RAS pathway mutations as a predictive biomarker for treatment adaptation in pediatric B-cell precursor acute lymphoblastic leukemia.

RAS pathway mutations have been linked to relapse and chemotherapy resistance in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive data on the frequency and prognostic value of subclonal mutations in well-defined subgroups using highly sensitive and quantitative methods are lacking. Targeted deep sequencing of 13 RAS pathway genes was performed in 461 pediatric BCP-ALL cases at initial diagnosis and in 19 diagnosis-relapse pairs. Mutations were present in 44.2% of patients, with 24.1% carrying a clonal mutation. Mutation frequencies were highest in high hyperdiploid, infant t(4;11)-rearranged, BCR-ABL1-like and B-other cases (50-70%), whereas mutations were less frequent in ETV6-RUNX1-rearranged, and rare in TCF3-PBX1- and BCR-ABL1-rearranged cases (27-4%). RAS pathway-mutated cells were more resistant to prednisolone and vincristine ex vivo. Clonal, but not subclonal, mutations were linked to unfavorable outcome in standard- and high-risk-treated patients. At relapse, most RAS pathway mutations were clonal (9 of 10). RAS mutant cells were sensitive to the MEK inhibitor trametinib ex vivo, and trametinib sensitized resistant cells to prednisolone. We conclude that RAS pathway mutations are frequent, and that clonal, but not subclonal, mutations are associated with unfavorable risk parameters in newly diagnosed pediatric BCP-ALL. These mutations may designate patients eligible for MEK inhibitor treatment.

Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: an international collaborative study.

Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4-7 and exons 1-8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case-control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2-7 (P=0.03), DEL 2-8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4-7 and DEL 1-8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

High-risk childhood acute lymphoblastic leukemia in first remission treated with novel intensive chemotherapy and allogeneic transplantation.

Children with acute lymphoblastic leukemia (ALL) and high minimal residual disease (MRD) levels after initial chemotherapy have a poor clinical outcome. In this prospective, single arm, Phase 2 trial, 111 Dutch and Australian children aged 1-18 years with newly diagnosed, t(9;22)-negative ALL, were identified among 1041 consecutively enrolled patients as high risk (HR) based on clinical features or high MRD. The HR cohort received the AIEOP-BFM (Associazione Italiana di Ematologia ed Oncologia Pediatrica (Italy)-Berlin-Frankfurt-Münster ALL Study Group) 2000 ALL Protocol I, then three novel HR chemotherapy blocks, followed by allogeneic transplant or chemotherapy. Of the 111 HR patients, 91 began HR treatment blocks, while 79 completed the protocol. There were 3 remission failures, 12 relapses, 7 toxic deaths in remission and 10 patients who changed protocol due to toxicity or clinician/parent preference. For the 111 HR patients, 5-year event-free survival (EFS) was 66.8% (±5.5) and overall survival (OS) was 75.6% (±4.3). The 30 patients treated as HR solely on the basis of high MRD levels had a 5-year EFS of 63% (±9.4%). All patients experienced grade 3 or 4 toxicities during HR block therapy. Although cure rates were improved compared with previous studies, high treatment toxicity suggested that novel agents are needed to achieve further improvement.

Outcome after first relapse in children with acute lymphoblastic leukemia: a report based on the Dutch Childhood Oncology Group (DCOG) relapse all 98 protocol.

BACKGROUND: We report on the treatment of children and adolescents with acute lymphoblastic leukemia (ALL) in first relapse. The protocol focused on: (1) Intensive chemotherapy preceding allogeneic stem cell transplantation (SCT) in early bone marrow relapse; (2) Rotational chemotherapy in late relapse, without donor; (3) Postponement of cerebro-spinal irradiation in late isolated CNS relapse; and (4) Treatment in very late bone marrow relapse with chemotherapy only. METHODS: From January 1999 until July 2006 all 158 Dutch pediatric patients with ALL in first relapse were recorded. Ninety-nine patients were eligible; 54 patients with early and 45 with late relapse. Eighteen patients had an isolated extra-medullary relapse; 69 patients had bone marrow involvement only. RESULTS: Five-years EFS rates for early and late relapses were 12% and 35%, respectively. For early relapses 5 years EFSs were 25% for patients transplanted; 0% for non-transplanted patients. For late relapses 5 years EFS was 64% for patients treated with chemotherapy only, and 16% for transplanted patients. For very late relapses EFS was 58%. CONCLUSIONS: Our data suggest the superiority of SCT for early relapse patients. For late relapses a better outcome is achieved with chemotherapy only using the rotational chemotherapy scheme. The most important factor for survival was interval between first CR and occurrence of the first relapse.

Apoptotic death of infiltrating cells in human cardiac allografts is regulated by IL-2, FASL, and FLIP.

