Lineage‐dependent skewing of loss of heterozygosity (LOH) of KRAS gene in a case of juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a clonal disease arising from abnormal hematopoietic stem cells, although the involvement of lymphoid lineage differs among reported cases. Here, we present a case of JMML with a KRAS G13D mutation. The mutation was detected in various hematopoietic lineages, including T and B lymphocytes and also in lineage^? CD34⁺CD38^? hematopoietic stem cells, showing a different percentage of affected cells in each lineage. Single cell‐based analysis of hematopoietic cells revealed the loss of wild‐type KRAS in a significant proportion of G13D‐harboring cells. The percentage of loss of heterozygosity (LOH)/non‐LOH cells showed lineage‐dependent skewing in hematopoietic cells. The loss of the wild‐type KRAS allele may be a common secondary genetic change in KRAS‐related JMML and may affect the differentiation behavior of early JMML progenitors.     

Wiskott-Aldrich syndrome with unusual clinical features similar to juvenile myelomonocytic leukemia

A male infant exhibited thrombocytopenia at birth, and later developed leukocytosis, monocytosis, and bloody stool. The bone marrow was hypercellular with dysplasia. Spontaneous granulocyte/macrophage-colony formation and hypersensitivity to granulocyte/macrophage-colony stimulating factor were confirmed by in vitro culture. These findings fulfilled most of the diagnostic criteria for juvenile myelomonocytic leukemia (JMML), with the exception of splenomegaly. However, no mutations in the PTPN11, RAS, and CBL genes, or clinical features of neurofibromatosis type 1, which are associated with JMML, were detected. The patient subsequently developed refractory eczema with undetectable serum IgM, which led to the consideration of Wiskott-Aldrich syndrome (WAS). Lack of WASP expression and a 4-nucleotide deletion mutation in WASP were identified. Approximately 20?% of patients with JMML show none of the abovementioned molecular abnormalities. Careful differential diagnosis, including the consideration of WAS, is, therefore, recommended in patients with clinical features and laboratory findings consistent with JMML.     

Juvenile myelomonocytic leukemia (JMML) with the hematologic phenotype of severe β thalassemia

A 3-year-old Filipino-American child with recurrent fever, splenomegaly, anemia, and thrombocytopenia, was found to have a hemoglobin F level of 76.9%. His reticulocyte count was elevated (4.3%), and erythroblasts were present in his peripheral blood. The child's erythrocytes were microcytic (MCV 66.9 fl) but his serum ferritin level was normal. His bone marrow at initial presentation demonstrated normal cellularity without an increase in blast cells. The disease progressed with worsening anemia, leukocytosis, and thrombocytopenia, with increased blasts in his marrow and the appearance of a mediastinal mass. His liver, spleen, and lymph nodes were found to be infiltrated with myeloblasts, supporting a diagnosis of juvenile myelomonocytic leukemia (JMML). Analysis of the child's Hb F showed a ^Gγ/^Aγ ratio of 2.2, which was within the characteristic range for JMML. A globin synthesis study using blood reticulocytes showed an α/non-α globin synthesis ratio of 2.24, typical of severe homozygous β thalassemia. Southern blot analysis of blood-leukocyte DNA from the patient and his parents demonstrated no apparent abnormality in the β-globin gene promoter or coding regions. The elevated level of Hb F in this child with JMML appeared to be part of an acquired Cooley's anemia-like hematologic phenotype. Am. J. Hematol. 58:67–71, 1998. © 1998 Wiley-Liss, Inc.     

