Primary central nervous system malignant lymphoma in a patient with rheumatoid arthritis receiving low-dose methotrexate treatment.

We report the first case of primary central nervous system lymphoma (PCNSL) developing in a patient with rheumatoid arthritis (RA) undergoing low-dose methotrexate therapy (LD-MTX). The characteristic clinical management and course in our experience of the present case illustrate the important points about PCNSL in methotrexate-associated lymphoproliferative disorders (MTX-LPD). The number of cases of MTX-LPD in RA patients may increase in the future, since current treatment strategies for RA recommend starting MTX use in early stage RA, and recent insights have tended to show an increase with higher doses.

Basophilic stippling in red blood cells in the bone marrow: indication for lead poisoning diagnosis.

A 40-year-old man presented at our hospital with anaemia that had been undiagnosed for 2 years. Blood tests, endoscopy, and contrast-enhanced computed tomography were performed, but a definitive diagnosis could not be made. A subsequent bone marrow biopsy revealed basophilic stippling in transformed red blood cells, which led to a differential diagnosis of lead poisoning. Additional tests revealed elevated levels of lead in the blood. Basophilic stippling is generally found on a peripheral blood smear in lead poisoning patients; however, in this case, basophilic stippling was found only on the bone marrow smear and not in the blood smear. Even if basophilic stippling is not found in the peripheral blood, lead poisoning cannot be excluded.

A case of fulminant Clostridium perfringens infection: Role of macroscopic examination of the serum and peripheral blood smears.

A 69-year-old man was brought to our hospital by ambulance with a fever. The translucent pink color of the serum sample suggested severe hemolysis. His blood pressure dropped rapidly, and he later suffered a cardiopulmonary arrest and died approximately 30 h after arriving at our hospital. The day after the patient's death, Clostridium perfringens was detected in the blood culture taken at the time of hospital admission. When serum sample shows translucent pink to red color and bacilli from bacteria is identified in peripheral blood smear, Clostridium perfringens should be considered and appropriate medical treatment should be initiated immediately.

Analysis of changes in CD20, CD55, and CD59 expression on established rituximab-resistant B-lymphoma cell lines.

Rituximab has markedly improved treatment results for B-cell lymphoma, but there are resistance problems similar to those of other chemotherapy drugs. With regard to the acquisition of rituximab resistance, there have been several reports describing the relation between rituximab and complement regulatory factors or CD20, but many points remain unclear. To further investigate acquisition of resistance to rituximab-related complement-dependent cytotoxicity (CDC), we established rituximab-resistant B-lymphoma cell lines (RAMOS) in vitro and then analyzed expression of CD20, CD55, and CD59 on these resistant cells by flow cytometry. With repeated exposure to a low concentration of rituximab and complement, RAMOS cells gradually acquired rituximab resistance, and selection and increase of CD55(bright) and CD59(bright) cell populations due to rituximab-related CDC were observed. With repeated exposure to a high concentration of rituximab and complement, RAMOS cells promptly acquired rituximab resistance, and CD20 expression of RAMOS cells was decreased. Not only selection of CD20(dim) cells but also down-modulation of CD20 caused by rituximab-related CDC appeared to cause the decrease in CD20 expression. We believe our findings will prove to be useful for prevention of or release from rituximab resistance in cases of B-cell lymphoma.

Pioglitazone inhibits the growth of human leukemia cell lines and primary leukemia cells while sparing normal hematopoietic stem cells.

