Cytotoxicity and exhaustion markers of chimeric antigen receptor T cells targeting BCMA in multiple myeloma cell lines between patients and healthy donors.

OBJECTIVES: Multiple myeloma (MM) accounts for 10% of hematologic malignancies. However, most of the patients suffered from relapsed/refractory disease. We would like to expand CAR T cell therapy to treat MM using our current platform. METHODS: BCMA CAR T lymphocytes were generated for volunteers or MM patients. The transduction efficiency was detected by the ddPCR technique. Immunophenotyping and exhaustion markers were monitored by flow cytometry. The efficacy of BCMA CAR T cells was tested using coculturing with BCMA CAR or mock, and the positive and negative targets, K562/hBCMA-ECTM and K562, respectively. RESULTS: BCMA CAR T cells were generated from consented volunteers or MM patients and could be detected CAR BCMA expression at a mean of 4.07 ± 1.95 or 4.65 ± 1.21 copies/cell, respectively. Those modified T cells were primarily effector memory T cells. Our BCMA CAR T cells could explicitly eradicate the K562/hBCMA-ECTM cell line while the K562 cell line survived. Interestingly, the BCMA CAR, mock T cells, and peripheral blood mononuclear cells from MM patients expressed similar levels of the exhaustion makers, TIM-3, LAG-3, and PD1. CONCLUSIONS: Our BCMA CAR T cells, mainly effector/effector memory, could eliminate BCMA-expressing cells in vitro and had similar levels of exhaustion markers among different populations.

Endothelial activation and stress index as a prognostic factor of diffuse large B-cell lymphoma: the report from the nationwide multi-center Thai Lymphoma Study Group.

Several prognostic models have been introduced to predict outcomes of patients with diffuse large B-cell lymphoma (DLBCL). Endothelial activation and stress index (EASIX) is a surrogate of endothelial dysfunction which has been shown to predict outcomes of patients with various hematologic malignancies. However, the prognostic implication of EASIX for DLBCL is limited and warrants exploration. We conducted a retrospective study enrolling adult DLBCL patients including a discovery cohort from the single-centered university hospital database and a validation cohort from the independent nationwide multi-center registry. EASIX scores were calculated using creatinine, lactate dehydrogenase, and platelet levels. The receiver operating characteristic curve analysis was used to determine optimal cutoff. Statistical analysis explored the impact of EASIX on survival outcomes. A total of 323 patients were included in the discovery cohort. The optimal EASIX cutoff was 1.07 stratifying patients into low (53.9%) and high EASIX (46.1%) groups. Patients with high EASIX had worse 2-year progression-free survival (PFS) (53.4% vs. 81.5%, p<0.001) and overall survival (OS) (64.4% vs. 88.7%, p<0.001) than patients with low EASIX. Multivariate analysis revealed that older age, bulky disease, impaired performance status, and high EASIX were associated with an unfavorable OS. In the validation cohort of 499 patients, the optimal EASIX cutoff was 1.04. Similar to the discovery cohort, high EASIX score was associated with high-risk diseases, worse PFS, and inferior OS. In conclusion, EASIX score was significantly associated with survival outcomes and may be used as a simple prognostic tool to better risk-classify DLBCL.

Outcomes of polatuzumab vedotin-containing regimens in real-world setting of relapsed and or refractory diffuse large B-cell lymphoma patients: a matched-control analysis from the Thai Lymphoma Study Group (TLSG).

Relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) is a challenging condition to treat, and there is an unmet clinical need for effective therapies. Recently, polatuzumab vedotin (Pola), an anti-CD79b antibody-drug-conjugate (ADC), combined with bendamustine-rituximab (BR), has been approved for R/R DLBCL patients. However, real-world data on Pola-based regimens in R/R DLBCL patients, especially in Thailand, are limited. This study aimed to evaluate the efficacy and safety of Pola-based salvage treatment in R/R DLBCL patients in Thailand. Thirty-five patients who received Pola-based treatment were included in the study, and their data were compared to 180 matched patients who received non-Pola-based therapy. The overall response rate (ORR) in the Pola group was 62.8%, with complete remission and partial remission rates of 17.1% and 45.7%, respectively. The median progression-free survival (PFS) and overall survival (OS) were 10.6 months and 12.8 months, respectively. The study found a significantly higher ORR in Pola-based salvage treatments compared to non-Pola-based therapy (62.8% vs. 33.3%). The survival outcomes were also significantly superior in the Pola group, with longer median PFS and OS than the control group. Grades 3-4 adverse events (AEs) were mainly hematological, and they were tolerable. In conclusion, this study provides real-world evidence of the efficacy and safety of Pola-based salvage treatment in R/R DLBCL patients in Thailand. The results of this study are promising and suggest that Pola-based salvage treatment could be a viable option for R/R DLBCL patients who have limited treatment options.

Incidence of anti-platelet factor4/polyanionic antibodies, thrombocytopenia, and thrombosis after COVID-19 vaccination with ChAdOx1 nCoV-19 in Thais.

BACKGROUND: The prevalence of anti-platelet factor 4 (PF4)/polyanionic antibodies occurring after vaccination with ChAdOx1 nCoV-19 is low. Most of these antibodies are not associated with vaccine-induced thrombotic thrombocytopenia. It remains unknown whether these antibodies are preexisting or occur as a result of vaccination. In this study, we demonstrated the incidence of anti-PF4/polyanionic antibodies, thrombocytopenia, and thrombosis after vaccination with ChAdOx1 nCoV-19 in a large cohort of Thais. METHODS: We conducted a prospective study in a cohort of health care workers and members of the general population who received COVID-19 vaccination with ChAdOx1 nCoV-19. Blood collection for complete blood count, D-dimer, and anti-PF4/polyanionic antibodies was performed before vaccination (day 0), day 10, and day 28 after vaccination. Anti-PF4/polyanionic antibodies were detected using enzyme-link immunosorbent assay (ELISA). Functional assay was performed for all positive ELISA tests. RESULTS: A total of 720 participants were included in the study. 214 participants received both the first and second doses, 91 participants received only the first, 51 received only the second, and 364 received the third booster dose of ChAdOx1 nCoV-19. Median age was 42 years (IQR, 34-53). 67% of participants were female. Three participants developed seroconversion, yielding an incidence of vaccination-induced anti-PF4/polyanionic antibodies of 0.42% (95% confidence interval 0.08, 1.23). Fourteen (1.9%) participants had preexisting anti-PF4/polyanionic antibodies before the vaccination but their optical density of anti-PF4/polyanionic antibodies did not significantly increase over time. None of the anti-PF4/polyanionic positive sera induced platelet aggregation. Abnormal D-dimer levels following vaccination were not different among the positive and negative anti-PF4/polyanionic groups (11.8% vs. 13.2%, p = 0.86). Thrombocytopenia occurred in one person with negative anti-PF4/polyanionic antibodies. No clinical thrombosis or bleeding occurred. CONCLUSION: We found a low incidence of seroconversion of anti-PF4/polyanionic antibodies after vaccination with ChAdOx1 nCoV-19 in Thais. Most of the anti-PF4/polyanionic antibodies were preexisting and did not significantly increase after vaccination with ChAdOx1 nCoV-19. Following vaccination, some participants with anti-PF4/polyanionic antibodies had elevated D-dimer levels, while only one developed thrombocytopenia and no thrombotic events were observed.

