Takashi Hisatomi

Retrospective and prospective studies of hepatitis B virus reactivation in malignant lymphoma with occult HBV carrier

BACKGROUND In surface antigen of hepatitis B virus (HBsAg)-positive carrier for anticancer treatment of malignant lymphoma, it is well recognized that reactivation of hepatitis B virus (HBV) occasionally occurs. However, there have been only a few studies of HBV reactivation in serum HBsAg-negative and hepatitis B core antigen (HBcAb)-positive occult HBV carriers. We looked at both retrospective and prospective studies to determine the prevalence, clinical course and risk factor of HBV reactivation during chemotherapy in lymphoma patients. PATIENTS AND METHODS Forty-eight of 127 (37.8%) lymphoma patients were HBsAg negative and HBcAb positive, and 24 of these patients were then given liver function tests and HBsAg tests monthly and serum HBV DNA every 3 months. RESULTS HBV reactivation was observed in two patients (4.1%) who had received intensive chemotherapy including steroid and rituximab. Immediate administration of entecavir therapy after elevation of HBV DNA level was conducted, and this resulted in reduction of it and improvement of liver function test. CONCLUSIONS Rituximab plus steroid-containing regimens may increase the risk of HBV reactivation in HBsAg-negative and HBcAb-positive lymphoma patients. More ambitious prospective studies are required to establish clinically useful or cost-effective follow-up methods for control of HBV reactivation in lymphoma patients with occult HBV infection.

NK314 potentiates antitumor activity with adult T-cell leukemia-lymphoma cells by inhibition of dual targets on topoisomerase IIα and DNA-dependent protein kinase

Adult T-cell leukemia-lymphoma (ATL) is an aggressive disease, incurable by standard chemotherapy. NK314, a new anticancer agent possessing inhibitory activity specific for topoisomerase IIα (Top2α), inhibited the growth of various ATL cell lines (50% inhibitory concentration: 23-70nM) with more potent activity than that of etoposide. In addition to the induction of DNA double-strand breaks by inhibition of Top2α, NK314 induced degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), resulting in impaired DNA double-strand break repair. The contribution of DNA-PK to inhibition of cell growth was affirmed by the following results: NK314 inhibited cell growth of M059J (a DNA-PKcs-deficient cell line) and M059K (a cell line with DNA-PKcs present) with the same potency, whereas etoposide exhibited weak inhibition of cell growth with M059K cells. A DNA-PK specific inhibitor, NU7026, enhanced inhibitory activity of etoposide on M059K as well as on ATL cells. These results suggest that NK314 is a dual inhibitor of Top2α and DNA-PK. Because ATL cells express a high amount of DNA-PKcs, NK314 as a dual molecular targeting anticancer agent is a potential therapeutic tool for treatment of ATL.

Mina53, a novel c-Myc target gene, is frequently expressed in lung cancers and exerts oncogenic property in NIH/3T3 cells.

PurposeMina53, whose expression is directly induced by c-Myc, is overexpressed in various cancers and plays an important role in cell growth. To clarify the involvement of Mina53 in lung cancers, we investigated its expression in human lung cancer tissues as well as in various lung cancer cell lines.MethodsMina53 expression was determined by real-time RT-PCR, western blotting, and immunohistochemistry using lung cancer cell lines, normal human bronchial epithelial cells, and lung cancer tissues. Biological effects of Mina53 were evaluated by soft agar colony formation assay and tumorigenicity in nude mice using Mina53-transfected NIH/3T3 cells. cDNA microarray analysis was performed to determine the gene alteration by Mina53 and confirmation was made using real-time RT-PCR with mina53 expression plasmid or mina53 shRNA-transfected NIH/3T3 cells.ResultsWe observed that 62% of patients evidenced overexpression of Mina53 from the early clinical stages of lung cancer. Differences according to gender, smoking status, or histologic type were not statistically significant. Forced expression of Mina53 in NIH/3T3 cells induced cell transformation, and mina53-transfected NIH/3T3 clones produced tumors in nude mice, demonstrating that Mina53 has oncogenic potential. cDNA microarray revealed that 254 genes had altered expression in a mina53-transfected NIH/3T3 clone. Mina53 regulates several genes related to cell adhesion and metabolism, which have also been reported to be regulated by c-Myc. Genes regulated by Mina53, but not by c-Myc included cytokine/growth factor related genes such as EGFR, IL-6, and HGF.ConclusionOur results suggest that Mina53 plays an important role in carcinogenesis and may be a target for cancer prevention.