Peroxisome proliferator-activated receptors (PPARs) compose a subfamily of nuclear hormone receptors functioning as transcriptional regulators. Originally, the PPARgamma ligand known as thiazolidinedione (TZD) was used for the treatment of diabetic patients. However, recent studies have shown that TZD also has an antitumor effect that inhibits cell growth in several types of human malignant neoplasms, including leukemia cell lines. Since pioglitazone is the only TZD currently available in clinics in Japan and the role of TZD in normal human hematopoietic cells or primary leukemia cells has not been previously reported, we investigated the effect of pioglitazone on human normal hematopoietic progenitor cells, primary leukemia cells, and leukemia cell lines (HL60, K562, U937, HEL, CEM, Jurkat, and NALM1). Pioglitazone inhibited the proliferation of leukemia cells in a dose-dependent manner. The viable cell numbers of HL60, K562, and Jurkat leukemia cell lines were profoundly reduced when the cells were cocultured with pioglitazone. Colony formation in the leukemia cell lines as well as the primary leukemia cells was significantly inhibited to 20-71% and 1-25% of that in control cultures by the addition of 100 and 300 microM of pioglitazone, respectively. However, the CFU-E and CFU-GM colonies of cells obtained from healthy volunteers were not altered in the presence of 100 microM of pioglitazone. Pioglitazone (300 microM) induced slight decrease of CFU-E and CFU-GM. BFU-E was more sensitive to pioglitazone than CFU-E and CFU-GM. Pioglitazone-induced growth inhibition in HL60 cells was associated with cell cycle arrest at the G1 phase, as has been reported for another TZD, troglitazone. Similar levels of PPARgamma protein were observed in both leukemia and normal bone marrow cells by Western blotting, suggesting that the expression of PPARgamma protein was not associated with the inhibitory potency of pioglitazone. In conclusion, our results suggest that pioglitazone may offer a new therapeutic approach to aid in the treatment of leukemia.

Quality of care associated with number of cases seen and self-reports of clinical competence for Japanese physicians-in-training in internal medicine.

BACKGROUND: The extent of clinical exposure needed to ensure quality care has not been well determined during internal medicine training. We aimed to determine the association between clinical exposure (number of cases seen), self- reports of clinical competence, and type of institution (predictor variables) and quality of care (outcome variable) as measured by clinical vignettes. METHODS: Cross-sectional study using univariate and multivariate linear analyses in 11 teaching hospitals in Japan. Participants were physicians-in-training in internal medicine departments. Main outcome measure was standardized t-scores (quality of care) derived from responses to five clinical vignettes. RESULTS: Of the 375 eligible participants, 263 (70.1%) completed the vignettes. Most were in their first (57.8%) and second year (28.5%) of training; on average, the participants were 1.8 years (range = 1-8) after graduation. Two thirds of the participants (68.8%) worked in university-affiliated teaching hospitals. The median number of cases seen was 210 (range = 10-11400). Greater exposure to cases (p = 0.0005), higher self-reports of clinical competence (p = 0.0095), and type of institution (p < 0.0001) were significantly associated with higher quality of care, using a multivariate linear model and adjusting for the remaining factors. Quality of care rapidly increased for the first 100 to 200 cases seen and tapered thereafter. CONCLUSION: The amount of clinical exposure and levels of self-reports of clinical competence, not years after graduation, were positively associated with quality of care, adjusting for the remaining factors. The learning curve tapered after about 200 cases.

Adult acute lymphoblastic leukemia with a rare b3a3 type BCR/ABL1 fusion transcript.

The Philadelphia chromosome (Ph) is the most frequent chromosomal abnormality detected in adult acute lymphoblastic leukemia (ALL). This chromosome forms the BCR/ABL1 fusion gene; thus, ABL1 exon a2 is generally used as a primer-binding region for the detection of the fusion transcript via reverse transcription polymerase chain reaction (RT-PCR). We observed a rare case of adult Ph-positive (Ph(+)) ALL, in which the BCR/ABL1 fusion transcript was not detected using the ABL1 exon a2 region primer. However, we were able to isolate a PCR product by RT-PCR with the BCR exon 13 (b2) and ABL1 exon a3 primers. Analysis of the sequence of the RT-PCR product revealed that the fusion point was between BCR exon 14 (b3) and ABL1 exon a3, and that the transcript lacked ABL1 exon a2. The patient achieved cytogenetic remission through combination chemotherapies, but relapse occurred before hematopoietic stem cell transplantation and the patient died 11 months after the initialization of chemotherapies. If the BCR/ABL1 fusion transcript is undetected with the ABL1 exon a2 region primer in Ph(+) ALL cases, an RT-PCR analysis that can detect the b3a3 type BCR/ABL1 fusion transcript should be considered to improve diagnosis.

Isolated oculomotor nerve palsy in Churg-Strauss syndrome.

A 30-year-old man with bronchial asthma complained of horizontal diplopia. Partial oculomotor nerve palsy with restrictions of elevation and adduction, and mydriasis was observed in the left eye. Cranial magnetic resonance imaging demonstrated an infarct lesion in the territory of the left superior median mesencephalic branch of the posterior cerebral artery. Based on bronchial asthma, hypereosinophilia, mononeuropathy multiplex, pulmonary eosinophilia and positive perinuclear antineutrophil cytoplasmic antibody in the serum, the patient was diagnosed as having Churg-Strauss syndrome. This is the first case of oculomotor nerve palsy due to midbrain infarction associated with Churg-Strauss syndrome.

