Involvement of folate and vitamin B12 deficiency in patients with normocytic anemia.

Objectives: Deficiencies in folate (FA) and vitamin B12 (VB12) are causes of macrocytic anemia. However, in clinical practice, FA and/or VB12 deficiency can occur in patients with normocytic anemia. This study aimed to determine the prevalence of FA/VB12 deficiency in patients with normocytic anemia and the importance of vitamin replacement therapy in these patients. Methods: We retrospectively reviewed electronic medical record information of patients whose hemoglobin and serum FA/VB12 concentrations were measured at the Department of Hematology (N=1,388) and other departments (N=1,421) of Fujita Health University Hospital. Results: In the Hematology Department, 530 (38%) patients showed normocytic anemia. Of these, 49 (9.2%) had FA/VB12 deficiency. Twenty of 49 (41%) patients had some hematological malignancies and 27 (55%) had benign hematological disorders. Of the nine patients who received vitamin replacement therapy, one showed a partial improvement in the hemoglobin concentration of ≥1 g/dL. Conclusions: In the clinical setting, the measurement of FA/VB12 concentrations in patients with normocytic anemia may be useful. Replacement therapy may be a treatment option to consider in patients with low FA/VB12 concentrations. However, physicians need to pay attention to the presence of background diseases, and the mechanisms of this situation require further investigation.

Correlation between serum albumin and serum zinc in malignant lymphoma.

Objectives: Zinc (Zn) is a cofactor for more than 200 enzymes within the human body. Zn deficiency can result in cell-mediated immune dysfunction. Furthermore, serum Zn levels have been reported to be associated with nutritional status, but this association has not been clarified in malignant lymphoma. This study aimed to examine the deficiency of serum Zn levels and clarify the factors that are correlated with serum Zn in malignant lymphoma. Methods: Initial malignant lymphoma was diagnosed in patients at Fujita Health University Hospital between April 2011 and March 2019. Based on the serum Zn levels, the study population was divided into "deficient" and "low or normal". For the serum Zn levels of patients undergoing pre-chemotherapy, laboratory parameters and nutritional factors were included. We compared these factors between the abovementioned two groups, and the serum Zn levels with its correlation factors were investigated. Results: A total of 77 patients (Deficient group, n=20 and Low or Normal group, n=57) were enrolled. Histology, hemoglobin, serum albumin levels, Glasgow Prognostic Score (GPS), neutrophile-lymphocyte ratio (NLR), prognostic nutrition index (PNI) and Controlling Nutritional Status (CONUT) were significantly different between the two groups. Of these parameters, only serum albumin level was significantly associated with serum Zn level (p=0.0024; estimated regression coefficient, 9.51; adjusted coefficient of determination, 0.28). Conclusions: Poor nutritional status at the initial diagnosis may have affected Zn deficiency in initial malignant lymphoma.

Truncated RUNX1 Generated by the Fusion of RUNX1 to Antisense GRIK2 via a Cryptic Chromosome Translocation Enhances Sensitivity to Granulocyte Colony-Stimulating Factor.

Fusions of the Runt-related transcription factor 1 (RUNX1) with different partner genes have been associated with various hematological disorders. Interestingly, the C-terminally truncated form of RUNX1 and RUNX1 fusion proteins are similarly considered important contributors to leukemogenesis. Here, we describe a 59-year-old male patient who was initially diagnosed with acute myeloid leukemia, inv(16)(p13;q22)/CBFB-MYH11 (FAB classification M4Eo). He achieved complete remission and negative CBFB-MYH11 status with daunorubicin/cytarabine combination chemotherapy but relapsed 3 years later. Cytogenetic analysis of relapsed leukemia cells revealed CBFB-MYH11 negativity and complex chromosomal abnormalities without inv(16)(p13;q22). RNA-seq identified the glutamate receptor, ionotropic, kinase 2 (GRIK2) gene on 6q16 as a novel fusion partner for RUNX1 in this case. Specifically, the fusion of RUNX1 to the GRIK2 antisense strand (RUNX1-GRIK2as) generated multiple missplicing transcripts. Because extremely low levels of wild-type GRIK2 were detected in leukemia cells, RUNX1-GRIK2as was thought to drive the pathogenesis associated with the RUNX1-GRIK2 fusion. The truncated RUNX1 generated from RUNX1-GRIK2as induced the expression of the granulocyte colony-stimulating factor (G-CSF) receptor on 32D myeloid leukemia cells and enhanced proliferation in response to G-CSF. In summary, the RUNX1-GRIK2as fusion emphasizes the importance of aberrantly truncated RUNX1 in leukemogenesis.