Clinico-pathological characteristics of p63 expression in B-cell lymphoma

A member of the family of p53‐related genes, p63 plays a role in regulating epithelial proliferation and differentiation programs, but the pathological and clinical meaning of p63 in B‐cell lymphoma has not been elucidated. We investigated the expression pattern of p63 in B‐cell malignancies, and evaluated the correlation between the expression of p63 and other germinal center markers. Ninety‐eight B‐cell lymphomas (28 FCL, 5 MCL, and 65 DLBCL) were analyzed by immunohistochemical examination for p63, bcl‐6, CD10 and MUM‐1 proteins, and for rearrangement of bcl‐2/IgH. Expression of p63 was observed in the nuclei of tumor cells obtained from 15 of 28 (54%) FCL, 22 of 65 (34%) DLBCL, but none of 5 MCL. In DLBCL, the expression of p63 and bcl‐6 showed a significant correlation (P < 0.02), but no correlation was observed between p63 and expression of CD10, MUM‐1, or bcl‐2/IgH rearrangement. RT‐PCR revealed that TAp63α‐type transcripts, a possible negative regulator of transcriptional activation of p21 promoter, were major transcripts in B‐cell lymphoma tissues. As for prognostic significance, only patients in the p63 positive group of FCL died, and in the non‐germinal center group, the p63 positive cases appeared to have inferior overall survival than other groups in DLBCL. Our preliminary results suggested that p63 expression is a disadvantageous factor for prognosis in this subgroup of B‐cell lymphomas. (Cancer Sci 2006; 97: 1050–1055)

Expression of Mina53, a novel c-Myc target gene, is a favorable prognostic marker in early stage lung cancer.

Mina53, a novel target gene product of c-Myc, is overexpressed in various malignancies. We previously demonstrated that Mina53 is overexpressed in lung cancer patients from the early clinical stages. In this paper, the association between disease prognosis and Mina53 expression in lung cancer patients is analyzed; we found that overexpression of Mina53 in lung cancer patients is associated with favorable prognosis. Statistical analysis using the Kaplan-Meier method showed that patients with negative staining for Mina53 had significantly shorter survival than patients with positive staining for Mina53, especially in stage I or with squamous cell carcinoma. Because the major cause of death in lung cancer patients after surgery is distant metastasis, the effect on cancer cell invasiveness was analyzed for the mechanisms involved in the association with favorable outcome. Overexpression of Mina53 in H226B, a lung squamous cell carcinoma cell line, inhibited cancer cell invasion. Transfection with mina53 shRNA increased the number of invading cells. These results suggest that Mina53 immunostaining is a useful prognostic marker--especially in the early stage of lung cancer--and that Mina53 negative patients should be managed particularly carefully after surgery.

Exon 19 of EGFR mutation in relation to the CA-repeat polymorphism in intron 1.

