The added values of multiplex reverse transcriptase-PCR followed by mutation screening in the initial evaluation of acute leukemia.

INTRODUCTION: This study investigates the benefits of using multiplex reverse transcriptase-PCR (RT-PCR) in addition to standard karyotyping during the initial evaluation of acute leukemia. METHODS: A total of 1114 consecutive specimens from patients with acute leukemia were tested using a commercial multiplex RT-PCR kit (HemaVision, DNA Diagnostic). NPM1 and CEBPA mutations were selectively tested in acute myeloid leukemia (AML) patients with multiplex RT-PCR negativity. RESULTS: In specimens with optimal cytogenetics, the frequency of recurrent translocations was 31.3%, and cryptic translocations were detected in 2.1% of samples. The concordance rate between karyotyping and multiplex RT-PCR was 97.5%. In addition to the established functions, we demonstrated the additional benefits of multiplex RT-PCR, including successful molecular characterization, even in cytogenetically suboptimal specimens (5.7%); detection of submicroscopic aberrations (1.0%); detection of rare but potentially significant translocations or variants (2.5%); selection of AML candidates for mutation analysis (68.3%); and finally exclusion of recurrent translocations in patients with acute lymphoblastic leukemia or mixed phenotype acute leukemia (22.5%). CONCLUSION: We reconfirmed the accuracy and reliability of multiplex RT-PCR for diagnosing acute leukemia and demonstrated additional advantages of this system for the initial evaluation of acute leukemia. Thus, multiplex RT-PCR is worth considering in diagnostic testing of acute leukemias.

Body fluid cellular analysis using the Sysmex XN-2000 automatic hematology analyzer: focusing on malignant samples.

INTRODUCTION: The majority of previous studies on body fluid (BF) mode of automatic hematology analyzer used nonmalignant BF samples. Here, we evaluated the BF mode on the recently launched Sysmex XN for counting blood cells, especially for malignant samples. METHODS: A total of 405 BF specimens including 125 malignant samples were analyzed using both the automated method and manual microscopy. RESULTS: In non-cerebrospinal fluids (CSF) samples, there was an agreement between two methods for WBC, RBC, polymorphonuclear, and mononuclear cell counts (R(2)  = 0.96, 0.94, 0.88, and 0.88, respectively). CSF samples showed slightly poorer correlations than other fluids. Exclusion of malignant samples significantly improved correlations in non-CSF samples, but not in CSF samples. High fluorescence-BF (HF-BF) cells were identified significantly more frequently in malignant samples compared to benign samples (17.8 and 4.15/100 WBC, respectively; P < 0.001). Receiver operating characteristic curve analysis demonstrated an HF-BF cell AUC of 0.791 using a cutoff value of 6.9/100 WBC for detecting malignant samples. CONCLUSION: The BF mode on the Sysmex XN could be an alternative method for the manual counts in the BF analysis with a few drawbacks. However, if a concentration of HF-BF cells is greater than the given threshold, microscopic examination should be subsequently performed.

FISH analysis of MLL gene rearrangements: detection of the concurrent loss or gain of the 3' signal and its prognostic significance.

INTRODUCTION: The rearrangement of the mixed-lineage leukemia (MLL) gene occurs through translocations and insertions involving a variety of partner chromosome genes. However, there are few studies on aberrant MLL signal patterns such as concurrent 3' MLL deletion. METHODS: A total of 84 patients with acute leukemia (AL) who had MLL rearrangements detected by florescence in situ hybridization (FISH) were enrolled in the study. The distribution of MLL fusion partner genes was analyzed, and aberrant MLL signals were evaluated. RESULTS: Seventy-seven (91.7%) patients had MLL rearrangements, involving previously described translocation partner genes (TPGs). Among these TPGs, the frequencies of MLLT3, AFF1, MLLT4, and ELL were 29.8%, 17.9%, 15.5%, and 13.1%, respectively. A high frequency of MLLT4 in our study was due to the high proportion of acute myeloid leukemia cases in pediatric and adult patients. Aberrant MLL signals were found in 18 patients: 11 (61.1%) with 3' MLL signal loss and 7 with 3' MLL signal gain. All cases with 3' MLL signal gain were due to an extra derivative partner chromosome. The median overall survival period of patients with 3' MLL gain was shorter than that in patients without aberrant MLL signal patterns. CONCLUSION: Aberrant MLL signals were frequently detected by FISH analysis. The 3' MLL gain was associated with poor prognosis in patients with AL. Therefore, it is important to detect aberrant MLL signal patterns using FISH analysis.

