Shan-Shan Chen

Imatinib mesylate versus allogeneic hematopoietic stem cell transplantation for patients with chronic myelogenous leukemia in the accelerated phase

The relative merits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and imatinib for chronic myelogenous leukemia in the accelerated phase (AP-CML) have not previously been evaluated. This cohort study was designed to compare the outcomes of imatinib (n = 87) versus allo-HSCT (n = 45) for AP-CML. A multivariate analysis of the total population revealed that a CML duration ≥ 12 months, hemoglobin < 100 g/L, and peripheral blood blasts ≥ 5% were independent adverse prognostic factors for both overall survival (OS) and progression-free survival (PFS). Both treatments resulted in similar survival in low-risk (no factor) patients, with 6-year event-free survival (EFS), OS, and PFS rates of more than 80.0%. Intermediate-risk (any factor) patients showed no difference in EFS and OS, but 6-year PFS rates were 55.7% versus 92.9% (P = .047) with imatinib versus allo-HSCT, respectively. Among high-risk (at least 2 factors) patients, imatinib was by far inferior to allo-HSCT, with 5-year EFS, OS, and PFS rates of 9.3% versus 66.7% (P = .034), 17.7% versus 100% (P = .008), and 18.8% versus 100% (P = .006), respectively. We conclude that allo-HSCT confers significant survival advantages for high- and intermediate-risk patients with AP-CML compared with imatinib treatment; however, the outcomes of the 2 therapies are equally good in low-risk patients. All trials were registered with the Chinese Clinical Trial Registry (www.chictr.org) as CHiCTR-TNC-10000955.

Expression patterns of WT1 and PRAME in acute myeloid leukemia patients and their usefulness for monitoring minimal residual disease.

Both WT1 and PRAME are highly expressed in acute myeloid leukemia (AML) patients. To assess the efficacy of their simultaneous detection for the purpose of monitoring minimal residual disease (MRD), we used real-time quantitative RT-PCR to quantify both WT1 and PRAME transcript levels in the bone marrow of 204 newly diagnosed AML patients, and 21 patients were serially monitored for a median of 11 months. The 22 normal bone marrow samples which served as controls collectively expressed low levels of WT1 and PRAME. An increase of >1-log over the upper limit of normal bone marrow was defined as positive. The positive rates of WT1 and PRAME for all patients were 79.2% and 55.4%, respectively. For the 108 patients lacking a specific fusion gene, the positive rate of WT1 was significantly higher than that of PRAME (76.9% vs. 35.2%, P<0.001). PRAME was positive in 9/25 WT1 (-) patients and the log increase of PRAME was higher than that of WT1 in 12/83 WT1 (+) patients. Dynamic patterns of WT1 and PRAME during follow-up showed that a consistent elevation or a rise over time to exceed the normal range predicted clinical relapse. The exception was that one patients WT1 significantly decreased at relapse compared to diagnosis. Therefore, a simultaneous detection of WT1 and PRAME might not only provide AML patients with either a positive or a more sensitive molecular marker for MRD monitoring. Moreover, it might also avoid false negativity in the case of expression alteration.

Characteristics of BCR–ABL kinase domain point mutations in Chinese imatinib-resistant chronic myeloid leukemia patients

To explore the characteristics of BCR–ABL kinase domain point mutations in Chinese chronic myeloid leukemia (CML) patients, a cohort of 127 patients with hematologic or cytogenetic resistance to imatinib are screened by direct sequencing. Mutations are found in 74 patients (58%). More patients with hematologic resistance show mutations compared to patients with cytogenetic resistance (70% vs 44%, p = 0.002), and more patients with acquired resistance present mutations compared to patients with primary resistance (68% vs 48%, p = 0.031). Frequencies of mutations were similar in early chronic phase (ECP), late chronic phase (LCP), accelerated phase, and blast phase (BP) patients (56%, 58%, 50%, and 69%, respectively; p > 0.05). Overall, 25 mutants are found in 21 amino acid sites, and four of them (I418V, E450A, E453L, and E455K) are first reported here. All patients with these four mutants either progress to or reenter the BP. The most frequent mutant is M244V, followed by Y253H, F359C/V/I, G250E, E255K, and T315I. Only seven patients (9%) have T315I mutants, and all showed hematologic resistance. Three of them were in the ECP and three in the LCP. Look-back studies show that mutants were detected 0–20 (median 7) months ahead of the appearance of clinical resistance in 15 tested patients with acquired resistance. ABL mutations are common in Chinese imatinib-resistant CML patients and are associated with clinical resistance. Chinese patients also seem to have unique profiles in the types and frequencies of some mutants, the disease phases of patients with T315I mutation, and the frequency of mutations in CP patients.