Rearrangement of VPS13B, a causative gene of Cohen syndrome, in a case of RUNX1-RUNX1T1 leukemia with t(8;12;21).

Variant chromosomal translocations associated with t(8;21) are observed in 3-4% of acute myeloid leukemia (AML) cases with a RUNX1-RUNX1T1 fusion gene. However, the molecular events that occur in variants of t(8;21) are not well characterized. In the present study, we report genetic features of a variant three-way translocation of t(8;12;21)(q22;p11;q22) in a patient with AML. In this patient, leukemia cells lacked azurophilic granules, which does not correspond with the classic features of t(8;21). RNA-seq analysis revealed that TM7SF3 at 12p11 was fused to VPS13B at 8q22 and VPS13B to RUNX1, in addition to RUNX1-RUNX1T1. VPS13B was located near RUNX1T1 and both were localized at the same chromosomal bands. The reading frames of TM7SF3 and VPS13B did not match to those of VPS13B and RUNX1, respectively. Disruption of VPS13B causes Cohen syndrome, which presents intermittent neutropenia with a left-shifted granulopoiesis in the bone marrow. Disruption of VPS13B may thus cause the unusual features of RUNX1-RUNX1T1 leukemia. Our case indicates that rearrangement of VPS13B may be additional genetic events in variant t(8;21).

The prognostic significance of EBV DNA load and EBER status in diagnostic specimens from diffuse large B-cell lymphoma patients.

Epstein-Barr virus (EBV)-encoded small RNA in situ hybridization (EBER-ISH) is a widely accepted method to evaluate EBV involvement in diffuse large B-cell lymphoma (DLBCL), although little is known regarding associations between EBV DNA load and the EBER status and whether EBV DNA load data provide additional clinical information. In this study, we quantified EBV DNA load in diagnostic specimens from DLBCL patients diagnosed at our hospital to evaluate clinical implications of EBV DNA load in diagnostic specimens as contrasted with EBER-ISH. Among 140 DLBCL patients without underlying immunodeficiency, 51 were evaluable for both EBER and EBV DNA load, 83 for EBER only and one for EBV DNA load only. The median EBV DNA load was 708 copies/µg. Although EBV DNA load was significantly higher for EBER-positive patients than for EBER-negative patients (p < 0.001), EBV DNA was detected in up to 72% of EBER-negative patients. Progression-free survival and overall survival were significantly worse for patients with EBV DNA load above 700 copies/µg than for those with EBV DNA load below 700 copies/µg (p = 0.009 and p = 0.003); they were also significantly worse for EBER-positive patients than for EBER-negative patients (p < 0.001 and p = 0.001). Even among EBER-negative patients, higher EBV DNA load conferred worse progression-free survival and overall survival (p = 0.041 and p = 0.013). These findings indicate that EBV DNA load in diagnostic specimens is not a simple surrogate for the EBER status and may be a potential biomarker associated with EBV involvement and prognosis in DLBCL. Copyright © 2015 John Wiley & Sons, Ltd.

TP53 mutations in older adults with acute myeloid leukemia.

The net benefits of induction therapy for older adults with acute myeloid leukemia (AML) remain controversial. Because AML in older adults is a heterogeneous disease, it is important to identify those who are unlikely to benefit from induction therapy based on information available at the initial assessment. We used next-generation sequencing to analyze TP53 mutation status in AML patients aged 60 years or older, and evaluated its effects on outcomes. TP53 mutations were detected in 12 of 77 patients (16 %), and there was a significant association between TP53 mutations and monosomal karyotype. Patients with TP53 mutations had significantly worse survival than those without (P = 0.009), and multivariate analysis identified TP53 mutation status as the most significant prognostic factor for survival. Neverthelsess, TP53-mutated patients had a 42 % chance of complete remission and a median survival of 8.0 months, which compares favorably with those who did not undergo induction therapy, even in the short term. These results suggest that screening for TP53 mutations at diagnosis is useful for identifying older adults with AML who are least likely to respond to chemotherapy, although the presence of this mutation alone does not seem to justify rejecting induction therapy.