Adult Kawasaki disease unrelated to epstein-barr virus and group A Streptococcus.

A 23-year-old Japanese woman with a fever and generalized skin eruptions was referred to our hospital in July 1999. At admission, her temperature was 38.9 degrees C, and she had fluctuating symptoms including erythema of the extremities, conjunctival hyperemia, strawberry tongue, and generalized skin eruptions, but lymphadenopathy was not verified. An initially elevated urine leukocyte count (more than 100 per high power field) later returned to normal range without antibiotic therapy. Adult Kawasaki disease was diagnosed on the basis of the above symptomology. Echocardiograph showed transient effusion in the pericardium. Using the Harada scoring system for treatment of Kawasaki disease, we gave the patient aspirin and did not administer intravenous immunoglobulin. The clinical course was uneventful, and on the day of discharge (day 22 after onset), the laboratory test results were nearly normal. Laboratory test results were negative for both Epstein-Barr virus and group A Streptococcus.

[Evaluation of prophylactic use of lamivudine in HBV-positive patients with malignant lymphoma undergoing chemotherapy].

A prospective evaluation was carried out on the effect of lamivudine administration as a prophylactic measure to prevent exacerbation of hepatitis in HBV carrier or chronic hepatitis B patients with malignant lymphoma undergoing chemotherapy. Eighteen patients were registered between 1997 and 2002 from institutions of the Research Group for the Treatment of Malignant Lymphoma. The patients' median age was 53 years old (39-73), and consisted of 8 males and 10 females. HBe-seroconversion had already occurred in 13 and liver biopsy had been performed in 8. No adverse effects of lamivudine were noted and the serum HBV-DNA content did not increase during lamivudine administration. Planned treatment courses could be completed in all patients. In 2, however, the viral load increased and the HBe antibody (Ab) value declined after the cessation of lamivudine, which were reversed to the normal ranges following the resumption of lamivudine. As for the overall outcome, 14 of the patients survived, and there were 4 fatalities due to malignant lymphoma. Serum HBeAb status may be regarded as a useful laboratory marker for deciding the safe cessation of lamivudine. An additional case is described, who had recovered from past HBV infection, but eventually succumbed to fulminant hepatitis after the cessation of lamivudine covering prolonged courses of chemotherapy. This illustrates a need for inclusion of such cases for the prophylactic use of lamivudine.

[Bronchoesophageal fistula in a patient with untreated malignant lymphoma].

Bronchoesophageal fistulae associated with lymphomas are generally associated with chemo-radiotherapy. We report here an unusual case of lymphoma with a therapy-unrelated bronchoesophageal fistula. Previously, only 10 similar cases have been reported. A 70-year-old male was diagnosed as having gastric diffuse large B-cell lymphoma in May 1998. In January 1999, he noted a cough after eating and drinking. Because of the presence of a febrile temperature, productive cough and dyspnea, he was referred to our hospital and diagnosed as having aspiration pneumonia. Antibiotics did not improve his symptoms. When tracheal intubation was performed with bronchoscopy, a bronchoesophageal fistula was revealed. Malignant lymphoma cells were found around the fistula in the biopsy specimen. The patient died of pneumonia after treatment with airway stenting and chemotherapy. Induction of necrosis by chemotherapy or low blood flow with stenting and dopamine probably caused enlargement of the fistula.

Purging in autologous hematopoietic stem cell transplantation using adenosine triphosphate (ATP) and 4-hydroperoxycyclophosphamide (4-HC).