Inhibition of C3 with pegcetacoplan results in normalization of hemolysis markers in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired hematologic disorder characterized by complement-mediated hemolysis. C5 inhibitors (eculizumab/ravulizumab) control intravascular hemolysis but do not prevent residual extravascular hemolysis. The newly approved complement inhibitor, pegcetacoplan, inhibits C3, upstream of C5, and has the potential to improve control of complement-mediated hemolysis. The PADDOCK and PALOMINO clinical trials assessed the safety and efficacy of pegcetacoplan in complement inhibitor-naïve adults (≥ 18 years) diagnosed with PNH. Patients in PADDOCK (phase 1b open-label, pilot trial) received daily subcutaneous pegcetacoplan (cohort 1: 180 mg up to day 28 [n = 3]; cohort 2: 270-360 mg up to day 365 [n = 20]). PALOMINO (phase 2a, open-label trial) used the same dosing protocol as PADDOCK cohort 2 (n = 4). Primary endpoints in both trials were mean change from baseline in hemoglobin, lactate dehydrogenase, haptoglobin, and the number and severity of treatment-emergent adverse events. Mean baseline hemoglobin levels were below the lower limit of normal in both trials (PADDOCK: 8.38 g/dL; PALOMINO: 7.73 g/dL; normal range: 11.90-18.00 g/dL), increased to within normal range by day 85, and were sustained through day 365 (PADDOCK: 12.14 g/dL; PALOMINO: 13.00 g/dL). In PADDOCK, 3 serious adverse events (SAE) led to study drug discontinuation, 1 of which was deemed likely related to pegcetacoplan and 1 SAE, not deemed related to study drug, led to death. No SAE led to discontinuation/death in PALOMINO. Pegcetacoplan was generally well tolerated and improved hematological parameters by controlling hemolysis, while also improving other clinical PNH indicators in both trials. These trials were registered at www.clinicaltrials.gov (NCT02588833 and NCT03593200).

MYH9 disorder: Identification and a novel mutation in patients with macrothrombocytopenia.

The diagnosis of MYH9 disorder is guided by recognizing granulocyte Döhle body-like inclusion bodies and large/giant platelets in the peripheral blood smear. Immunofluorescence study of nonmuscle myosin heavy chain IIA is a sensitive screening method for diagnosis of MYH9 disorder. The diagnosis can then be confirmed by genetic analysis. A total of 67 patients with macrothrombocytopenia were included, of which 11 patients (16%), aged 4 months to 22 years, were ultimately diagnosed with MYH9 disorder. One novel mutation in exon 30 at c.4338T>C (p.F1446A) was detected. This mutation was associated with nonhematologic manifestations presenting in late adolescence with cataracts, hearing loss, and hematuria.

Incidence of thromboembolism in patients with COVID-19: a systematic review and meta-analysis.

BACKGROUND: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, the incidence of thromboembolism has been increasingly reported. The aim of this systematic review was to explore the incidence of venous and arterial thromboembolism among COVID-19 patients requiring hospitalization. METHODS: Medline, Embase, Scopus, and grey literature were searched until June 2020. Observational studies reported on the incidence of venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT) or arterial thromboembolism (ATE) were included. The pool incidences and their 95% confidence intervals (CI) were calculated using the random-effects model. RESULTS: A total of 36 studies were included. In the intensive care unit (ICU) setting, the pooled incidence of VTE was 28% (95% CI, 22-34%). Subgroups based on compression ultrasound (CUS) screening revealed a higher incidence of DVT in the CUS screening group than in the no CUS screening group (32% [95% CI, 18-45%] vs. 6% [95% CI, 4-9%]). The pooled incidence of ATE in ICU was 3% (95% CI, 2-5%). In the non-ICU setting, the pooled incidence of VTE was 10% (95% CI, 6-14%,). CONCLUSIONS: The incidence of VTE in COVID-19 patients was higher in the ICU setting than in the non-ICU setting, and also significantly higher in studies that incorporated the CUS screening protocol. The incidence of ATE in the ICU setting was low. VTE prophylactic measures should be given to all hospitalized patients diagnosed with COVID-19.

Event free survival at 24 months is a strong surrogate prognostic endpoint of peripheral T cell lymphoma.