Epidermal growth factor receptor (EGFR) mutations in lung cancer enhance tyrosine kinase activity and increase sensitivity to the EGFR tyrosine kinase inhibitor, gefitinib. Mutation analysis of the EGFR gene is therefore indispensable for predicting gefitinib response. We investigated a CA‐repeat polymorphism in the EGFR gene related to EGFR mutations. Because an increasing number of CA‐repeats at intron 1 of the EGFR gene has been reported to reduce transcription activity, we examined the relationship between EGFR mutations and this CA‐repeat polymorphism. EGFR mutations at exon 19 were closely associated with shorter CA‐repeat length in the shorter allele, but this was not the case for EGFR mutations at exons 18 or 21. Increased intrinsic EGFR mRNA expression in non‐cancerous lung tissues from lung adenocarcinoma patients was also significantly associated with shorter CA‐repeat length. A higher frequency of EGFR mutations at exon 19 was associated with shorter CA‐repeat length only in patients with high levels of EGFR mRNA expression. To determine the phenotypes of cells possessing shorter CA‐repeats, an in vitro study using human bronchial epithelial cells with different CA‐repeat lengths was performed; more rapid cell growth and activated EGF/EGFR signaling were found more often in the cells having both shorter CA‐repeats and increased EGFR mRNA expression. These results suggest that CA‐repeat length in the EGFR gene may be a genetic factor related to cancer in the case of EGFR mutations at exon 19. The mechanism likely involves enhanced intrinsic expression of EGFR mRNA and activated EGF/EGFR signaling that accompany shorter CA‐repeats. (Cancer Sci 2008; 99: 1180–1187)

Non-germinal cell phenotype and bcl-2 expression in primary adrenal diffuse large B-cell lymphoma

Primary adrenal lymphoma is a rare subtype observed in only 0.83% of all non-Hodgkin lymphoma [1], although the adrenal gland may be involved in up to 25% of NHL cases, by autopsy [2]. The immunophenotype of primary adrenal lymphoma is usually B-cell, with a few T-cell types reported [1,3 – 6]. About 60% of primary adrenal lymphoma patients have bilateral involvement and two-thirds show adrenal insufficiency. Previous reports found that most primary adrenal lymphoma patients belonged to high-risk group by IPI classification, associated with poor prognosis with only 4 months over all survival [1]. It was recently shown that diffuse large B-cell lymphoma (DLBCL) can be divided into three prognostically important subgroups: germinal center B-cell-like (GCB)/DLBCL, activated B-cell-like DLBCL, and type 3, based on gene expression profiles using a cDNA microarray [7,8]. Germinal center B-cell-like DLBCL has a better prognosis than either activated B-cell or type 3. Hans et al. proposed that the immunohistochemical expression pattern of CD10, Bcl-6 and MUM-1 could classify cases of DLBCL into GCB and non-germinal center B-cell type (non-GCB), and predict survival similar to the cDNA microarray [9]. The majority of primary adrenal lymphoma cases are diffuse large B-cell lymphoma, but no precise immunohistochemical examination has been reported. We examined the phenotype of primary adrenal diffuse large B-cell lymphoma (PA-DLBCL) using immunohistochemistry for CD10, Bcl-6, MUM-1 and bcl-2. We found four cases (1.0%) of primary adrenal lymphoma among 390 cases of NHL diagnosed at Saga University Hospital from 1997 to 2006. PAL was diagnosed when the extra nodal site was the only adrenal gland mass, or when the bulk of disease was confined to site of adrenal gland [10] and tissue sample could be obtained only from adrenal mass. The four cases included three men and one woman from 48 to 78 years old, and CT or MRI showed bilateral adrenal masses in all cases. These tumors appeared to be complex masses with variable density in CT scan, no apparent hemorrhage or fibrotic change, and detectable normal adrenal glands. Adrenal insufficiency was present in Case 2 and 4, and bulky mass (410 cm) was observed in Case 2 and 4. Only Case 3 was stage IE, the other cases being stage IV. Case 1 showed B-symptom (fever), a high level of lactate dehydrogenase (LDH) was observed in Case 1, 2 and 4, and a high level of soluble IL-2 receptor (sIL-2R) were present in all cases. International prognostic index (IPI) was high or high-intermediate in three of these cases. The definitive diagnostic procedures for histopathological diagnosis were resection (Case 1 and 4) and ultrasound scan guided needle biopsy (Case 2 and 3). Hematoxylin and eosin staining showed that all cases demonstrated diffuse proliferation of large lymphoid cells [Figure 1(a)]. Case 2 included pleomorphic features, with most of these cells noncleaved/centroblastic morphology; Case 2 and 4 had fibrosis in various degrees; and Case 4 had

