Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca 2+ -dependent gap junction intercellular communication / 中华医学杂志(英文版)

BACKGROUND@#Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown.@*METHODS@#Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca 2+ -related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance.@*RESULTS@#Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca 2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments.@*CONCLUSIONS@#Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.

Myeloid sarcoma: an uncommon presentation of myeloid neoplasms; a case series of 4 rare cases reported in a tertiary care institute

Myeloid sarcoma (MS) is a rare extramedullary neoplasm of myeloid cells, which can arise before, concurrently with, or following hematolymphoid malignancies. We report 04 such cases of MS, diagnosed in this institute over a period of 6 years, during various phases of their respective myeloid neoplasms/leukemias. These cases include MS occurring as a relapse of AML (Case 1), MS occurring as an initial presentation of CML (Case 2), MS occurring during ongoing chemotherapy in APML (Case 3), and MS presenting as a progression of MDS to AML (Case 4). In the absence of relevant clinical history and unemployment of appropriate immunohistochemical (IHC) studies, these cases have a high risk of being frequently misdiagnosed either as Non-Hodgkin's Lymphoma (NHL) or small round cell tumors or undifferentiated carcinomas, which may further delay their management, making an already bad prognosis worse. This case series has been designed to throw light on the varied presentation of MS and the lineage differentiation of its neoplastic cells through the application of relevant IHC markers along with their clinical correlation.

Fatal toxoplasmosis in an immunosuppressed domestic cat from Brazil caused by Toxoplasma gondii clonal type I / Toxoplasmose fatal em um gato doméstico imunossuprimido do Brasil causada por Toxoplama gondii clonal tipo I

Abstract The objective of the study was to report on a fatal case of feline toxoplasmosis with coinfection with the feline leukemia virus (FeLV). A domestic cat (Felis silvestris catus) presented intense dyspnea and died three days later. In the necropsy, the lungs were firm, without collapse and with many white areas; moderate lymphadenomegaly and splenomegaly were also observed. The histopathological examination showed severe necrotic interstitial bronchopneumonia and mild necrotic hepatitis, associated with intralesional cysts and tachyzoites of Toxoplasma gondii that were positive by anti-T. gondii immunohistochemical (IHC) evaluation. The bone marrow showed chronic myeloid leukemia and the neoplastic cells were positive by anti-FeLV IHC evaluation. DNA extracted from lungs was positive for T. gondii by PCR targeting REP-529. T. gondii was characterized by PCR-RFLP and by the microsatellites technique. ToxoDB-PCR-RFLP #10, i.e. the archetypal type I, was identified. Microsatellite analysis showed that the strain was a variant of type I with two atypical alleles. This was the first time that a T. gondii clonal type I genotype was correlated with a case of acute toxoplasmosis in a host in Brazil.

Resumo O objetivo deste estudo foi relatar um caso de toxoplasmose felina fatal com coinfecção com o vírus da leucemia felina (FeLV). Um gato doméstico (Felis silvestris catus) apresentou intensa dispneia e morreu três dias depois. Na necropsia, observaram-se pulmões firmes, não colabados e com múltiplas áreas brancas, além de linfoadenomegalia e esplenomegalia moderadas. No exame histopatológico, evidenciaram-se broncopneumonia intersticial necrótica acentuada e hepatite necrótica discreta associada a cistos e taquizoítas de T. gondii intralesionais positivos na imuno-histoquímica (IHC) anti-T. gondii. Evidenciou-se ainda, na medula óssea, leucemia mieloide crônica com IHC anti-FeLV positiva nas células neoplásicas. O DNA extraído dos pulmões foi positivo para T. gondii por meio da PCR-REP-529. T. gondii foi caracterizado por PCR-RFLP e pela técnica de microssatélites. Foi identificado o genótipo ToxoDB-PCR-RFLP #10, i.e., o arquétipo tipo I. A análise por microssatélites mostrou que a cepa era uma variante do tipo I, com dois alelos atípicos. Esta é a primeira vez que T. gondii clonal tipo I foi relacionado com um caso agudo de toxoplasmosis em um hospedeiro no Brasil.