Event free survival at 24 months (EFS24) has been described as a powerful predictor for outcome in several subtypes of B cell lymphoma. However, it was limitedly described in T cell lymphoma. We explored the implication of EFS24 as a predictor marker for peripheral T cell lymphoma (PTCL). We reviewed 293 systemic PTCL patients at 13 nationwide major university hospitals in Thailand from 2007 to 2014. The median event free survival (EFS) and overall survival (OS) of PTCL patients in our cohort was 16.3 and 27.7 months with corresponding 2-year EFS and 2-year OS of 45.8% and 51.9%, respectively. A total of 118 patients achieved EFS24 (no events during the first 24 mo). Patients who achieved EFS24 had better OS than patients who did not (2-y OS 92% vs 18.8%; HR, 0.1; P < .001). The standardized mortality ratio of patients achieving EFS24 was 18.7 (95% CI, 14.6-22.8). Multivariable analysis demonstrated performance status, histologic subtype, remission status, and EFS24 achievement as independent predictors for OS. Our study affirmed the value of EFS24 as a powerful prognostic factor for PTCL. Further validation in prospective study setting is warranted.

Non-Hodgkin lymphoma in South East Asia: An analysis of the histopathology, clinical features, and survival from Thailand.

Systemic reports on the descriptive epidemiology of non-Hodgkin lymphoma (NHL) from Southeast Asia are scarce. A nationwide multi-institutional registry was conducted to compare the histopathology, clinical features, and survival of Thai adult patients with NHL using large registries, especially those from Far East Asia (FEA). Using a web-based registry system, 13 major medical centers from the 4 geographic regions of Thailand prospectively collected, from 2007 to 2014, the diagnostic pathology, according to the World Health Organization classification, 2008, clinical features and survival of 4056 patients who were newly diagnosed with NHL. The median age of the patients was 56 years (range, 16-99 years). The male-to-female ratio was 1.3:1. From the total of 4056 patients, T/NK-cell lymphoma (TNKCL) accounted for 12.6% of cases, and 5.1% had human immunodeficiency virus-associated lymphoma. The four leading histological subtypes were diffuse large B-cell lymphoma, not otherwise specified (58.1%); follicular lymphoma (5.6%); extranodal mucosa-associated lymphoid tissue lymphoma (5.2%); and peripheral T-cell lymphoma, not otherwise specified (4.0%). With a median follow-up duration of 46.1 months, the median overall survival of B-cell NHL was significantly longer than that of patients with TNKCL (76.5 vs 28.8 months, P = .0001). Compared to FEA, the Thai registry had an approximately one-half lower relative frequency of TNKCL; the prevalence of extranodal mucosa-associated lymphoid tissue lymphoma was much lower than in Korea, and the frequency of extranodal TNKCL, nasal type, was strikingly low compared to China. It is concluded that while the median age of Thai patients with NHL was approximately a decade younger than for Caucasians, the long-term survival rates for most histological subtypes were comparable. While the histological distribution generally complied with the characteristic Asian features, some differences from FEA were observed.

Atypical central retinal artery occlusion as the first presentation of POEMS syndrome: a case report.