ETV6-LPXN fusion transcript generated by t(11;12)(q12.1;p13) in a patient with relapsing acute myeloid leukemia with NUP98-HOXA9.

ETV6, which encodes an ETS family transcription factor, is frequently rearranged in human leukemias. We show here that a patient with acute myeloid leukemia with t(7;11)(p15;p15) gained, at the time of relapse, t(11;12)(q12.1;p13) with a split ETV6 FISH signal. Using 3'-RACE PCR analysis, we found that ETV6 was fused to LPXN at 11q12.1, which encodes leupaxin. ETV6-LPXN, an in-frame fusion between exon 4 of ETV6 and exon 2 of LPXN, did not transform the interleukin-3-dependent 32D myeloid cell line to cytokine independence; however, an enhanced proliferative response was observed when these cells were treated with G-CSF without inhibition of granulocytic differentiation. The 32D and human leukemia cell lines each transduced with ETV6-LPXN showed enhanced migration towards the chemokine CXCL12. We show here for the first time that LPXN is a fusion partner of ETV6 and present evidence indicating that ETV6-LPXN plays a crucial role in leukemia progression through enhancing the response to G-CSF and CXCL12.

NUP214-RAC1 and RAC1-COL12A1 Fusion in Complex Variant Translocations Involving Chromosomes 6, 7 and 9 in an Acute Myeloid Leukemia Case with DEK-NUP214.

DEK-NUP214 gene fusion in acute myeloid leukemia (AML) is associated with poor prognosis. It is most often a sole translocation and more rarely observed as complex chromosomal forms. We describe an AML case with complex karyotype abnormalities involving chromosome bands 6p23, 6q13, 7p22, and 9q34. RNA sequencing analysis revealed that exon 17 of NUP214 (9q34) was fused to exon 2 of RAC1 (7p22). We also detected that the 5'-end of intron 1 of RAC1 was fused with the antisense strand of intron 5 of COL12A1 (6q13). RT-PCR analysis confirmed the expression of DEK-NUP214, NUP214-RAC1, RAC1-COL12A1, NUP214, and RAC1. These results suggest that the 5'- and 3'-ends of NUP214 from the breakpoint in the same locus were fused to RAC1 and DEK, respectively, and the 5'-end of RAC1 was fused to COL12A1. The reading frame of NUP214 was not matched with RAC1; however, high expression of the RAC1 protein was detected by Western blotting. This study identifies the variant complex fusion genesNUP214-RAC1 and RAC1- COL12A1 in a case of AML.

Prognostic significance of Epstein-Barr virus DNA detection in pretreatment serum in diffuse large B-cell lymphoma.

It is still a matter of debate whether detection of Epstein-Barr virus (EBV) DNA in pretreatment serum has clinical implications for diffuse large B-cell lymphoma. For this study, we measured EBV DNA load in pretreatment serum from 127 diffuse large B-cell lymphoma patients without any underlying immunodeficiency to evaluate its effects on clinical manifestations and prognosis. Anthracycline-based chemotherapy in combination with rituximab was given as initial therapy for 119 patients (94%). Epstein-Barr virus DNA was detected in 15 patients (12%), who were older (P = 0.005) and tended to be at a more advanced disease stage (P = 0.053). They showed significantly worse progression-free survival (PFS) and overall survival (OS) than other patients (P < 0.001 each). This effect remained significant (P = 0.004 and P = 0.027, respectively) after adjustment for age, lactate dehydrogenase, performance status, stage, and extranodal sites. The status of EBV-encoded small RNA in situ hybridization was known for 123 patients; 6 of 8 positive patients (75%) and 9 of 115 negative patients (8%) had detectable EBV DNA in pretreatment serum. While patients positive for EBV-encoded small RNA had significantly worse PFS and OS than negative patients (P = 0.001 and P = 0.029, respectively), EBV DNA detection in pretreatment serum was associated with poorer PFS and OS even for the 115 patients negative for EBV-encoded small RNA (P < 0.001 each). These findings suggest that EBV DNA detection in pretreatment serum may have an adverse prognostic impact for patients with diffuse large B-cell lymphoma.