Clinical characteristics of 15 children with juvenile myelomonocytic leukaemia who developed blast crisis: MDS Committee of Japanese Society of Paediatric Haematology/Oncology

Juvenile myelomonocytic leukaemia (JMML) is a rare haematopoietic stem cell disease of early childhood, which can progress to blast crisis in some children. A total of 153 children diagnosed with JMML were reported to the Myelodysplastic Syndrome Committee in Japan between 1989 and 2007; 15 of them (9·8%) had 20% or more blasts in the bone marrow (blast crisis) during the disease course. Blast crisis occurred during observation without therapy (n = 3) or with oral 6‐mercaptopurine treatment (n = 9) and in relapse after haematopoietic stem cell transplantation (HSCT; n = 3). Six patients had a complex karyotype (5 including monosomy 7) and an additional three patients had isolated monosomy 7 at blast crisis. Seven patients received HSCT after blast crisis and four of them achieved remission. Eleven out of the 15 patients died; the cause of death was disease progression in 10 patients and transplant‐related complication in one patient. In summary, patients with blast crisis have poor prognosis and can be cured only by HSCT. The emergence of monosomy 7 and complex karyotype may be characteristic of blast crisis in a substantial subset of children.     

HLA-mismatched haploidentical transplantation using low-dose anti-thymocyte globulin (ATG: thymoglobulin)

Objectives: To clarify optimal strategies for human leukocyte antigen (HLA)-mismatched haploidentical hematopoietic stem cell transplantation (HSCT).

Methods: Twelve patients who underwent HSCT from a haploidentical related donor using low-dose thymoglobulin were analyzed retrospectively. Thymoglobulin was added to conditioning regimens at 2.5?mg/kg/day for 2 days (days ?4 and ?3). Prophylaxis against graft-versus-host disease (GVHD) was performed with cyclosporine and methotrexate.

Results: The median age of the patients was 33 years. Six patients had previous allogeneic HSCT, and HSCT was performed in non-remission for nine patients. All patients but one, who died due to early infection, achieved neutrophil engraftment at a median of 17 days with complete donor-type chimerism. Acute and chronic GVHD were observed in six and five patients, respectively, but no patients died of GVHD-associated complication. No one developed cytomegalovirus disease, but Epstein–Barr virus-related lymphoproliferative disorder was observed in one patient. Long-term survival in remission without immunosuppressive agents are observed in two patients who underwent HSCT in remission, but the majority of patients who underwent HSCT in non-remission experienced disease progression.

Conclusion: Haploidentical HSCT could be performed with thymoglobulin at 5?mg/kg, with the balance between GVHD and relapse rate. The dose reduction of thymoglobulin may be considered for advanced hematological malignancy.     

Correction of Severe Myelofibrosis,Impaired Platelet Functions and Abnormalities in a Patient with Gray Platelet Syndrome Successfully Treated by Stem Cell Transplantation

Abstract

Gray platelet syndrome (GPS) is an inherited disorder. Patients harboring GPS have thrombocytopenia with large platelets lacking α-granules. A long-term complication is myelofibrosis with pancytopenia. Hematopoietic stem cell transplant (HSCT) could be a curative treatment. We report a male GPS patient with severe pancytopenia, splenomegaly and a secondary myelofibrosis needing red blood cells transfusion. He received an HSCT from a 10/10 matched HLA-unrelated donor after a myeloablative conditioning regimen. Transfusion independence occurred at day+21, with a documented neutrophil engraftment. At day+ 180, we added ruxolitinib to cyclosporine and steroids for a moderate chronic graft versus host disease (GVHD) and persistent splenomegaly. At day+240 GVHD was controlled and splenomegaly reduced. Complete donor chimesrism was documented in blood and marrow and platelets functions and morphology normalized. At day+ 720, the spleen size normalized and there was no evidence of marrow fibrosis on the biopsy. In GPS, HSCT may be a curative treatment in selected patients with pancytopenia and myelofibrosis.     

Juvenile myelomonocytic leukemia characterized by cutaneous lesion containing Langerhans cell histiocytosis-like cells

