. The patient did not show any increase of HBV DNA or a worsening of his hepatitis. Thus, lamivudine may be a promising treatment for HBsAg-positive patients receiving myeloablative chemotherapy and autologous stem cell transplantation.

Acquired Pelger-Huët anomaly in association with concomitant tacrolimus and fluconazole therapy following allogeneic bone marrow transplantation.

A 38-year-old Japanese woman with severe aplastic anemia received an allogeneic bone marrow transplant from her serologically HLA-identical father. Cyclosporine and methotrexate were administered to prevent graft-versus-host disease (GVHD). However, grade III acute GVHD developed on day 44, which was successfully treated with methylprednisolone and tacrolimus. Fluconazole therapy was started for oral candidiasis on day 112, but she complained of headache soon after. In addition to glycosuria and increased serum creatinine levels, Pelger-Huët anomaly of granulocytes was found in her blood, which disappeared after discontinuation of tacrolimus. Transient occurrence of Pelger-Huët cells may be associated with tacrolimus toxicity due to drug interaction with fluconazole.

Fatal cytomegalovirus interstitial pneumonia following autologous peripheral blood stem cell transplantation. Fukuoka Bone Marrow Transplantation Group.

A 52-year-old Japanese woman suffering from AML (FAB classification M4) in her first remission received an autologous peripheral blood stem cell transplant (APBSCT). She was seropositive for CMV prior to APBSCT. Her post-APBSCT course was complicated with CMV-associated disease and hemophagocytic syndrome. Finally, CMV interstitial pneumonia developed and death ensued. Even after APBSCT, there can be a short period of immune deficiency resembling that occurring following allogeneic or autologous BMT. CMV infection must be considered in the differential diagnosis in cases of unexplained fever or pneumonia following APBSCT.

Donor lymphocyte transfusion for the treatment of Epstein-Barr virus-associated lymphoproliferative disorder of the brain.

EBV-associated lymphoproliferative disorder (LPD) is a rare but serious complication in marrow transplant recipients. A 31-year-old Japanese woman in the second chronic phase of CML received an allogeneic BMT from her HLA 2-locus-incompatible 62-year-old father. Around day +200, she developed EBV-LPD of the right parieto-temporal lobe which caused slowly progressive left hemiparesis. Two courses of donor lymphocyte transfusions (DLT) of 10(6)CD3+ T cells/kg of body weight failed to suppress her central nervous system (CNS) EBV-LPD. The patient died of recurrent blastic crisis of CML. This case suggests that DLT may be ineffective for the treatment of CNS EBV-LPD.

Successful foscarnet therapy for mucocutaneous infection with herpes simplex virus in a recipient after unrelated bone marrow transplantation.

A 36-year-old Japanese man who received an unrelated bone marrow transplant (BMT) developed severe mucocutaneous infection with herpes simplex virus (HSV) type 1 during oral acyclovir prophylaxis. The lesions progressed despite treatment with intravenous acyclovir and vidarabine. The HSV isolates were sensitive acyclovir, vidarabine and foscarnet in vitro, but peripheral CD3- or CD19-positive cells were barely detectable even 4 months after transplant. A 12-day course of treatment with foscarnet led to a rapid improvement. Foscarnet therapy should be considered for all severe HSV infections following BMT, regardless of whether or not the HSV isolates are sensitive to acyclovir.

Serum concentration of the soluble interleukin-2 receptor for monitoring acute graft-versus-host disease.

Soluble interleukin-2 receptors (sIL-2R) are elevated in various disorders involving the activation of T cells. We measured serial serum concentrations of sIL-2R in 30 patients receiving allogeneic BMT to evaluate the usefulness of sIL-2R as a parameter for acute GVHD. In the 17 patients who developed acute GVHD, the sIL-2R concentration rose significantly on day 3 following transplantation, preceding the occurrence of acute GVHD. This change was not seen in the 13 patients without acute GVHD. The serum concentration of sIL-2R decreased as the acute GVHD subsided. The peak concentration of serum sIL-2R correlated with the severity of the acute GVHD. Simultaneous measurement of tumor necrosis factor alpha (TNF alpha) showed a significant rise in patients with acute GVHD, that became evident earlier than the sIL-2R elevation. TNF alpha concentrations also decreased following treatment of the acute GVHD. However, significant rise in TNF alpha were also seen in the early phase of allogeneic BMT in patients who did not develop acute GVHD. Our data suggest that the serum concentrations of sIL-2R as well as TNF alpha might reflect the severity of acute GVHD, and that the serum sIL-2R concentration might be a sensitive and practical indicator for acute GVHD.

Cyclosporine combined with methylprednisolone or methotrexate in prophylaxis of moderate to severe acute graft-versus-host disease.

To evaluate the efficacy of cyclosporine (CYA) regimens in preventing moderate to severe acute graft-versus-host disease (GVHD), 25 patients received immunosuppressive therapy consisting of either CYA and methylprednisolone or CYA and methotrexate (MTX) and the incidence and severity of acute GVHD was compared. These patients had leukemia or myelodysplastic syndrome (MDS) and received bone marrow transplants (BMT) from genotypically HLA-identical siblings. The incidence of grade I-IV acute GVHD in patients on the CYA/methylprednisolone regimen was 64% (7 of 11) compared with 50% (7 of 14) in those on the CYA/MTX regimen. Five of 11 patients with the CYA/methylprednisolone regimen developed moderate to severe acute GVHD (grade II-IV), fatal in 3 cases. No patient on the CYA/MTX regimen developed moderate to severe acute GVHD. Engraftment was faster in the CYA/methylprednisolone group than in the CYA/MTX group. The incidence of toxicity observed soon after BMT was comparable between groups. The CYA/MTX regimen may be superior to the CYA/methylprednisolone regimen for preventing moderate to severe acute GVHD.