BACKGROUND: POEMS syndrome is a plasma cell disorder, which clinically manifests from paraneoplastic syndrome: polyneuropathy, organomegaly, endocrinopathy, monoclonal plasma cell disorder, and skin changes. The most common ocular manifestation is optic disc swelling, whereas other ocular manifestations; cystoid macular edema, serous macular detachment, venous sinus thrombosis, infiltrative orbitopathy, uveitis, neovascularization of the disc, peripapillary choroidal neovascularization and optic disc drusen, had also been reported. CASE PRESENTATION: A 52-year-old Thai man presented with 5-day sudden painless visual loss in the left eye. Ocular examination revealed visual acuity of 20/20 and no light perception in the right and left eye, respectively. Right fundoscopic examination was significant for hyperemic generalized optic disc swelling. Left fundoscopic examination revealed opaque and edematous entire retina giving the appearance of central retinal artery occlusion (CRAO) along with pallid "chalky white" optic disc swelling. Fluorescein angiography showed profound leakage of bilateral optic nerve heads and arteriolar filling defect in macular area along with leakage of small retinal arterioles in the left eye. Indocyanine green angiography demonstrated choroidal filling defect in the left eye only. Neuroimaging showed enhancement and luminal narrowing of left internal carotid artery, early subacute watershed infarctions in the left cerebral hemisphere and pachymeningeal enhancement. Cerebrospinal fluid analysis revealed high protein level with normal opening pressure. Intravenous methylprednisolone was initially started without any benefit. After extensive investigations, diagnosis of "POEMS syndrome" was made based on polyneuropathy, elevated lambda light chain level, elevated plasma vascular endothelial growth factor (VEGF), hepatomegaly, spinal sclerotic bone lesions, and thrombocytosis. Furthermore, sural nerve biopsy demonstrated neuropathy and positive VEGF staining. He was treated with eight cycles of bortezomib, cyclophosphamide and dexamethasone (BorCyDex). Polyneuropathy and thrombocytosis had remarkably improved after 2nd cycle, whereas, visual impairment had shown no recovery. Hepatomegaly was significantly reduced after the completion of BorCyDex. Our case eventually received autologous hematopoietic stem cell transplantation with high dose melphalan. CONCLUSIONS: To our knowledge, we illustrated the first patient given CRAO as the first presentation and ocular finding ever reported in POEMS syndrome. Both cerebral and ocular infarctions were presumably the result of VEGF-induced cranial vasculopathy as evidenced by neuroimaging.

Secondary central nervous system relapse in diffuse large B cell lymphoma in a resource limited country: result from the Thailand nationwide multi-institutional registry.

Secondary central nervous system (CNS) relapse is a serious and fatal complication of diffuse large B cell lymphoma (DLBCL). Data on secondary CNS (SCNS) relapse were mostly obtained from western countries with limited data from developing countries. We analyzed the data of 2034 newly diagnosed DLBCL patients enrolled into the multi-center registry under Thai Lymphoma Study Group from setting. The incidence, September 2006 to December 2013 to represent outcome from a resource limited pattern, management, and outcome of SCNS relapse were described. The 2-year cumulative incidence (CI) of SCNS relapse was 2.7 %. A total of 729, 1024, and 281 patients were classified as low-, intermediate-, and high-risk CNS international prognostic index (CNS-IPI) with corresponding 2-year CI of SCNS relapse of 1.5, 3.1, and 4.6 %, respectively (p < 0.001). Univariate analysis demonstrated advance stage disease, poor performance status, elevated lactate dehydrogenase, presence of B symptoms, more than one extranodal organ involvement, high IPI, and high CNS-IPI group as predictive factors for SCNS relapse. Rituximab exposure and intrathecal chemoprophylaxis offered no protective effect against SCNS relapse. At the time of analysis, six patients were alive. Median OS in SCNS relapsed patients was significantly shorter than relapsed patients without CNS involvement (13.2 vs 22.6 months) (p < 0.001). Primary causes of death were progressive disease (n = 35, 63.6 %) and infection (n = 9, 16.7 %). In conclusion, although the incidence of SCNS relapse in our cohort was low, the prognosis was dismal. Prophylaxis for SCNS involvement was underused even in high-risk patients. Novel approaches for SCNS relapse prophylaxis and managements are warranted.

FIRST IDENTIFICATION OF HEMOGLOBIN LANSINGRAMATHIBODI [α87(F8)His → Gln; CAC>CAG (HBA1: c.264C>G)] IN A THAI FAMILY WITH SPURIOUS HYPOXEMIA.