In this study, we show potent in vitro purging induced by adenosine triphosphate (ATP) for leukemic cells. The treatment of murine L1210 leukemic cells with 2mM of ATP in vitro for 3h was able to reduce the number of leukemic clonogenic cells by about one order of magnitude presumably by changing the permeability of the leukemic cell membrane. Furthermore, the incubation of L1210 cells with ATP (2mM) and low dose 4-hydroperoxycyclophosphamide (4-HC, 2 microg/ml) for 3h resulted in at least a four-log reduction of clonogenic L1210 cells. Only a slight degree of toxicity to pluripotent hematopoietic stem cells (CFU-S) was observed in both treatment protocols. To determine the efficacy of pharmacological purging by ATP, we designed a murine system to mimic the conditions expected in the clinical setting of autologous transplantation using simulated partial remission marrow (SPRM) which was prepared by mixing normal marrow cells and L1210 cells at a ratio of 9:1. After the SPRM cells were incubated in vitro at a concentration of 1 x 10(6)/ml with both ATP (2mM) and low dose 4-HC (2 microg/ml) for 3h, 5 x 10(4) of the cells were then injected into lethally irradiated 9 weeks male BDF1 mice. All the mice given untreated-SPRM died of leukemia by day 27, whereas none of the recipients transplanted treated-grafts had died by day 70, thus suggesting that the combination use of ATP and 4-HC may be a potentially effective way to purge leukemic cells in autologous stem cell transplantation. The mechanism of the selective killing of leukemic cells is assumed that 4-HC is effectively incorporated into leukemic cells by increasing the permeability of the cell membrane by ATP. Taken together, this simple and rapid procedure is able to purge leukemic cells from autologous bone marrow grafts.

Requirement of soluble factors produced by bone marrow stromal cells on the growth of novel established human myeloma cell line.

The growth of myeloma cells is believed to be mediated by functional interactions between tumor cells and the marrow environment involving the action of several cytokines. We report on the establishment and characterization of a new human myeloma cell line (TAB1) that can be long-term maintained in the presence of conditioned medium of bone marrow stromal cells (BMCM) and a BMCM independent variant, C2-2. Both cell lines have plasma cell morphology and express plasma cell antigens (CD38, PCA-1 and immunoglobulin kappa light chain). In the absence of BMCM, TAB1 cells undergoing apoptosis were observed. Among the adherent molecules tested, these cells expressed VLA-4, ICAM-1 and H-CAM, but not VLA-5, suggesting that these were mostly immature plasmacytes. Introduction with exogenous IL-6 and/or GM-CSF, which were detected in BMCM, partially supported the proliferation of TAB1 cells. Treatment with anti-IL-6 antibody partially inhibited the proliferation of TAB1 cells cultured with BMCM. These findings strongly suggest that TAB1 required at least two or more factors on their growth in vitro; IL-6 was one of the factors necessary for cell growth. Further studies are required to clarify the precise molecules which support TAB1 cell growth in combination with IL-6, however, TAB1 and its variant C2-2 cells may offer an attractive model to unravel novel molecular mechanisms involved in bone marrow stroma-dependent growth of myeloma cells.

Spontaneous clinical and cytogenetic remission of aplastic anemia in a patient with del(13q).

We describe the case of a 64-year-old Japanese man with pancytopenia. Bone marrow biopsy findings were consistent with aplastic anemia. The patient was treated by transfusions without immunosuppressive therapy. Chromosome analysis of bone marrow cells at 6 months after onset showed a 46,XY,del(13) (q14q22) karyotype. The pancytopenia resolved gradually over the next 5 years; chromosome analysis of bone marrow cells at that time yielded normal findings. To our knowledge, this is the first report of spontaneous hematologic and cytogenetic remission of aplastic anemia. These findings suggest that the abnormal clone with deletion of the long arm of chromosome 13 was not sufficient for clonal evolution in aplastic anemia in this case.

Different clones of t(1;12)/t(12;12) involving the ETV6 gene in a case of acute myeloid leukemia.

We describe here a case of a 77-year-old Japanese man who developed acute myeloid leukemia (AML-M2). Chromosome analysis of the bone marrow blast cells showed 46,XY,del(7q) at onset, and after relapse, two clones, 46,XY,t(1;12) and 46,XY,del(7q),t(12;12), were present. Fluorescence in situ hybridization analysis confirmed that each clone with the 12p abnormality involved the ETV6 gene. These findings suggest that the ETV6 gene rearrangements in this case were apparently independent of contribution to leukemogenesis, because this cytogenetic aberration appeared as a secondary change. To our knowledge, this is the first report of two different clones with ETV6 gene rearrangements in the same patient.

Dose-intensified CHOP (double-CHOP) followed by consolidation with high-dose chemotherapy for high and high-intermediate risk aggressive non-Hodgkin's lymphomas.