Lenalidomide in combination with dexamethasone induced rhabdomyolysis in a multiple myeloma patient treated with pravastatin

A 77-year-old Japanese man was diagnosed with multiple myeloma (MM, IgG-j type, Durie-Salmon stage I, ISS stage I in 2007). He was additionally diagnosed with hypercholesterolemia, and had been administered pravastatin since February 2009. He was treated with a single course of melphalan plus prednisolone, intermittently administered with dexamethasone (DEX), which was followed by 12 courses of melphalan plus DEX. As his symptoms aggravated, he was treated with thalidomide plus DEX from August 2009 to August 2010. This treatment was discontinued due to peripheral neuropathy. From 8 November 2010, we began treating the patient with lenalidomide 25 mg daily plus DEX. He had additionally been on treatment regimens of allopurinol for 4 years, mecobalamin for 1 year, sulfamethoxazole trimethoprim for 1 year, brotizolam for 9 months, warfarin for 2 months, and fluconazole for 3 weeks. On the night of November 13, he developed sudden myalgic pains in both upper arms, weakness of the extremities, abasia, and fever (38.1 C), and was thus immediately admitted to our hospital. His leukocyte count was 5.0 9 10/L, hemoglobin was 7.8 g/dL, and platelets count were 14.5 9 10/L. Biochemical analysis revealed BUN 14.5 mg/dL, creatinine 1.49 mg/dL, AST 71 IU/L, ALT 47 IU/L, LDH 299 IU/L, Na 139 mEq/L, K 3.4 mEq/L, creatine kinase (CK) 3,445 IU/L. Isozymes of CK-MB, -MM, and -BB were 1, 99, and 0%, respectively. Urine myoglobin was 153,000 ng/mL (normal 0–4 ng/mL). His thyroid function was normal (TSH 2.13 lIU/ml, free T4 1.1 ng/ml). He was diagnosed with rhabdomyolysis based on the following symptoms: pain in the proximal limb muscles, faintness, CK increase, myoglobinuria, and slightly impaired renal function. Neurologic examination revealed no tetraparesis. Computed tomography of the head revealed no acute infarction or hemorrhage. We discontinued oral administration of lenalidomide and pravastatin on the day of hospitalization and initiated hydration (1.5 L/day). Although he experienced myalgic pain in both thighs on day 2 and CK increased to 16,126 IU/L, the symptoms were alleviated promptly. On day 10, both the muscular symptoms and laboratory test values returned to normal and he was discharged on day 36. Rhabdomyolysis occurs secondary to the breakdown of skeletal muscle, which leads to the release of intracellular substances into the bloodstream and can be induced by various factors, such as external injury, exercise stress, heatstroke, dehydration, hypokalemia, hypothyroidism, infectious disease, and medication. Among the latter, statins are among the best-known pharmaceutical triggers. Standard statin doses are associated with very low rates of rhabdomyolysis incidence: fewer than 1 in 10,000 patients are treated. However, when combined with other factors such as use of higher doses, concomitant drugs, age, and renal impairment, the incidence of muscle toxicity is higher [1]. It is known that rhabdomyolysis can occur in a concentration-dependent manner, especially when a patient is administered statins concomitantly with a CYP450-mediated medication [1]. Lenalidomide is an imunomodulatory drug related to thalidomide. 25 mg of lenalidomide plus DEX was given safely in Japanese patients with relapsed or refractory MM [2]. This is the first case of rhabdomyolysis among more than 1,500 registered patients according to a C. Urata M. Yoshimura (&) H. Itamura T. Hisatomi Y. Kubota N. Fukushima E. Sueoka S. Kimura Division of Hematology, Respiratory Medicine, and Oncology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan e-mail: tanakam8@cc.saga-u.ac.jp

Successful reduced-intensity umbilical cord blood transplant for fulminant hemophagocytic syndrome in an adult with pre-existing rheumatoid arthritis and autoimmune hemolytic anemia

Hemophagocytic syndrome (HPS) is a potentially fatal syndrome of dysregulated immune activation characterized by severe clinical manifestations such as pancytopenia, high fever and hepatosplenomega...