We report, for the first time, hemoglobin (Hb) Lansing-Ramathibodi [α87(F8)His → Gln; CAC>CAG (HBA1: c.264C>G)] in four members of a Thai family presented with low measured oxygen saturation by pulse oximetry (SpO2), with discrepancy between low SpO2 and normal calculated oxygen saturation by arterial blood gas analysis, and no cyanosis or methemoglobinemia. The causative mutation is located in HBA1 whereas in previous reports of Hb Lansing the mutation is on HBA2, including that in a Japanese individual. The index and a male sibling also co-inherited Hb Pakse, a non-deletional α-thalassemia 2, resulting in mild reticulocytosis. Correct Hb identification is crucial for genetic counselling and, thereby, avoiding unnecessary investigation and treatment for spurious hypoxemia.

The use of hematocrit level for predicting the efficiency of peripheral blood CD34(+) cell collection after G-CSF Mobilization in Healthy Donors.

Recently, peripheral blood stem cell (PBSC) has been widely used and replaced bone marrow (BM) as the stem cell source in allogeneic hematopoietic stem cell transplantation (HSCT) because of a more rapid engraftment, easier accessibility, and lower risk of donor complications. We, therefore, report the predicting factors for the high PBSC harvest yields in 50 healthy donors. Among the 50 donors, median collected CD34(+) cell number was 4.6 × 10(6/) kg (1.5-16.3 × 10(6) /kg). Number of circulating CD34+ cells and hematocrit (HCT) level increased parallelly whereas peripheral CD34+ cell numbers were decreased with increasing donor age. In univariate analysis, HCT level≥ 35.5% at the time of PBSC collection was significantly associated with high PBSC number (≥ 5.0 × 10(6) cells/kg) and donor aged <30 years was significantly associated with collected CD34+ cells ≥ 6.0 × 10(6) /kg, P = 0.03. HCT level ≥35.5% was an independent parameter for high WBC count (≥50 × 10(9) /L), P < 0.05. None of donor who had both HCT < 35.5% and WBC < 50 × 10(9) /L had circulating CD34+ cells ≥ 5.0 × 10(6) /kg. Platelet count ≥ 200 × 10(9) /L was found significantly in donors with WBC ≥ 40 × 10(9) /L (P = 0.03) and HCT ≥ 35.5%, P < 0.05. Collected PBSC number tended to be higher in our donors with high levels of HCT, WBC, and platelet. We also found that HCT and platelet levels in our donors decreased after receiving G-CSF administration compared with the initial complete blood counts (CBC) results. We, therefore, concluded that HCT level at the time of initiation leukapheresis was an important predictor for PBSC collection yields.

Adding MYC/BCL2 double expression to NCCN-IPI may not improve prognostic value to an acceptable level.

BACKGROUND: MYC/BCL2 double expression (DE) is associated with poor prognosis in patients with diffuse large B-cell lymphoma (DLBCL) receiving rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP). This study aimed to determine whether the addition of DE to the National Comprehensive Cancer Network Internal Prognostic Index (NCCN-IPI) could improve the prediction of disease progression in patients with DLBCL treated with R-CHOP. METHODS: This confirmatory prognostic factor study retrospectively recruited patients with newly diagnosed DLBCL between January 1, 2014, and January 31, 2018, at Ramathibodi Hospital (RA) and Thammasat University Hospital (TU). The follow-up period ended on July 1, 2022. Tumors expressing MYC ≥ 40% and BCL2 ≥ 50% were classified as DE. We calculated the hazard ratios (HR) for progression-free survival (PFS) from the date of diagnosis to refractory disease, relapse, or death. Discrimination of the 5-year prediction was based on Cox models using Harrell's concordance index (c-index). RESULTS: A total of 111 patients had DE (39%), NCCN-IPI (8%), and disease progression (46%). The NCCN-IPI adjusted HR of DE was 1.6 (95% confidence interval [CI]: 0.9-2.8; P = 0.117). The baseline NCCN-IPI c-index was 0.63. Adding DE to the NCCN-IPI slightly increased Harrell's concordance index (c-index) to 0.66 (P = 0.119). CONCLUSIONS: Adding DE to the NCCN-IPI may not improve the prognostic value to an acceptable level in resource-limited settings. Multiple independent confirmatory studies from a large cohort of lymphoma registries have provided additional evidence for the clinical utility of DE.