Cutaneous myeloid sarcoma associated with chronic myeloid leukemia

Abstract: Myeloid sarcoma is an extramedullary tumor of malignant myeloid cells often associated with acute myeloid leukemia, chronic myeloproliferative disorders and myelodysplastic syndromes. The skin is one of the most commonly affected sites. We report a rare case of cutaneous myeloid sarcoma associated with chronic myeloid leukemia.

Circulating endothelial cells are increased in chronic myeloid leukemia blast crisis

We measured circulating endothelial precursor cells (EPCs), activated circulating endothelial cells (aCECs), and mature circulating endothelial cells (mCECs) using four-color multiparametric flow cytometry in the peripheral blood of 84 chronic myeloid leukemia (CML) patients and 65 healthy controls; and vascular endothelial growth factor (VEGF) by quantitative real-time PCR in 50 CML patients and 32 healthy controls. Because of an increase in mCECs, the median percentage of CECs in CML blast crisis (0.0146%) was significantly higher than in healthy subjects (0.0059%, P<0.01) and in the accelerated phase (0.0059%, P=0.01). There were no significant differences in the percentages of CECs in chronic- or active-phase patients and healthy subjects (P>0.05). In addition, VEGF gene expression was significantly higher in all phases of CML: 0.245 in blast crisis, 0.320 in the active phase, and 0.330 in chronic phase patients than it was in healthy subjects (0.145). In conclusion, CML in blast crisis had increased levels of CECs and VEGF gene expression, which may serve as markers of disease progression and may become targets for the management of CML.

Imatinib mesylate as first‑line therapy in patients with chronic myeloid leukemia in accelerated phase and blast phase: A retrospective analysis.

INTRODUCTION: Imatinib is a bcr‑abl tyrosine kinase inhibitor which has revolutionized the treatment for chronic myeloid leukemia (CML). Even though there is much data on CML chronic phase, there is limited data on imatinib‑naïve advanced phase CML. MATERIALS AND METHODS: We retrospectively analysed 90 patients with advanced phase CML (accelerated phase [AP]: 51 and blast crisis [BC]: 39), patients who received imatinib as frontline therapy. RESULTS: The median age of presentation in CML‑AP and CML‑BC were 32 years (12‑61) and 39 years (8‑59), respectively. Imatinib at 600 mg/day was initiated within 2 weeks of diagnosis. Median time to complete hematological response in both CML‑AP and CML‑BC was 3 months (CML‑AP: 1‑9 months and CML‑BC: 1‑14 months). At 6 months 30 (59%) CML‑AP and 15 (38%) CML‑BC patients achieved major cytogenetic response (MCyR), of them 24 (47%) and 10 (25.6%) being the complete cytogenetic response, respectively. At a median follow‑up of 41 months, the median overall survival in CML‑AP was 61 months, but in CML‑BC it was 14 months. The median progression‑free survival and event‑free survival were 30 months and 23 months in CML‑AP and 14 and 12 months in CML‑BC, respectively. On univariate analysis, performance status (PS), spleen size, and MCyR predicted survival in AP, whereas in BC, platelet count, PS, and early MCyR were predictive. Non‑hematologic and hematologic adverse events were observed in 80% and 60% of patients, respectively. Dose was reduced in 10% of patients for grade IV toxicity and interrupted in 30% for grade III toxicity. CONCLUSION: Front‑line imatinib is an option in advanced phases of CML especially in CML‑AP in low‑resource countries, where stem cell transplantation and alternate TKIs are not available.

Factors Related to Decreased Bone Mineral Density in Childhood Cancer Survivors

The risk of osteoporosis or osteopenia is known to increase after childhood cancer treatment. The purpose of this study was to evaluate patterns of bone mineral density (BMD) and to identify factors related to the decreased BMD in childhood cancer survivors. We studied 78 patients (34 boys, 44 girls) treated for childhood cancer. Twenty (25.7%) patients had lumbar BMD (LBMD) standard deviation score (SDS) lower than -2. Nineteen (24.4%) patients had femur neck BMD (FNBMD) SDS lower than -2. The patients treated with hematopoietic stem cell transplantation had lower LBMD SDS (-1.17 +/- 1.39 vs -0.43 +/- 1.33, P = 0.025). The risk of having LBMD SDS < -2 was higher in the patients treated with glucocorticoid (GC) for graft-versus-host disease (GVHD) (36.6% vs 13.5%; odds ratio [OR], 3.7; P = 0.020). In multivariate logistic regression analysis, longer duration of GC treatment for GVHD (OR, 1.12; 95% confidence interval [CI], 1.05-1.20) and lower body mass index (BMI) SDS (OR, 0.59; 95% CI, 0.36-0.95) were associated with decreased LBMD SDS. These findings suggest that prolonged GC use and reduction in BMI are risk factors for decreased BMD in childhood cancer survivors. Anticipatory follow-up and appropriate treatment are necessary, especially for the patients with risk factors.