Morphologic detection of blast cells in the cerebrospinal fluid at diagnosis of adult acute lymphoblastic leukemia appears to be associated with adverse prognosis.

INTRODUCTION: Appropriate treatment of central nervous system (CNS) involvement in adult acute lymphoblastic leukemia (ALL) is important for patient prognosis, but the diagnostic criteria of CNS involvement has not been established. METHODS: The significance of blast cells in the cerebrospinal fluid (CSF) at diagnosis was evaluated in 81 adults newly diagnosed with ALL. Patients with unequivocal morphologic evidence of lymphoblasts in the cytocentrifuged CSF slide were considered to have CNS involvement regardless of the leukocyte count. The outcomes of the patients were analyzed. RESULTS: Four of the 81 patients (5%) had detectable blast cells, and three of these four patients had less than five leukocytes/µL of CSF. One-year event-free survival (EFS) was 25.0% and 53.2% (P = 0.008) and overall survival (OS) was 50.0% and 68.8% (P = 0.001) in patients with and without CNS involvement, respectively. CNS involvement had prognostic impact on EFS (P = 0.047) and OS (P = 0.009) after adjusting for sex, age, leukocyte count, Philadelphia chromosome status, and immunophenotype. CONCLUSION: This study suggests that morphologic detection of blast cells in the CSF at diagnosis of adult ALL, regardless of the leukocyte count, is associated with adverse prognosis.

Differential diagnosis of myelofibrosis based on WHO 2008 criteria: acute panmyelosis with myelofibrosis, acute megakaryoblastic leukemia with myelofibrosis, primary myelofibrosis and myelodysplastic syndrome with myelofibrosis.

INTRODUCTION: The aim of this study was to characterize clinicopathological features of acute panmyelosis with myelofibrosis (APMF), acute megakaryoblastic leukemia with myelofibrosis (AMKL-MF), primary myelofibrosis (PMF) and myelodysplastic syndrome with myelofibrosis (MDS-MF) in order to provide the keys to the differential diagnosis of bone marrow (BM) fibrosis. METHODS: We compared age, gender, splenomegaly, serum lactate dehydrogenase level, blood cell counts, blast counts in peripheral blood (PB) and BM, megakaryocyte counts, BM cellularity, dysplasia, and the karyotypes of patients with APMF (n = 6), AMKL-MF (n = 7), PMF (n = 44), and MDS-MF (n = 44). RESULTS: APMF showed hyperplasia of all three lineages, increase in megakaryocyte count with dysplasia and frequent abnormal karyotypes. AMKL-MF was associated with elevated BM blast counts, decreased BM megakaryocyte count with rare megakaryocytic dysplasia and chromosome 21 abnormality. PMF patients displayed splenomegaly, rare blasts in PB/BM, and JAK2 V617F mutation. MDS-MF patients showed pancytopenia, dysplasia in all three lineages and recurrent chromosomal abnormalities involving chromosome 5,7,12, and 17. CONCLUSIONS: Although differential diagnosis among APMF, AMKL-MF, PMF, and MDS-MF is very challenging due to the overlapping clinical and morphological features, meticulous investigation of the patient with respect to splenomegaly, blood cell count, PB and BM findings, and karyotype will serve as a guide to correct diagnosis.

Detection of ABL1 kinase mutations in Philadelphia-positive patients exhibiting an inadequate molecular response using restriction fragment mass polymorphism and its clinical significance: a single-center experience in Korea.