The fate of patients with acute myeloid leukemia not undergoing induction chemotherapy.

Advances in chemotherapy for acute myeloid leukemia (AML) have resulted in the exclusion of patients not undergoing induction chemotherapy from research studies. To examine in detail the clinical experience of such patients, we retrospectively analyzed the outcomes of consecutive patients diagnosed with AML at our hospital from 2004 to 2012. Of 158 AML patients, 43 (27 %) did not undergo induction chemotherapy. Their median survival duration was 1.5 months, with 11, six, and four patients surviving more than 3, 6, and 12 months, respectively. As expected, their survival was worse than that of those treated with intensive or less-intensive induction therapy (14, 74, and 47 % at 1 year, and 0, 40, and 10 % at 4 years, respectively). Low white blood cell count at AML diagnosis and prior history of myelodysplastic syndrome were significantly associated with longer survival. Our findings suggest that modern supportive care measures do not prolong survival for AML patients not undergoing induction chemotherapy, although certain patients show relatively long survival. These data should prove helpful in discussing treatment pathways with patients for cases in which palliative or supportive therapy alone may be a viable treatment option.

A varicella outbreak in B-cell lymphoma patients receiving rituximab-containing chemotherapy.

Varicella, characterized by a vesicular rash, occurs primarily in young children. Although older individuals can also be affected or vaccinated, outbreaks among adults are rare. We investigated a small outbreak of varicella in B-cell lymphoma patients for elucidation of risk factor of the disease. We experienced four cases of varicella after an index herpes zoster case. All varicella cases were confirmed varicella zoster virus (VZV) infection by PCR. All varicella cases occurred in diffuse large B-cell lymphoma patients receiving rituximab-containing chemotherapy. On the other hand, only three of the 18 non-varicella patients in the same room were receiving rituximab-containing chemotherapy (P = 0.005). All varicella patients had detectable serum anti-varicella zoster virus IgG antibodies before chemotherapy. Even in the presence of neutralizing antibodies to the virus, lymphoma patients treated with rituximab-containing chemotherapy can possibly become re-infected with varicella. These findings suggest that zoster patients should be strictly isolated in hematology and oncology ward, and prophylactic acyclovir should be considered for such patients when exposed to zoster/varicella.

Differences in outcome for consecutive patients with diffuse large B-cell lymphoma before and after the advent of rituximab: a single-center experience.

The beneficial effect of rituximab for first-line treatment of diffuse large B-cell lymphoma (DLBCL) has been demonstrated by several randomized controlled trials. To clarify whether results for selected patient populations also apply to unselected patients, we analyzed long-term outcomes for all the 277 consecutive adults diagnosed with de novo DLBCL in a single center between 1998 and 2008. The study population included 147 and 130 patients diagnosed before (Cohort A) and after the advent of rituximab (Cohort B). Progression-free survival (PFS) was significantly better for Cohort B than for Cohort A (P = 0.005). For patients age 60 or younger, PFS did not differ significantly between Cohort A and Cohort B (P = 0.329), but for patients over 60, Cohort B showed superior PFS (P = 0.002). Patients with high or high-intermediate risk according to the International Prognostic Index score showed less improvement in PFS than did those with low or low-intermediate risk primarily because of still unfavorable outcomes of patients with poor performance status. These results indicate that the advent of rituximab has significantly improved outcome for unselected patients with DLBCL, and that improvement was greater for older patients. Further investigations are warranted in the hope of improving outcomes for younger patients with DLBCL.

A novel RUNX1-C11orf41 fusion gene in a case of acute myeloid leukemia with a t(11;21)(p14;q22).