Different kinetic patterns of BCR-ABL transcript levels in imatinib-treated chronic myeloid leukemia patients after achieving complete cytogenetic response

Bone marrow BCR‐ABL transcript levels were monitored serially by real‐time quantitative PCR in 46 imatinib‐treated chronic myeloid leukemia patients after achieving complete cytogenetic response (CCyR) for a median of 42 months (range: 9–53). Of 41 patients in continuous CCyR, 32 and nine could achieve a ≥3‐log (MMoR group) or 2‐ to 3‐log reduction (non‐MMoR group), respectively. The MMoR group had a significantly lower recurrence rate of Ph+ metaphase than the non‐MMoR group (6/32 vs. 7/9, P = 0.002), which was not significantly different between patients first achieving CCyR within or after 12 months of imatinib treatment (7/27 vs. 6/14, P = 0.086). Five patients suffered cytogenetic or hematological relapse. For all 46 patients, a >2‐log reduction but not time when CCyR was first achieved was related to a lower relapse rate (1/42 vs. 4/4, P < 0.001). We concluded that the depth of BCR‐ABL reduction after CCyR is more critical than when CCyR is first achieved. The kinetic pattern of BCR‐ABL transcript is a good predictor of disease stability.

MPL W515L/K mutations in 343 Chinese adults with JAK2V617F mutation-negative chronic myeloproliferative disorders detected by a newly developed RQ-PCR based on TaqMan MGB probes.

Acquired mutations in the juxtamembrane region of MPL (W515L or W515K), the receptor for thrombopoietin, have been reported in patients with primary essential thrombocythemia (ET) or primary myelofibrosis (PMF). The mutations were detected by the newly developed real‐time quantitative PCR (RQ‐PCR) with TaqMan MGB probes and followed by the sequencing analysis. DNA samples were from 343 Chinese adults with JAK2V617F mutation‐negative chronic myeloproliferative disorders (cMPDs). Reference curves were obtained using cloned fragments of MPL containing either the wild‐type or MPL W515L or MPL W515K mutated sequence; the predicted sensitivity level was at least 0.5%(0.1–0.5%) for MPL W515L and 0.5%(0.2–0.5%) for MPL W515K mutant allele in a wild‐type background. The detection rates of MPL W515 mutations were 3.5% in 199 ET patients (7/199), 12.5% in 24 PMF patients (3/24) and 5.6% in 36 cMPD‐unclassed patients (2/36), respectively. No MPL W515 mutations were detected in 32 polycythemia vera (PV) patients, 40 chronic myeloid leukaemia (CML) patients, 12 hypereosinophilic syndrome (HES) patients and 29 normal volunteers. The mean calculated burden of MPL mutant alleles using RQ‐PCR for MPL W515L/K was 24.88 ± 14.80% (range, 1.10–56.32%). MPL W515L/K patients presented lower haemoglobin levels, compared with the patients with JAK2V617F mutation‐positive cMPDs (p < 0.01). The results demonstrated that RQ‐PCR was a reliable and sensitive method for large‐scale screening of the MPL W515L/K mutation in patients suspected to have a cMPD. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy. Copyright

Aberrant expression of CKLF-like MARVEL transmembrane member 5 (CMTM5) by promoter methylation in myeloid leukemia

CMTM5 has been shown to exhibit tumor suppressor activities, however, its role in leukemia is unclear. Herein we firstly reported the expression and function of CMTM5 in myeloid leukemia. CMTM5 was down-regulated, or undetectable, in leukemia cell lines and bone marrow cells from leukemia patients with promoter methylation. Ectopic expression of CMTM5-v1 strongly inhibited the proliferation of K562 and MEG-01 cells. In addition, significant negative correlations were observed between CMTM5 and three leukemia-specific fusion genes (AML1-ETO, PML-RARα and BCR/ABL1). CMTM5 expression was up-regulated in patients who had undergone treatment. Therefore, CMTM5 may be involved in the pathomechanism of myeloid leukemias.