INTRODUCTION: ABL1 kinase mutations represent a major mechanism of imatinib resistance in Philadelphia-positive (Ph+) patients. There is a paucity of data on ABL1 kinase mutations in Ph+ patients in Korea. METHODS: We used restriction fragment mass polymorphism (RFMP) analysis to detect ABL1 kinase mutations in blood or bone marrow specimens from 80 Ph+ patients. RESULTS: Fifty-seven patients met the criteria for inadequate molecular response (IMR). ABL1 kinase mutations were found in 2.6% of patients with chronic-phase chronic myelogenous leukemia (CML), 25.0% of accelerated-phase CML, 66.7% of blast-phase CML, and in 58.3% with Ph+ acute lymphoblastic leukemia. Twelve mutations were identified: 7 T315I, 2 E255V, 1 E255K, 1 F359V, and 1 Y253H. The majority of mutation-positive patients showed an unfavorable clinical course and often had an extra Ph or additional chromosomal abnormalities. Mutations were detected in two patients who had very low or absent BCR-ABL1 normalized ratios. CONCLUSION: Mutation analysis should be performed in Ph+ patients exhibiting an IMR to imatinib. RFMP analysis is helpful for revising therapeutic strategies because it can sensitively detect clinically relevant ABL1 kinase mutations with high frequencies.

Automated digital cell morphology identification system (CellaVision DM96) is very useful for leukocyte differentials in specimens with qualitative or quantitative abnormalities.

INTRODUCTION: The CellaVision DM96 system (CellaVision AB, Lund, Sweden) was developed as one of the automated digital cell morphology analyzer for determining leukocyte differential counts in peripheral blood smears (PBS) and we evaluated this system. METHODS: A total of 308 PB samples with abnormalities were analyzed in this study. For each sample, manual differential counts were performed by two independent technologists, and the CellaVision DM96 system was applied in duplicate. Correlations between the two methods and ability of this system to identify six abnormalities were assessed. RESULTS: The correlation coefficients between two methods were consistently high, ranged from 0.864 to 0.992. The sensitivity, specificity, positive predictive value, negative predictive values of this system for the identification of abnormalities were consistently high, especially for blasts (98.2%, 99.2%, 96.6%, 99.6%). When the instrument was ordered to count 300 or 500 cells from the operator, better performance was demonstrated than 100 cells in the leukopenic samples by sacrificing only 40 s/slide in average. CONCLUSIONS: The CellaVision DM96 system is useful in the clinical laboratory providing comparative accuracy compared with manual counts in samples with abnormalities. In leukopenic samples, report quality can be improved by ordering to count 300 or 500 cells from the operator without severe prolongation of turnaround time.

Application of an immune-magnetic cell sorting method for CD138-positive plasma cells in FISH analysis of multiple myeloma.

INTRODUCTION: Interphase fluorescence in situ hybridization (FISH) analysis of multiple myeloma (MM) may indiscriminately count signals of nonplasma cells, thus decreasing specificity and sensitivity. We aimed to evaluate the usefulness of an immune-magnetic sorting method for plasma cells in FISH analysis of MM and define optimal sample preparation conditions. METHODS: Plasma cells were purified using EasySep(®) CD138 Positive Selection Cocktail and Magnetic Nanoparticles (Invitrogen). We compared FISH results with and without plasma cell purification for three sample preparation methods: direct harvest, 24-h culture, and 96-h culture with interleukin-4 in five newly diagnosed MM patients. Archived fixed bone marrow cells of 17 MM patients were also studied. RESULTS: The percentage of abnormal cells identified was significantly higher with plasma cell purification than without purification (median, 88.0%; range, 84.0-100.0%vs. 15.0%, 12.5-29.5%, respectively). The three sample preparation methods showed comparable results. Immune-magnetic sorting also significantly increased the percentage of abnormal cells identified in FISH analysis of archived fixed bone marrow cells (P < 0.001). CONCLUSIONS: Immune-magnetic CD138-positive cell sorting significantly increased the percentage of abnormal cells identified in FISH analysis of MM samples for all sample preparation methods. This method could also be applied for retrospective FISH analysis of archived fixed bone marrow cells.