Philadelphia chromosome detection in chronic myeloid leukemia: Utility of phytohemagglutinin-stimulated peripheral blood culture.

Background: The conventional cytogenetic approach to demonstrate Philadelphia (Ph) chromosome at times does not yield enough number of metaphases or are of suboptimal quality. Further, the rapid molecular tests have completely pushed this simple technique into disrepute. Aims: This study aimed to evaluate usefulness of phytohemagglutinin (PHA)-stimulated peripheral blood culture for detection of Ph chromosome in chronic myeloid leukemia (CML) patients. Materials and Methods: Fifty-six patients, including 11 newly diagnosed cases of CML and 45 patients of CML on imatinib therapy showing the presence of Ph chromosome in unstimulated samples, were included in the study. Cytogenetic analysis was done on unstimulated samples, i.e. bone marrow aspirate, 24- and 48-h peripheral blood culture, and compared with PHA-stimulated 72-h peripheral blood culture. Results: The preparations from PHA-stimulated peripheral blood culture samples in all 56 patients yielded high number of good-quality metaphases. All the 11 (100%) newly diagnosed patients and 39/45 (87%) of the patients on imatinib therapy showed the presence of Ph chromosome in PHA-stimulated samples. Addition of PHA-stimulated 72-h peripheral blood culture preparation can be of use for increasing the diagnostic yield in cases of CML with suboptimal results on conventional cytogenetics from bone marrow aspirate sample.

Optimizing second-line therapy for chronic myeloid leukemia.

Treatment of chronic myeloid leukemia has evolved from symptom control to long-term disease-free survival with cure potentially round the corner. This required faster, deeper, and longer response. Optimizing treatment decisions therefore requires clear understanding of and strict implementation of guidelines for shift from imatinib. In patients who are resistant to or intolerant of imatinib, second-line TKIs have to be selected carefully. Currently available data show comparable efficacy between nilotinib and dasatinib. With a better safety profile (especially with respect to grade 3 or 4 hematologic toxicity and clinically relevant non-hematologic toxicities), nilotinib becomes the preferred choice in most instances.

Cytogenetic & molecular analyses in adult chronic myelogenous leukaemia patients in north India.

Background & objectives: Chronic myelogenous leukaemia (CML) is the commonest leukaemia in Asia. There is a paucity data on cytogenetic and molecular analyses of Indian CML patients. This apparently reflects the low availability of cytogenetic and molecular techniques in our country. This study aimed to document various types of BCR-ABL fusion transcripts in different phases of CML and to compare the Ph chromosome positivity/negativity vis-a-vis BCR-ABL fusion transcripts in adult CML patients. Methods: Between June 2004 and February 2009, 208 patients were diagnosed as CML in chronic phase (CP), accelerated phase (AP) and blast crisis (BC), according to standard criteria. Cytogenetic and molecular genetic analyses were performed in all patients. Various types of BCR-ABL hybrid transcripts were compared with phases of CML and cytogenetic abnormalities. Results: Among 208 CML patients, b3a2 BCR-ABL transcripts were most commonly detected (66.82%) followed by b2a2 (28.84%), b3a2 + b2a2 (3.36%), b3a2 + e19a2 (0.48%) and b2a2 + e19a2 (0.48%). b3a2 transcripts were more frequently detected than b2a2 transcripts, in the whole group of 208 as well as in 183 CML-CP patients (P<0.0001). Ph chromosome was positive in 135 of 139 patients with b3a2 transcripts and 56 of 60 patients with b2a2 transcripts, difference not being significant. Additional cytogenetic abnormalities detected in 3.8 per cent patients in CML-CP and 44 per cent patients in CMLAP/ BC, did not show predilection for any BCR-ABL transcript type. Interpretation & conclusions: This study documents higher Ph positivity (96.15%) by cytogenetic analysis among CML patients, as confirmed by qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in a large patient group from north India. Both the techniques contribute towards understanding the disease biology, and have important implications for diagnosis and management of CML patients.