We present a 1-year-old boy who developed a cutaneous lesion on the trunk and hepatosplenomegaly. Laboratory examination showed leukocytosis with peripheral blasts, atypical monocytosis, anemia, hyper IgG, and a mild elevation of C-reactive protein. Clinical features and skin biopsy findings matched the diagnostic criteria of both juvenile myelomonocytic leukemia (JMML) and Langerhans cell histiocytosis (LCH). Histopathology revealed atypical mononuclear cells that had infiltrated around vessels throughout the dermis in a skin biopsy specimen. These cells were CD1a (+), S-100 (+), CD68 (+), CD207 (−), lysozyme (+), and myeloperoxidase (−). The diagnosis of JMML was confirmed by detection of spontaneous colony formation and granulocyte–macrophage colony-stimulating factor hypersensitivity in vitro, and a somatic NRAS point mutation. Transplantation of bone marrow from an HLA-matched unrelated donor was performed, and the marrow was successfully engrafted. The cutaneous lesion and hepatosplenomegaly were improved at the time of discharge. It is often difficult to distinguish between JMML and LCH-like infiltrates by assessing clinical and light microscopic features of various cutaneous lesions. In the current case, molecular biological analysis enabled us to develop a precise diagnosis.     

Genome-wide single-nucleotide polymorphism array-based karyotyping in myelodysplastic syndrome and chronic myelomonocytic leukemia and its impact on treatment outcomes following decitabine treatment

Decitabine is a hypomethylating agent with proven clinical efficacy in myelodysplastic syndrome (MDS). The current study analyzed the role of single nucleotide polymorphism array (SNP-A)-based karyotyping in prediction of clinical outcome in MDS or chronic myelomonocytic leukemia (CMML) patients following decitabine therapy. A total of 61 MDS/CMML patients treated with decitabine were evaluated with Genome-Wide Human SNP 6.0 Array using DNAs derived from marrow samples. The primary endpoint was the best response rate including complete (CR) and partial response (PR) with overall (OS) and event-free survival (EFS) as secondary endpoints. Best response was noted in 14 patients (26.4 %) out of 53 evaluated patients including 12 CR and two PR with median follow-up of 21.6 months. A total of 81 abnormal SNP lesions were found in 25 out of 61 patients (41.0 %). The patients carrying abnormal SNP lesions showed an inferior CR/PR rate (p?=?0.002) and showed a trend of worse OS (p?=?0.02 in univariate, p?=?0.09 in multivariate) compared to those without SNP lesions, but not were associated with inferior EFS. The presence of abnormal SNP lesions in MDS was associated with adverse outcomes following decitabine therapy. Further study is strongly warranted to establish the role of SNP-A karyotyping in MDS.     

RAS diseases in children

RAS genes encode a family of 21 kDa proteins that are an essential hub for a number of survival, proliferation, differentiation and senescence pathways. Signaling of the RAS-GTPases through the RAF-MEK-ERK pathway, the first identified mitogen-associated protein kinase (MAPK) cascade is essential in development. A group of genetic syndromes, named “RASopathies”, had been identified which are caused by heterozygosity for germline mutations in genes that encode protein components of the RAS/MAPK pathway. Several of these clinically overlapping disorders, including Noonan syndrome, Noonan-like CBL syndrome, Costello syndrome, cardio-facio-cutaneous (CFC) syndrome, neurofibromatosis type I, and Legius syndrome, predispose to cancer and abnormal myelopoiesis in infancy. This review focuses on juvenile myelomonocytic leukemia (JMML), a malignancy of early childhood characterized by initiating germline and/or somatic mutations in five genes of the RAS/MAPK pathway: PTPN11, CBL, NF-1, KRAS and NRAS. Natural courses of these five subtypes differ, although hematopoietic stem cell transplantation remains the only curative therapy option for most children with JMML. With whole-exome sequencing studies revealing few secondary lesions it will be crucial to better understand the RAS/MAPK signaling network with its crosstalks and feed-back loops to carefully design early clinical trials with novel pharmacological agents in this still puzzling leukemia.     