A rare case of Lennert's type peripheral T-cell lymphoma with t(14;19)(q11.2;q13.3).

Although most patients with peripheral T-cell lymphoma (PTCL) show clonal rearrangement of T-cell receptor genes, few PTCLs show recurrent chromosomal abnormalities. We describe here a rare chromosomal rearrangement, t(14;19)(q11.2;q13.3), in a Lennert's lymphoma, a variant of PTCL, not otherwise specified. Sequential fluorescence in situ hybridization assays showed that the breakpoint in 19q13.3 was located distal to the BCL3 and PVRL2 genes, both of which may be candidate proto-oncogenes. These findings suggest that another gene is involved in the pathogenic characteristics observed in this patient with Lennert's lymphoma.

Prevalence of abnormal in vitro closure time using the Platelet Function Analyzer-100 in chronic kidney disease patients and analysis of associated factors.

AIM: Platelet Function Analyzer- 100 evaluates platelet function by determining time to occlusion of an aperture in a membrane coated with collagen and epinephrine (CEPI) or collagen and ADP (CADP) during the flow of citrated whole blood. We sought to determine prevalence of abnormal in vitro closure time (CT) in chronic kidney disease (CKD) patients and to analyze associated factors. MATERIALS AND METHODS: CEPI-CT (normal, 82 - 182 sec in Korean), CADP-CT (normal, 62 - 109 sec), CBC, serum creatinine (Cr) and blood urea nitrogen (BUN) were measured in CKD patients, 30 with Stage I, 36 with Stage II, 30 with Stage III, 56 with Stage IV, 283 with Stage V (79 with pre-dialysis Stage V, 130 on chronic hemodialysis (CHD), and 74 on chronic peritoneal dialysis (CPD)). Estimated glomerular filtration rate (eGFR) was calculated with a MDRD equation. RESULTS: Abnormal CEPI-CT and CADP-CT occurred in < 15% of Stage I - III, 20% of Stage IV, and 41% and 54%, respectively, of Stage V patients. There were no differences in prevalence of abnormal CEPI-CT and CADP-CT among predialysis Stage V, CHD and CPD patients. CEPI-CT and CADP-CT were correlated with BUN, Cr and platelet counts in predialysis patients, and with platelet counts in dialysis patients, and CEPI-CT was correlated with BUN, Cr in CPD patients. Neither, however, was correlated with age, gender, hemoglobin or hematocrit. CONCLUSION: Prevalence of abnormal in vitro CT increases as stage worsens in CKD patients. In vitro CT is correlated with BUN, Cr and platelet counts in predialysis and total CKD patients.

Direct international normalized ratio determination using multicalibrators is more responsive than the conventional method for measuring prothrombin time.

Direct international normalized ratio (INR) determination using certified INR plasmas was shown to improve precision and accuracy. We evaluated the utility of a multicalibrator in determining INR. INR values were measured in 493 blood samples from patients subjected to anticoagulation therapy (320) and control subjects (173). Study was performed using CA-7000 coagulation analyzer (Sysmex, Japan) with Thromborel S (Dade Behring, Germany). Direct INR values were obtained using PT-Multi Calibrator (Dade Behring) composed of five lyophilized calibrant plasmas. Conventional INR values were calculated from mean normal prothrombin time and instrument/reagent-specific international sensitivity index (ISI). We compared the difference between the INR results obtained with the two methods. The mean INR value of direct INR method was significantly higher than that of conventional method. The differences in values (direct INR - conventional INR) generated using the two methods increased in proportion to the INR values. Elevation of INR was observed in data obtained with the direct INR method, compared with conventional INR values. Accordingly, we conclude that direct INR method is more responsive than conventional method in determining INR.

Establishment of a reference interval for natural killer cell activity through flow cytometry and its clinical application in the diagnosis of hemophagocytic lymphohistiocytosis.