Nilotinib as first-line therapy for chronic myeloid leukemia.

Tyrosine Kinase Inhibitors brought a revolution in the management of chronic myeloid leukemia. Long term disease free survival became a reality for the majority of patients. With the identification of imatinib resistance and its implications, roles of newer targeted therapy molecules came into focus. Nilotinib data has matured and shows the fulfillment of earlier promise - even in first line therapy. This review provides insight into the place of this molecule in the first line management of chronic myeloid leukemia.

Chronic myelogenous leukemia in sickle cell/beta0-thalassemia.

Sickle cell/beta 0 -thalassemia (S/β0 -thal) is a compound heterozygous state for βS and β0 thalassemia. There are rare reported cases of patients with sickle cell disease who developed hematological neoplasms including myeloid and lymphoid conditions; however, to the best of our knowledge, chronic myelogenous leukemia (CML) occurring in S/β0 -thal has been reported in one case and this is the second such report.

Reacción adversa cutánea al Imatinib durante el tratamiento de la leucemia mieloide crónica / Cutaneous adverse reaction to imatinib in the treatment of chronic myeloid leukemia

El imatinib es el tratamiento de elección para pacientes con leucemia mieloide crónica (LMC). Su objetivo es una proteína de fusión BCR-ABL con actividad kinasa y se la considera una droga con buena tolerancia; sin embargo, pueden presentarse efectos adversos que incluyen múltiples manifestaciones a nivel cutáneo, hematológico, gastrointestinal y cardiológico. La toxicidad en piel es un efecto adverso frecuente del tratamiento con imatinib, que se presenta con una frecuencia de hasta el 66% y es dependiente de la dosis administrada. Presentamos una paciente añosa con LMC que recibió tratamiento con imatinib 800 mg/día y desarrolló una erupción maculopapulosa, pruriginosa, asociada a episodio de insuficiencia cardíaca congestiva.

Avaliação laboratorial da doença residual mínima na leucemia mielóide crônica por Real-Time PCR: [revisão] / Evaluation diagnosis of minimal residual disease in chronic myeloid leukemia by Real-Time PCR: [review]

A leucemia mielóide crônica (LMC) representa 15 por cento das leucemias e apresenta três fases: crônica, acelerada e crise blástica. A partir da análise citogenética, pode ser identificado o cromossomo Philadelphia, característico da LMC. O transplante de células-tronco é o único tratamento curativo, mas é acompanhado de altas taxas de morbimortalidade, dificultando sua aplicação. A doença residual mínima é de grande importância para avaliar a resposta ao tratamento, tanto na verificação de doença residual, quanto na identificação de pacientes com alto risco de recaída. Muitas técnicas específicas têm sido introduzidas para detectar as translocações ou os produtos do cromossomo Philadelphia. A mais sensível é a Real-Time PCR, que detecta uma célula leucêmica em 10(5) células normais. O objetivo deste trabalho foi realizar uma revisão bibliográfica sobre a LMC, dando ênfase à utilização da técnica por Real-Time PCR.

Chronic myeloid leukemia (CML) represents about 15 percent of all leukemias and has three phases: the chronic phase, accelerated phase and blast crisis. After cytogenetic analysis, the Philadelphia chromosome, characteristic of CML, can be identificated. Stem cell transplantation is the only curative treatment for CML, but it is accompanied by high levels of morbimortality, difficulting its application. The minimal residual disease is very important for the evaluation of the response to treatment, to verify the residual disease and also to identify patients with a high risk of relapse. Many specific techniques have been introduced for the detection of translocations or products of the Philadelphia chromosome; the most sensitive being Real-Time PCR which detects 1 leukemia cell in 10(5) normal cells. The aim of this study was to perform a bibliographic review of CML, with emphasis on the utilization of the Real-Time PCR technique.