Acute myeloid leukemia with t(19;21)(q13;q22) and marked eosinophilia

Dear Editor, Translocation (19;21)(p13;q22) has only once been previously reported, in one case of radiation-associated acute myeloid leukemia (AML) [1]. Here, we report the first case of AML with t(19;21)(q13;q22) and marked eosinophilia. A 58-year-old man was admitted because of severe anemia. His white blood cell (WBC) count was 6.7 × 10/L, with 42% eosinophils, 1% myeloblasts, and 1.5% erythroblasts (Fig. 1a). The hemoglobin concentration was 49 g/L, and the platelet count was 196 × 10/L. Bone marrow aspiration showed myeloid dysplasia with 4.8% myeloblasts and 22.4% eosinophils (Fig. 1b). The patient was diagnosed temporarily with myelodysplastic syndrome, unclassifiable (MDS-U). Cytogenetic analysis revealed the karyotype 45, X, -Y, t(19;21)(p13;q22) in 17/20 of the metaphases examined (Fig. 1c). Fluorescence in situ hybridization analysis showed three RUNX1 signals and two RUNX1T1 signals in 37% of cells, but no signals corresponding to RUNX1-RUNX1T1 gene fusion (Fig. 1d). As the fusion transcript termed RUNX1AMP19 has been isolated from an AML patient with a t(19;21)(q13;q22) [2], RT-PCR of RUNX1-AMP19 fusion gene was performed; however, the transcripts were not detected. As the patient’s symptoms were improved by transfusions, he was discharged from hospital and closely monitored for disease progression. The peripheral blood (PB) eosinophil count diminished over time. Two months later, anemia and thrombocytopenia progressed. Transformation to AML was confirmed by the increasing blast counts in the bone marrow (64%; Fig. 1e). After two courses of ineffective chemotherapy, pancytopenia and transfusion dependency remained because normal hematopoiesis was suppressed by increasing numbers of blasts. Thereafter, the patient developed septic shock due to Klebsiella pneumonia infection. His clinical condition recovered after intensive treatment for the infection, but, 2 weeks later, the patient died from pontine hemorrhage due to severe thrombocytopenia. In the present case, because the duration from diagnosis of MDS to diagnosis of AML was very short (2 months), the marked eosinophilia could be considered one of the accompanying symptoms at the onset of AML. The decrease in the proportion of eosinophils observed thereafter could have been due to immature blasts outnumbering eosinophils. Although RUNX1-AMP19 fusion transcript was cloned from an AML patient with a t(19;21)(q13;q22) [2], this fusion gene was not detected in the present patient. Identification of chimeric gene in our case is an interesting subject in the future. Both the first reported case [1] and the present case showed loss of sex chromosome (LOS). LOS is one of the most common recurrent cytogenetic abnormalities observed in t(8;21) AML [3]. Several studies suggest that LOS leads to decreased expression of the GM-CSF receptor, located on the pseudoautosomal regions of the sex chromosomes, and is an additional event required for transformation to full-blown leukemia [4–6]. Thus, LOS could also be an important event for the leukemic transformation in t(19;21) AML. As interleukin (IL)-3Rα, GM-CSFRα, and IL-5Rα chain share a commonβ subunit (cβ) [7, 8], and IL-5 is a major regulator of eosinophils, it is feasible that insufficient expression of GM-CSFRα could induce greater occupation of the cβ by IL-5Rα, * Yasushi Kubota kubotay@cc.saga-u.ac.jp