Recently, the Histiocyte Society revised the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) to include low or absent natural killer (NK) cell activity, according to local laboratory reference. The aim of this study was to establish reference interval for functional NK-cell activity in 63 healthy Korean individuals using a flow-cytometric assay. We used peripheral blood mononuclear cells (PBMCs) as effector cells and Fluorescein isothiocyanate-labeled K562 cells as target cells. NK-cell activity was calculated using the following equation: NK-cell activity (%) = (test lysis - spontaneous lysis) x 100/(maximum lysis - spontaneous lysis). NK-cell activity was analyzed in 13 known HLH patients and 16 suspected non-HLH patients using a flow-cytometric assay. The mean (+/-SD) cytotoxicity of PBMCs from healthy individuals was 20.9 +/- 5.3% and the reference interval was 11.8-31.9%. The mean NK-cell activity of HLH patients (8.3 +/- 8.9%) was significantly lower (P = 0.001) than that of non-HLH patients (20.1 +/- 7.8%). The sequential changes in NK-cell activity in the HLH group corresponded to clinical and laboratory findings following treatment. We successfully developed a functional NK-cell activity test for use in the clinical laboratory and obtained a reference interval of NK-cell activity from healthy donors. This assay, and associated reference interval, was used to analyze 30 clinically relevant specimens and the results were shown to be well correlated.

Allogeneic hematopoietic cell transplantation for myelodysplastic syndrome: prognostic significance of pre-transplant IPSS score and comorbidity.

We analyzed the clinical significance of pre-transplant International Prognostic Scoring System (IPSS) score and comorbidity in 68 patients who underwent allogeneic hematopoietic cell transplantation (HCT) for myelodysplastic syndrome (MDS) (n=48) or acute myeloid leukemia evolved from MDS (n=20) between December 1995 and January 2008 in a single institute. During a median follow-up period of 41.0 months (range, 3.2-132.0 months), 27 patients died, and 7 relapsed. The 5-year probabilities of overall survival (OS) and event-free survival (EFS) were 60.0 and 57.4%, respectively, and the 5-year cumulative incidences of non-relapse mortality (CINRM) and relapse were 32.7 and 9.9%, respectively. OS, EFS, and CINRM were significantly different according to pre-transplant IPSS score and presence of pre-transplant comorbidity, which were independent risk factors along with Karnofsky performance score in multivariate analyses. In conclusion, pre-transplant IPSS score and comorbidity may stratify the risk of post transplant outcomes in MDS.

Quantitative Epstein-Barr virus viral load monitoring in pediatric liver transplantation.

The aim of this study was to determine whether the implementation of the quantitative Epstein-Barr virus polymerase chain reaction (qEBV-PCR) test in 2003 decreased the incidence of posttransplant lymphoproliferative disease (PTLD) and PTLD-related mortality. Of the 128 children who underwent liver transplantation between January 1994 and May 2007, 110 (85.9%) survived. Patients were divided into pre (1994 to 2002; n = 86) and post (2003 to 2007; n = 42) EBV-PCR groups. There were no between-group differences in mean age, percentage of patients < 12 months old, or seronegative for EBV. The incidence rates of primary EBV infection in the pre- and post-EBV-PCR groups were 14.0% and 33.3%, respectively (P < .05). In contrast, the pre- and post-EBV-PCR groups showed similar incidences of symptomatic EBV infection (31.3% vs 35.7%; P = .625) and PTLD (10.5% vs 9.5%; P = .869), but different survival rates (80.2% vs 97.6%; P < .001). Five of nine PTLD patients in the pre-EBV-PCR group died of PTLD, but there was no PTLD-related mortality in the post-EBV-PCR group, indicating that PTLD-related mortality decreased after qEBV-PCR monitoring. These findings suggested that frequent EBV viral load monitoring and subsequent modulation of immunosuppression can reduce PTLD and PTLD-related mortality among pediatric liver transplant patients.

Robotic tumor-specific mesorectal excision of rectal cancer: short-term outcome of a pilot randomized trial.