El coactivador de receptores nucleares RAC3 tiene un rol protector de la Apoptosis inducida por distintos estímulos / RAC3 nuclear receptor co-activator has a protective role in the apoptosis induced by different stimuli

RAC3 pertenece a la familia de coactivadores de receptores nucleares p160, y se encuentra sobreexpresado en varios tumores. Demostramos previamente que RAC3 es coactivador del factor de transcripción anti-apoptótico NF-kB. En este trabajo investigamos su rol en la apoptosis inducida por H2O2 en una línea celular no tumoral derivada de riñón embrionario humano (HEK293), y por el ligando inductor de apoptosis relacionado a TNF (TRAIL) en una línea de leucemia mieloide crónica humana (K562), naturalmente resistente a la muerte por este estímulo. Observamos que las células tumorales K562 poseen niveles altos de RAC3 comparados con las células no tumorales HEK293. La sobreexpresión normal de coactivador o por transfección, inhibe la apoptosis mediante una disminución de la activación de caspasas, translocación del factor inductor de apoptosis (AIF) al núcleo, aumento de la actividad de NF-kB y las quinasas AKT y p38 y disminución de la quinasa ERK. Lo opuesto fue observado por disminución de RAC3 mediante la técnica de ARN interferente (RNAi) en K562, aumentando así la apoptosis inducida por TRAIL. Estas evidencias sugieren que una sobreexpresión de RAC3 contribuye al desarrollo de tumores, participando en las cascadas que controlan la muerte celular por mecanismos no estrictamente dependientes de hormonas esteroideas y/o de acetilación, constituyendo esto un posible blanco de ataque para el tratamiento de tumores.

RAC3 belongs to the family of p160 nuclear receptors coactivators and it is over-expressed in several tumors. We have previously shown that RAC3 is a NF-kB coactivator. In this paper, we investigated the role of RAC3 in cell-sensitivity to apoptosis, using H2O2 in the human embryonic kidney cell line (HEK293), and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in a human chronic myeloid leukemia cell line (K562) naturally resistant to TRAIL. We observed that the tumoral K562 cells have high levels of RAC3 if compared with the non-tumoral HEK293 cells. The normal or transfected coactivator over-expression inhibits apoptosis through a diminished caspase activity and AIF nuclear translocation, increased NF-kB, AKT and p38, and decreased ERK activities. In contrast, inhibition of RAC3 by siRNA induced sensitivity of K562 to TRAIL-induced apoptosis. Such results suggest that over-expression of RAC3 contributes to tumor development through molecular mechanisms that do not depend strictly on acetylation and/or steroid hormones, which control cell death. This could be a possible target for future tumor therapies.

Impaired cell motility in chronic myeloid leukemic granulocytes related to altered cytoskeletal pattern.

The bactericidal activity of polymorphonuclear leucocyte (PMNL) against infection stimulates cytoskeletal changes accompanied with alteration in adhesion and locomotion. Microfilaments, the motile apparatus is known to regulate these changes by polymerization of monomeric G-actin to fibrous F-actin. PMNL from chronic myeloid leukemia (CML) patients have been reported to be defective in locomotion in response to synthetic peptide, n-formyl-methionyl-leucyl-phenylalanine (fMLP) but the mechanism leading to defective locomotion and their spatial reorganization remains unclear. Therefore, in order to study the cause of defective motility of PMNL from CML patients the spatial distribution and reorganization of microfilaments and microtubules in response to fMLP have been examined by transmission electron (TEM) and scanning electron microscopy (SEM). Under SEM, the PMNL-CML surface appeared smoother with reduced ruffling resulting in rounding off cells with lesser polarized morphology. Unstimulated PMNL from normal as well as CML subjects showed shorter and fewer microtubules and evenly distributed microfilaments as compared to fMLP stimulated PMNL. It is proposed that the cause of defective locomotion was due to reduced surface activity as a consequence of altered cytoskeletal configuration. This phenomenon seems to be related to impaired functional appendages and as a whole led to the defective cell motility and hence reduced chemotaxis in PMNL from CML patients.

Extramedullary blast crisis in chronic myeloid leukemia--a case report.

Presence of focal poliferation of myeloblasts at an extramedullary site even when peripheral blood/bone marrow blast count is less than 20% in a case of chronic myeloid leukaemia leads to a diagnosis of blast crisis. A case of focal extramedullary blast crisis with chronic myeloid leukaemia is reported here.