Prevalence and clinical outcomes of germline variants among patients with myeloid neoplasms.

AIMS: Myeloid neoplasms (MNs) with germline predisposition have been recognised as a distinct entity. Emerging evidence suggests that sporadic myelodysplastic syndromes may also harbour undetected germline predispositions. We investigated germline alterations in a cohort of 122 adult Thai MNs. METHODS: MN patients were recruited and tested for germline variants using deep targeted next-generation sequencing. The germline variant was filtered using American College of Medical Genetics classifications and then evaluated for the association with clinical characteristics and outcomes. RESULTS: Our findings revealed pathogenic/likely pathogenic germline alterations in 12 (10%) of the patients. These germline lesions were commonly found in the DNA damage response pathway (n=6, 50%). We also identified novel deleterious FANCA A1219GfsTer59 variants in two patients diagnosed with secondary acute myeloid leukaemia (sAML) from aplastic anaemia and AML with myelodysplasia related. Among sAML, individuals with germline mutations had inferior overall survival compared with those with wild-type alleles (2 months vs 12 months) with HR 4.7 (95% CI 1.0 to 20), p=0.037. Therefore, the presence of pathogenic or likely pathogenic mutations may be linked to inferior survival outcomes. CONCLUSIONS: Our study highlighted that the prevalence of germline predisposition in Southeast Asian populations is comparable to that in Caucasians. This underscores the importance of germline genetic testing within the Asian population.

Committed change of real-time quantitative PCR to droplet digital PCR for monitoring BCR::ABL1 transcripts in tyrosine kinase inhibitor treated CML.

OBJECTIVES: We performed a feasibility study of an FDA-approved commercial ddPCR assay to measure BCR::ABL1 in CML patients treated using TKI therapy. METHODS: Assay performance of standard RQ-PCR and commercially available FDA-approved ddPCR were compared to measure BCR::ABL1 p210 transcripts in RNA samples from 100 CML patients who received TKI therapy. RESULTS: %BCR::ABL1/ABL1IS levels obtained from both methods were not statistically significant difference after normalization with batch-specific conversion factor (p = 0.0651). The correlation and agreement of %BCR::ABL1/ABL1IS between the two assays were high. Molecular response stratification data showed 56% concordance between RQ-PCR and ddPCR, and 37% higher residual disease detection using ddPCR. Furthermore, 21.21% (7/33) of RQ-PCR undetectable samples were detected by ddPCR, representing high sensitivity to quantify the low abundance of BCR::ABL1 transcripts. CONCLUSION: ddPCR was proven to be a highly sensitive method with the potential to overcome some limitations of traditional RQ-PCR, and has the potential of being a valuable tool for monitoring BCR::ABL1 transcripts in CML during TKI therapy. (163 words).

A customized mass array panel for BCR::ABL1 tyrosine kinase domain mutation screening in chronic myeloid leukemia.

Introduction: The therapeutic strategy and management of chronic myeloid leukemia (CML) have rapidly improved with the discovery of effective tyrosine kinase inhibitors (TKIs) to target BCR::ABL1 oncoprotein. However, nearly 30% of patients develop TKI resistance due to acquired mutations on the tyrosine kinase domain (TKD) of BCR::ABL1. Methods: We customized a mass array panel initially intended to detect and monitor the mutational burden of hotspot BCR::ABL1 TKD mutations accumulated in our database, including key mutations recently recommended by European LeukemiaNet. Additionally, we extended the feasibility of using the assay panel for the molecular classification of myeloproliferative neoplasms (MPNs) by incorporating primer sets specific for analyzing JAK2 V617F, MPL 515 K/L, and CALR types 1 and 2. Results: We found that the developed mass array panel was superior for detecting and monitoring clinically significant BCR::ABL1 TKD mutations, especially in cases with low mutational burden and harboring compound/polyclonal mutations, compared with direct sequencing. Moreover, our customized mass array panel detected common genetic alterations in MPNs, and the findings were consistent with those of other comparable assays available in our laboratory. Conclusions: Our customized mass array panel was practicably used as a routine robust assay for screening and monitoring BCR::ABL1 TKD mutations in patients with CML undergoing TKI treatment and feasible for analyzing common genetic mutations in MPNs.