Frequency and allele burden of CALR mutations in Chinese with essential thrombocythemia and primary myelofibrosis without JAK2V617F or MPL mutations

CALR mutations are detected in about 50% of persons of predominately European descent with essential thrombocythemia (ET) or primary myelofibrosis (PMF) with wild-type alleles of JAK2 and MPL. We studied 1088 Chinese with diverse myeloproliferative neoplasms including ET (N=234) and PMF (N=50) without JAK2(V617F) or MPL exon 10 mutations. CALR mutation was detected in 53% (95% CI, 46-60%) of subjects with ET and 56% (95% CI, 41-70%) of subjects with PMF. 152 CALR mutations were identified clustering into 15 types including deletions (N=8), insertions (N=3) and complex indels (N=4). We also identified 9 new mutations. Mean (±SD) mutant allele burden was 31±12% (range, 0.5-69%). Persons with PMF had higher CALR mutant allele burdens than those with ET (38±8% vs. 29±12%; P<0.001). Amongst persons with CALR mutations, those with PMF had different clinical features from those with ET. These data may be useful for diagnosing ET and PMF in Chinese who are about 40% of all persons with ET and PMF and for monitoring therapy-response. They also highlight similarities and differences in CALR mutations between Chinese and persons of predominately European descent with these diseases.

A rapid, highly accurate method for quantifying CALR mutant allele burden in persons with myeloproliferative neoplasms

Abstract Background Calreticulin (CALR) mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2V617F. Consequently rapid, sensitive, and specific methods to detect and quantify these mutations are needed. Methods We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2V617F negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. Results We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML, or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5–5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions, and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5–5%) in a wild-type background. Conclusions PCR amplification followed by fragment length analysis is a rapid, sensitive, and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PMF and for estimating mutant allele burden.

The clinical value of the quantitative detection of four cancer-testis antigen genes in multiple myeloma

BackgroundCancer-testis (CT) antigen genes might promote the progression of multiple myeloma (MM). CT antigens may act as diagnostic and prognostic markers in MM, but their expression levels and clinical implications in this disease are not fully understood. This study measured the expression levels of four CT antigen genes in Chinese patients with MM and explored their clinical implications.MethodsReal-time quantitative polymerase chain reaction (qPCR) was used to quantify the expression of MAGE-C1/CT7, MAGE-A3, MAGE-C2/CT10 and SSX-2 mRNA in 256 bone marrow samples from 144 MM patients.ResultsIn the newly diagnosed patients, the positive expression rates were 88.5% for MAGE-C1/CT7, 82.1% for MAGE-C2/CT10, 76.9% for MAGE-A3 and 25.6% for SSX-2. The expression levels and the number of co-expressed CT antigens correlated significantly with several clinical indicators, including the percentage of plasma cells infiltrating the bone marrow, abnormal chromosome karyotypes and the clinical course.ConclusionMAGE-C1/CT7, MAGE-A3, MAGE-C2/CT10 and SSX-2 expression levels provide potentially effective clinical indicators for the auxiliary diagnosis and monitoring of treatment efficacy in MM.

High frequency of IKZF1 deletions in Chinese adult patients with acute lymphoblastic leukemia detected by multiplex real-time quantitative PCR

Objectives: To examine the incidence and clinical dynamics of IKZF1 alterations in Chinese adult patients with ALL. Methods: Samples were studied from 328 newly diagnosed adult ALL patients at Peking University People’s Hospital from 2007 to 2012 by multiplex real-time quantitative PCR, multiplex fluorescent PCR and sequence analysis for four types of IKZF1 deletions. Albumin (ALB) was the plasmid standard. Results: All correlation coefficients for amplified ALB(albumin) and IKZF1 Δ4-7 deletion plasmids were above 0.99, and the sensitivity of detection was at least ten copies. About 36.3% (95% [CI], 31.1-41.7%) of 328 untreated cases showed IKZF1-deletion. The median IKZF1-deletion copies/ALB copy was 85.8% (0.1%697.9%). 27 IKZF1-deletion-positive patients (219 samples in total) were followed up after treatment, among them, 18 patients in hematologic remission continued to be tested negative within 8-66 weeks. In 5 relapsed cases, elevated levels of IKZF1 deletion were detected when relapse occurred. In 4 non responders, IKZF1 deletion maintained at high level. Patient with B-cell ALL subtype had much higher IKZF1-deletion rate (40.4%) than those with T-cell ALL subtype (2.8%, P<0.01). In particular, it was higher in common B-cell ALL group than those with other subtypes. Conclusion: This assay is reliable and sensitive. It is useful in diagnosis and monitoring minimal residual disease in adult ALL. High frequency of IKZF1 deletions occur in adult patients with common B-cell ALL. Correspondence to: Guo-Rui Ruan, Peking University People’s Hospital and Institute of Hematology 11 Xi-Zhi-Men, South Street, 100044 Beijing, China, Tel: 8610-88324672, Fax: 8610-88324672 # Li-Xin Wu and Jiao Zhou contributed equally to the work.