The RUNX1 locus, which encodes a transcription factor that is essential for normal hematopoiesis, is a frequent location of chromosomal rearrangements in human hematological malignancies. We report the case of a 78-year-old man with acute myeloid leukemia (AML), M1 subtype (French-American-British classification), with a t(11;21)(p14;q22). Fluorescence in situ hybridization showed a split signal for RUNX1, which indicated that RUNX1 was involved in this translocation. Using 3'-rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction analyses, we found that RUNX1 was fused to C11orf41 on 11p14 and detected two in-frame C11orf41-RUNX1 fusion transcripts. One was a fusion between exon 5 of RUNX1 and exon 13 of C11orf41, and the other was between exon 6 of RUNX1 and exon 13 of C11orf41. This suggested that the RUNX1 breakpoint was in intron 6 and had generated alternative fusion splice variants. A reciprocal C11orf41-RUNX1 fusion was not detected. Thus, we identified C11orf41 as a novel fusion partner of RUNX1 in AML.

Severe hepatitis associated with varicella zoster virus infection in a patient with diffuse large B cell lymphoma treated with rituximab-CHOP chemotherapy.

Severe disseminated varicella zoster virus (VZV) infection rarely occurs in patients who are not recipients of hematopoietic stem cell transplantation. This report concerns severe disseminated VZV infection in a diffuse large B cell lymphoma (DLBCL) patient treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP). The patient was an 82-year-old male with DLBCL who had a history of type II diabetes mellitus. He incurred VZV infection with severe hepatitis and disseminated intravascular coagulopathy after three courses of R-CHOP. When the VZV infection occurred, anti-VZV IgG was not detected and lymphopenia was observed. We initiated treatment with acyclovir, immunoglobulin, and thrombomodulin alpha, and rescued this patient. We suggest that the use of chemotherapy for immune-suppressed elderly lymphoma patients may involve the risk of severe VZV infection.

Isolation of human mAbs that directly modulate FMS-related tyrosine kinase 3 signaling.

FMS-related tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase that plays important roles in hematopoiesis, including early progenitors and dendritic cell development. FLT3 is expressed at high levels in 70-100% of cases of AML and in virtually all cases of B-lineage acute lymphoblastic leukemia. FLT3 is regarded as a molecular target in the development of novel therapies for acute leukemia patients. Currently, many small-molecule FLT3 inhibitors have been developed, but clinical trials have resulted in limited antileukemia effects because of off-target toxicities and drug resistance. The development of anti-FLT3 Abs might overcome these difficulties and enhance the antileukemia efficacy of FLT3 inhibitors. In the present study, we demonstrate the isolation of novel human mAbs against FLT3 with antagonistic or agonistic activities. An antagonistic Ab, designated A2, continuously inhibits FLT3 ligand (FL)-induced phosphorylation of FLT3 and MAPK. A2 cooperatively induces apoptosis with daunorubicin, even in the presence of FL. An agonistic Ab, designated 3E6, surprisingly induces the phosphorylation of FLT3 and MAPK, and supports the growth of a factor-dependent cell line independently of FL addition. In addition, A2 showed complement-dependent cytotoxicity activity, but was devoid of Ab-dependent cell mediated cytotoxicity. Finally, we evaluated Ab internalization in a cell line. Immunofluorescence and flow cytometry analyses revealed that A2 is efficiently internalized. Collectively, these data demonstrate that A2 is a potent human Ab that might be capable of delivering cytotoxic reagents and that has antagonistic effects on FLT3 signaling. In addition, 3E6 might be a potential scaffold for novel dendritic cell-based immunotherapies.

CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.

In the mammalian testis, spermatogenesis is initiated from a subset of stem cells belonging to undifferentiated type A spermatogonia. In spite of the biologic significance of undifferentiated type A spermatogonia, little is known about their behavior and properties because of a lack of specific cell surface markers. Here we show that CDH1 (previously known as E-cadherin) is expressed specifically in undifferentiated type A spermatogonia in the mouse testis. Histologic analysis showed that CDH1-positive cells had all the characteristics of undifferentiated type A spermatogonia. Whole-mount immunohistochemistry showed that CDH1-positive cells made clusters mainly comprising one, two, four, or eight cells. They survived after administration of the cytotoxic agent busulfan to mice, and then regenerated seminiferous epithelia. Transplantation experiments showed that only CDH1-positive cells had colonizing activity in the recipient testis. Our data clearly demonstrated that spermatogenic stem cells reside among undifferentiated type A spermatogonia, which express CDH1.