Does leukocytosis remain a predictive factor for survival outcomes in patients with acute promyelocytic leukemia receiving ATRA plus a chemotherapy-based regimen? A prospective multicenter analysis from TALWG.

INTRODUCTION: Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) with a unique clinical presentation and prognosis. This study aimed to investigate the epidemiology, clinical characteristics, treatments, and clinical outcomes of Thai APL patients dominantly treated with all-trans-retinoic acid (ATRA) combined with a chemotherapy-based therapy. METHODS: This was an eight-year prospective, observational study from nine academic hospitals in the Thai Acute Leukemia Working Group (TALWG) of the Thai Society of Hematology, which included newly diagnosed Thai APL patients, aged 18 years or older. The web-based registration collected baseline charateristic, and clinical outcomes. RESULTS: From 992 newly diagnosed AML patients, 79 APL patients were enrolled in this study. Almost all subjects were de novo APL (94.9%), while the others were therapy-related APL. The commonest clinical presentation was disseminated intravascular coagulation (38%). One-third of the patients were categorized as high risk according to the initial WBC. Almost all patients received ATRA combined with idarubicin regimen. The complete response rate was as high as 95.7%, which translated into excellent four-year overall survival (OS) (75.6%) and four-year leukemia-free survival (LFS) (75.4%). The multivariate analysis demonstrated that the older age and WBC count >20 × 109/L conferred a significantly unfavorable OS with the hazard ratios of 3.03 (95% confidence interval [CI]: 1.14-8.05) and 4.18 (95%CI: 1.69-10.35), respectively. Similarly, these two parameters remained independent of the poor prognosis factors for LFS. CONCLUSION: This report confirmed that APL had a favorable prognosis. However, advanced age and high WBC count >20 × 109/L contributed to a worse outcome. ABBREVIATIONS: APL; acute promyelocytic leukemia; ATRA; all-transretinoic acid; CR; complete remission; DS; differentiation syndrome; ECOG; Eastern Cooperative Oncology Group; ED; early death; HR; hazard ratio; IQR; interquartile range; LFS; leukemia-free survival; OS; overall survival; WBC; white blood cell.

Comparison of Molecular International Prognostic Scoring System (M-IPSS) and Revised International Prognostic Scoring System (R-IPSS) in Thai patients with myelodysplastic neoplasms.

INTRODUCTION: Risk stratification is essential for treatment decision in myelodysplastic neoplasms (MDS). Molecular international prognostic scoring system (M-IPSS) has been recently developed combining somatic mutations and clinical information being used in the revised international prognostic scoring system (R-IPSS). OBJECTIVE: We aimed to explore the performances of M-IPSS and R-IPSS in Thai patients with MDS. METHOD: MDS patients were stratified into risk categories using R-IPSS and M-IPSS scores. The performance of both models were evaluated for prognostic prediction. RESULTS: One hundred and sixty-two MDS patients were recruited from the multicenter study. Survival analysis revealed that both R-IPSS and M-IPSS were good prediction models with the Concordance Index (C-index) of 0.71 (95% Confidence interval [CI] 0.64-0.78) and 0.75 (95% CI 0.69-0.80), respectively (p = 0.22). Comparing the two, 13 of 162 (8%) cases were re-staged between lower and higher risks which would have affected treatment decisions. CONCLUSION: Our study showed that R-IPSS score can be used for risk stratification in most Thai patients. A prediction model using somatic mutations specifically in Asian patients should be formulated in the future.