Quantitative assessment of PTPN11 or RAS mutations at the neonatal period and during the clinical course in patients with juvenile myelomonocytic leukaemia

To evaluate minimal residual disease (MRD) after chemotherapy and haematopoietic stem cell transplantation in juvenile myelomonocytic leukaemia (JMML), a locked nucleic acid‐allele specific quantitative polymerase chain reaction (LNA‐AS‐qPCR) was developed for 13 patients (four types of PTPN11 mutation and four types of RAS mutation). The post‐transplant MRD detected by LNA‐AS‐qPCR analysis was well correlated with chimerism assessed by short tandem repeat PCR analysis. Non‐intensive chemotherapy exerted no substantial reduction of the tumour burden in three patients. There was no significant difference in the quantity of RAS mutant DNA after spontaneous haematological improvement in 4 patients with NRAS or KRAS 34G > A during a 2‐ to 5‐year follow‐up. PTPN11, NRAS, or KRAS mutant DNA was detected from Guthrie card dried blood in five of seven patients (who were aged <2 years at diagnosis) at a level of 1·0–6·5 × 10^?1 of the values at diagnosis. Accordingly, these five patients might have already reached a subclinical status at birth. Considering the negative correlation between mutant DNA level in neonatal blood spots and age at diagnosis, JMML patients with a larger tumour burden at birth appeared to show earlier onset.     

Venetoclax + hypomethylating agents combined with dose-adjusted HAG for relapsed/refractory acute myeloid leukemia: Two case reports

Rationale:Some acute myeloid leukemia (AML) patients are unresponsive to treatment or have remission followed by worsening of disease (known as relapsed/refractory AML [R/RAML]) after standardized treatment. The CAG/HAG regimen is not often used clinically because heterogenous patient responses, resistance, and hematopoietic bone marrow dysfunction have been reported with its use. We present 2 cases of R/RAML treated with a new combined therapy (venetoclax+ hypomethylating agents [HMAs]) in which the HAG dose was adjusted and effective in the first course of treatment.Patient characteristics:Case 1 involved a 23-year-old man who had suffered from AML for >4 years, and his FLT3 mutation status was positive at the initial diagnosis. After the first course of treatment with the standard-dose “Da” plan, the patient experienced complete remission. During the subsequent courses of treatment, the patient experienced 6 recurrences and was treated with the “ID Ara-C + MIT + sidaaniline” and “CAG + sidaaniline” regimens. However, the disease did not respond. Case 2 involved a 26-year-old man who received chemotherapy with the “Da,” “ID Ara-C,” “decitabine + half-dose CAG,” and “HAE” regimens. In this patients, remission could not be achieved. Reintroduction of the “ia” scheme also failed after treatment in our hospital.Diagnosis:Two patients were diagnosed with R/RAML.Interventions:The patient in case 2 received chemotherapy interventions, whereas the patient in case 1 refused to receive medical services at our hospital.Outcomes:The patient in case 1 was discharged after complete response treatment due to economic reasons and relapsed 2 months later. The patient ultimately died of infection and heart failure. The patient in case 2 is receiving a second cycle of chemotherapy.Lessons:We recommend the “venetoclax + HMAs combined with dose-adjusted CAH/HAG” regimen as an effective treatment for adult R/RAML.     

Plasmatic KRAS Kinetics for the Prediction of Treatment Response and Progression in Patients With KRAS-mutant Lung Adenocarcinoma

IntroductionKRAS is the most common driver mutation in lung cancer. ctDNA-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. Monitoring KRAS mutational load in ctDNA may be useful in the management of the patients.MethodsConsecutive patients diagnosed with KRAS mutant lung adenocarcinoma in the tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. KRAS mutations in plasma were quantified using digital PCR and correlated with mutations in tumor and with radiological response and progression.ResultsTwo hundred and forty-five plasma samples from 56 patients were analyzed. The rate of detection of KRAS mutations in plasma in our previously characterized KRAS-mutant cases was 82% overall, reaching 96% in cases with more than 1 metastatic location. The dynamics of KRAS mutational load predicted response in 93% and progression in 63% of cases, 33 and 50 days respectively in advance of radiological evaluation. Progression-free survival for patients in whom ctDNA was not detectable in plasma after treatment initiation was significantly longer than for those in whom ctDNA remained detectable (7.7 versus 3.2 months; HR: 0.44, p = 0.004).ConclusionsThe detection of KRAS mutations in ctDNA showed a good correlation with that in tumor biopsy and, in most cases, predicted tumor response and progression to chemotherapy in advance of radiographic evaluation. The liquid biopsies for ctDNA-based molecular analyses are a reliable tool for KRAS testing in clinical practice.     