BACKGROUND: Laparoscopic colorectal resection has become popular. The recently developed da Vinci Surgical System promises to facilitate endoscopic surgery and overcome its disadvantages. This study therefore aimed to compare the short-term results between robotic tumor-specific mesorectal excision (R-TSME) using the da Vinci Surgical System and conventional laparoscopic tumor-specific mesorectal excision (L-TSME) in rectal cancer patients. METHODS: Between April 2006 and February 2007, 36 patients were randomly assigned to receive R-TSME or L-TSME. During the study, 18 patients underwent robotic low anterior resection using the da Vinci Surgical System, and 18 patients had conventional laparoscopic low anterior resection. Patient characteristics, perioperative clinical results, complications, and pathologic details were compared between the two groups. RESULTS: The patient characteristics were not significantly different between the two groups. The mean operating time, hemoglobin change, and conversion rate were not significantly different between the groups. Complications were treated conservatively and did not require surgical intervention in the R-TSME group. The average length of stay was 6.9 +/- 1.3 days in the R-TSME group and 8.7 +/- 1.3 days in the L-TSME group (p < 0.001). The specimen quality of the R-TSME group was acceptable. CONCLUSION: Tumor-specific mesorectal excision was performed safely and effectively using the da Vinci Surgical System and the perioperative outcomes were acceptable.

Clinical effect of imatinib added to intensive combination chemotherapy for newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia.

Clinical impact of imatinib was evaluated in 20 patients (median age, 37 years; range, 15-67 years) with newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), who were administered with induction chemotherapy of daunorubicin, vincristine, prednisolone, and L-asparaginase, along with imatinib 600 mg/day during remission induction and 400 mg/day during consolidation courses. One patient died on day 14 from septic shock, while the remaining 19 achieved complete remission (CR). In total, 15 patients underwent allogeneic hematopoietic cell transplantation (HCT) during first CR. After median follow-up period of 799 days, six patients experienced recurrence; two with early recurrence within 100 days, one with leptomeningeal recurrence at 11 month, and three with post-HCT recurrence. Eight patients died. Median CR duration (821 days) and median patient survival (894 days) in the study were significantly longer by 2.9- and 2.3-fold, respectively, when compared to those of 18 historical patients treated with same regimen of combination chemotherapy without imatinib. Toxicities of the combined treatment were manageable and included grade 4 myelosuppression (n = 20) and reversible > or = grade 3 hyperbilirubinemia (n = 4). Beneficial clinical effects were observed when imatinib was added to combination chemotherapy in patients with newly diagnosed Ph+ ALL. Further studies with larger number of patients are necessary.

Treatment of relapsed acute lymphoblastic leukemia after allogeneic bone marrow transplantation with chemotherapy followed by G-CSF-primed donor leukocyte infusion: a prospective study.

Donor leukocyte infusion (DLI) alone has very limited efficacy for patients with acute lymphoblastic leukemia (ALL) who have relapsed after allogeneic bone marrow transplantation (BMT). We, therefore, prospectively tested the efficacy of cytoreductive chemotherapy (intermediate-dose cytarabine+idarubicin+etoposide) followed immediately by G-CSF-primed DLI (Chemo-DLI) in 10 relapsed ALL patients after allogeneic BMT. Seven achieved complete remission (CR) at a median of 25 days (19-73 days) after DLI. Of these seven CR patients, only one remains alive in CR 907 days after DLI. Two CR patients died in CR of graft-versus-host disease. The remaining four CR patients relapsed at a median of 153 days (120-991 days) after DLI. One is alive with leukemia at post-DLI day 1217. The median survival duration after DLI was 175 days (15-1217 days). In summary, although Chemo-DLI for relapsed ALL after allogeneic BMT induced a relatively high CR rate, durable remissions were rare. Although our data should be interpreted cautiously considering the small number of patients, these results suggest that poor outcome of DLI in relapsed ALL may be primarily due to intrinsic resistance to graft-versus-leukemia effect rather than to the rapid pace of the disease.