Patients with aggressive non-Hodgkin's lymphoma (NHL) showing several risk factors have a poor prognosis. In such patients, conventional chemotherapy results in a low complete response rate and a high relapse rate. We performed a prospective trial of intensive induction chemotherapy followed by high-dose consolidation therapy to determine the feasibility of such an approach. We treated 39 patients with aggressive poor risk NHL with double-CHOP (D-CHOP) chemotherapy followed by high-dose therapy (HDT) with or without peripheral blood stem cell transfusion (PBSCT). The median age of the patients was 54 years (range 17-65). Twenty-seven were considered to be at high (H) and 12 were at high-intermediate (H-I) risk according to the age-adjusted International Prognostic Index. Thirty-four patients (87%) responded (complete response: 74%, partial response: 13%) to D-CHOP chemotherapy. Of the 34 responders, 24 received HDT followed by PBSCT, 7 received high-dose methotrexate, and 3 refused consolidation therapy. At a median follow-up period of 26 months, the estimated 3-year overall survival rate and event-free survival rate was 64 and 51%, respectively. Our data suggest that dose-intensified D-CHOP followed by consolidation HDT is safe and appears efficacious in aggressive NHL patients with H-I and H risk.

Adenosine triphosphate (ATP) enhances the antitumor effect of etoposide (VP16) in lung cancer cells.

We have previously reported that adenosine triphosphate (ATP) enhances the cytotoxic effects of 4-hydroperoxycyclophosphamide (4HC) on leukemia cells without affecting the normal hematopoietic stem cells. Increased cell membrane permeability induced by ATP may cause high incorporation of 4HC into leukemia cells, ultimately leading to cell death. In the present study, we show that ATP has cytotoxicity against PC14, a lung adenocarcinoma cell line. When PC14 cells were cultured with 1, 3, and 5 mM ATP, colony number significantly decreased to 91.0, 48.8, and 2.3% respectively, compared to untreated controls. Additionally, ATP enhanced the antitumor effects of etoposide (VP16) in PC14 and another lung adenocarcinoma cell line, A549. With 5, 25, and 50 mM VP16, the percentage of colony numbers compared to control was 95.5, 75.8, and 61.3% in PC14 and 86.0, 65.0, and 57.1% in A549 cells, respectively. In the presence of 3 mM ATP, however, the colony number of PC14 was further limited to 49.6, 34.1, and 24.4% of the untreated level in 5, 25, and 50 mM VP16, respectively. When A549 cells were incubated with 1 mM ATP, the proportion of clonogeneic cells significantly fell to 62.5, 41.7, and 31.7% in 5, 25, and 50 mM VP16, respectively. With 3 and 5 mM of ATP, uptake of [3H]VP16 in PC14 cells increased respectively to 8.9- and 14.1-fold of the negative controls. These results suggest that ATP itself has antitumor effects on lung cancer cells and enhances the cytotoxicity of VP16 through the increased uptake of VP16 into the cells. The combined use of ATP and antitumor agents such as VP16 may have the potential to improve the therapeutic index in human lung carcinoma.

Mutational analysis of IkappaBalpha in hematologic malignancies.

The activation of the NF-kappaB family of transcription factors plays a crucial role in oncogenesis. The IkappaB family has the ability to retain the NF-kappaB in an inactive complex in the cytoplasm. Recently, mutations of the IkappaBalpha gene were found in Hodgkin's lymphoma, which allows NF-kappaB proteins to translocate into the nucleus in an active form. In this report, we describe a mutational analysis of IkappaBalpha for primary tumor cells obtained from patients with a variety of hematologic malignancies (acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, hairy cell leukemia, adult T-cell leukemia, and mantle cell lymphoma) as well as 15 leukemia, lymphoma, and myeloma cell lines (HL60, U937, HEL, K562, NALM1, Jurkat, JM, MOLT4, Raji, KS1, OKM2T, OKM3T, F6T, Su9T01, and C2-2). RT-PCR, followed by direct sequencing, was performed and all samples expressed IkappaBalpha. One missense mutation was identified in a primary effusion lymphoma cell line, KS1. However, NF-kappaB (p65) protein was absent from the nucleus of KS1 immunohistochemically, suggesting that the mutation did not alter the function of IkappaBalpha in this case. Taken together, although it is not clear whether normal IkappaBalpha protein was expressed in hematologic malignancies, mutations of IkappaBalpha could be rare events in these diseases, except for Hodgkin's lymphoma. Alterations of other members of NF-kappaB/ IkappaB family proteins might act on the development of hematologic malignancies.