Somatic mosaicism for oncogenic NRAS mutations in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. Somatic mutations in genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and CBL, have been identified in more than 70% of children with JMML. In the present study, we report 2 patients with somatic mosaicism for oncogenic NRAS mutations (G12D and G12S) associated with the development of JMML. The mutated allele frequencies quantified by pyrosequencing were various and ranged from 3%-50% in BM and other somatic cells (ie, buccal smear cells, hair bulbs, or nails). Both patients experienced spontaneous improvement of clinical symptoms and leukocytosis due to JMML without hematopoietic stem cell transplantation. These patients are the first reported to have somatic mosaicism for oncogenic NRAS mutations. The clinical course of these patients suggests that NRAS mosaicism may be associated with a mild disease phenotype in JMML.     

Late-onset hemorrhagic cystitis after haploidentical hematopoietic stem cell transplantation in patients with advanced leukemia: differences in ATG dosage are key

Late-onset hemorrhagic cystitis (LOHC) is a common complication following allogeneic hematopoietic stem cell transplantation (HSCT), but its cause remains obscure. More attention to risk factors for LOHC is needed. We retrospectively analyzed patients with advanced leukemia who had been treated with ATG-containing conditioning regimens to evaluate the influence of different doses of ATG on LOHC after haploidentical HSCT. Seventy-five patients undergoing haploidentical HSCT from January 2003 to February 2011 were evaluated. A total of 39 patients receiving transplantation before June 2008 were treated with high-dose ATG (10 mg/kg), whereas 36 patients received low-dose ATG (6 mg/kg) thereafter. LOHC occurred in 16.7 % of the patients with low-dose ATG, and in 38.5 % of the patients with high-dose ATG (P = 0.027). Univariate analysis showed high-dose ATG, male gender and cytomegalovirus reactivation to be significant risk factors for LOHC. However, only high-dose ATG (HR 2.96, 95 % CI 1.143–7.663, P = 0.025) and male gender (HR 4.033, 95 % CI 1.355–12.008, P = 0.012) were independent risk factors, as shown by multivariate analysis. In the setting of haploidentical HSCT, we concluded that LOHC is more prevalent in recipients of high-dose ATG and male patients. Future studies should focus on immune reconstitution and virus infection after haploidentical HSCT with 6 mg/kg or 10 mg/kg ATG.     

Haemopoietic cell transplantation in children with juvenile myelomonocytic leukaemia

Seven children, 11 months to 5.9 years of age, with juvenile myelomonocytic leukaemia (JMML) underwent allogeneic haemopoietic cell transplantation (HCT) using related or unrelated donor bone marrow or umbilical cord blood. All patients had active disease at time of transplant despite chemotherapy in five patients and chemotherapy and splenectomy in one patient prior to HCT conditioning. All patients received cyclophosphamide and TBI, with the addition of busulphan in two cases. Engraftment was documented in all cases. Notably, six of seven patients relapsed after allogeneic HCT with one achieving a return to full donor chimaerism after cyclosporin A (CSA) withdrawal. Two patients are alive in remission, 23+ and 30+ months after transplant. The role of allogeneic HCT in patients with JMML is discussed. A cooperative multicentre trial is needed to establish the optimal therapy for these patients.     

Successful treatment of JMML relapsed after unrelated allogeneic transplant with cytoreduction followed by DLI and interferon-alpha: evidence for a graft-versus-leukemia effect in non-monosomy-7 JMML

Relapse is the major cause of treatment failure after allogeneic transplantation of children with juvenile myelomonocytic leukemia (JMML), and the role of post-transplant immunomodulation is poorly understood. We report a 12-month-old child with JMML relapsed after unrelated marrow transplantation who received cytoreduction followed by donor lymphocyte infusion (DLI) with improvement, and after addition of interferon-alpha (IFN) achieved complete donor chimerism. He was weaned from IFN and has maintained complete remission for 19 months. This is the first published report of a patient with non-monosomy-7 JMML responding to post-transplant immunomodulation and suggests a role for DLI plus IFN in these patients.     

Efficacy of the hypomethylating agents as frontline, salvage, or consolidation therapy in adults with acute myeloid leukemia (AML)

The hypomethylating agents (HAs), azacitidine and decitabine, have emerged as an alternative to initial and salvage therapy in patients with acute myeloid leukemia (AML). Little is known about how AML responds to hypomethylating agents after standard therapy, and the activity of these agents in a real-world setting is not well studied. We retrospectively examined data for 75 consecutive AML patients at Wake Forest from 2002 to 2011 treated with HAs either as first-line (n?=?34), salvage (n?=?28), or consolidation (n?=?13) therapy. We collected data on age, gender, race, Charlson comorbidity index (CCI), cytogenetics, type of treatment, complete remission (CR), complete remission with incomplete count recovery (CRi), and survival. Statistical analysis was performed using Kaplan–Meier estimates and Cox proportional hazards models. Frontline response rate (CR + CRi) was 26.5 %, and median overall survival (OS) was 3.4 months (95 % CI 1.3–7.4), with 18 % alive at 1 year. In the salvage cohort, the response rate was significantly lower compared to frontline (3.6 versus 26.5 %, p?=?0.017). Despite the reduced response, OS from time of HA treatment was longer than frontline at 8.2 months (CI 4.8–10.3). In the consolidation cohort, OS was 13.8 months (CI 8.0–21.6) with one patient in remission more than 30 months from diagnosis. These data suggest that prior cytotoxic therapy decreases marrow response rates to HAs but not survival. Furthermore, use of hypomethylating agents for consolidation resulted in a median overall survival over 1 year in a cohort of older patients. This suggests that hypomethylating agents have activity in all phases of AML treatment.     

Impact of MLL5 expression on decitabine efficacy and DNA methylation in acute myeloid leukemia

Hypomethylating agents are widely used in patients with myelodysplastic syndromes and unfit patients with acute myeloid leukemia. However, it is not well understood why only some patients respond to hypomethylating agents. We found previously that the effect of decitabine on hematopoietic stem cell viability differed between Mll5 wild-type and null cells. We, therefore, investigated the role of MLL5 expression levels on outcome of acute myeloid leukemia patients who were treated with decitabine. MLL5 above the median expression level predicted longer overall survival independent of DNMT3A mutation status in bivariate analysis (median overall survival for high vs. low MLL5 expression 292 vs. 167 days; P=0.026). In patients who received three or more courses decitabine, high MLL5 expression and wild-type DNMT3A independently predicted improved overall survival (median overall survival for high vs. low MLL5 expression 468 vs. 243 days; P=0.012). In transformed murine cells, loss of Mll5 was associated with resistance to low-dose decitabine, less global DNA methylation in promoter regions, and reduced DNA demethylation upon decitabine treatment. Together, these data support our clinical observation of improved outcome in decitabine-treated patients who express MLL5 at high levels, and suggest a mechanistic role of MLL5 in the regulation of DNA methylation.     

Acute parvovirus B19 infection mimicking juvenile myelomonocytic leukemia

An 11-month-old patient with parvovirus infection mimicking juvenile myelomonocytic leukemia (JMML) is presented. The patient's history, presenting physical and laboratory features, was suggestive of JMML and consisted of fever, hepatosplenomegaly, lymphadenopathy, desquamation of the skin, anemia, leukocytosis with monocytosis and trilineage dysplastic findings of the peripheral blood and bone marrow. However, positive IgM titers for parvovirus B19 followed by seroconversion, negative cytogenetics and the benign follow-up of the patient suggested acute parvovirus infection as an etiologic factor for development of dysplastic features in the patient, and thus is recommended for consideration in the differential diagnosis of MDS. Although parvovirus B19 infection mimicking MDS has previously been shown in two patients with spherocytosis and one with subclinical immune deficiency; to our knowledge, the present report is the first describing the association of acute parvovirus B19 infection with dysplastic features mimicking myelodysplasia (MDS) in a child without a demonstrable underlying hematolymphoid disorder.