INTRODUCTION: In vitro studies have shown that apoptotic cell death is triggered by a IL-2-dependent activation of the Fas-FasL pathway and that this pathway can be inhibited by FLIP. METHODS: To define whether FLIP regulates apoptotic death of graft infiltrating T-cells during IL-2-mediated rejection, we analyzed endomyocardial biopsies (EMB) from cardiac allograft recipients for CD3, DNA strand breaks (TUNEL assay), FLIP (mRNA and protein), and FasL mRNA expression. RESULTS: Apoptosis was present in CD3+ T-cell infiltrates. The number of TUNEL-stained mononuclear cells was inversely correlated with FLIP mRNA expression levels (P=.09). FLIP protein was present in 5% to 10% of the infiltrating cells and was constitutively produced by cardiomyocytes irrespective of the rejection grade. Rejection biopsies had elevated IL-2 and FasL mRNA expression levels compared to the expression levels before and after acute rejection (P=.03 and P=.11), while FLIP mRNA expression levels were significantly decreased during rejection (P=.05). CONCLUSION: Our results indicate that during the IL-2-induced rejection process, infiltrated T cells become more sensitive to apoptosis.

Intragraft interleukin 2 mRNA expression during acute cellular rejection and left ventricular total wall thickness after heart transplantation.

OBJECTIVE: To assess whether diastolic graft function is influenced by intragraft interleukin 2 (IL-2) messenger RNA (mRNA) expression in rejecting cardiac allografts. DESIGN: 16 recipients of cardiac allografts were monitored during the first three months after transplantation. The presence of IL-2 mRNA in endomyocardial biopsies (n = 123) was measured by reverse transcriptase polymerase chain reaction. To determine heart function, concurrent M mode and two dimensional Doppler echocardiograms were analysed. RESULTS: Histological signs of acute rejection (International Society for Heart and Lung Transplantation (ISHLT) rejection grade > 2) were strongly associated with IL-2 mRNA expression (IL-2 mRNA was present in 12 of 20 endomyocardial biopsies (60%) with acute rejection and in 24 of 103 endomyocardial biopsies (23%) without acute rejection, p = 0.002). No significant relation was found between either histology or IL-2 mRNA expression alone and the studied echocardiographic parameters. However, stratification of the echocardiographic data into those of patients with and those without acute rejection showed that during acute rejection IL-2 mRNA expression was significantly associated with increased left ventricular total wall thickness (mean change in total wall thickness was +0.22 cm in patients with IL-2 mRNA expression versus -0.18 cm in patients without IL-2 mRNA expression, p = 0.048). CONCLUSIONS: An increase in left ventricular total wall thickness precedes IL-2 positive acute rejection after heart transplantation. Thus, cardiac allograft rejection accompanied by intragraft IL-2 mRNA expression may be indicative of more severe rejection episodes.

Failure to down-regulate intragraft cytokine mRNA expression shortly after clinical heart transplantation is associated with high incidence of acute rejection.

BACKGROUND: Brain-death, ischemia and reperfusion damage have been implicated as initial factors that lead to a cascade of immunologic events that result in allograft rejection in experimental animals. Cytokines are thought to play a central role in this process. Therefore, we evaluated intragraft cytokine mRNA expression at an early stage after clinical heart transplantation and related these data to ischemia, immunosuppression, and rejection. METHODS: We sampled endomyocardial biopsies at 30 minutes (EMB 0) and at 1 week (EMB 1) after transplantation from 20 cardiac allograft recipients. Intragraft monocyte chemoattractant protein (MCP-1) and basic fibroblast growth factor (bFGF) mRNA expression levels were quantitatively measured using competitive template Reverse-transcriptase polymerase chain reaction (RT-PCR). RESULTS: We measured significantly lower MCP-1 and bFGF mRNA expression levels in EMB 1 compared with EMB 0 (MCP-1, p = 0.006; bFGF, p = 0.019). We found no direct correlation between the cytokine mRNA expression levels in EMB 0 or EMB 1 and ischemic times, induction therapy, or cyclosporine whole-blood trough levels. Patients with a high incidence of acute rejection episodes (>2 in the first year) had higher bFGF mRNA expression levels (p = 0.009) and comparable MCP-1 mRNA expression levels (p = 0.378) at 1 week, compared with patients with a lower rejection incidence. The MCP-1 and bFGF mRNA expression levels in the first week were not associated with the development of graft vascular disease in the first year post-transplant. CONCLUSIONS: We found a significant decrease of intragraft MCP-1 and bFGF mRNA expression levels in the first post-operative week. Patients with a high incidence of acute rejection had higher bFGF mRNA expression levels in their first week biopsy. Therefore, we conclude that patients who fail to down-regulate their bFGF mRNA expression early after transplantation are at higher risk for acute rejection.

Intragraft platelet-derived growth factor-alpha and transforming growth factor-beta1 during the development of accelerated graft vascular disease after clinical heart transplantation.

This study was to determine whether the growth factors platelet-derived growth factor-alpha (PDGF-alpha) and transforming growth factor-beta1 (TGF-beta1) contribute to the development of graft vascular disease (GVD) after clinical heart transplantation. We analysed intragraft PDGF-alpha and TGF-beta1 messenger RNA (mRNA) expression levels by competitive template reverse transcriptase polymerase chain reaction (RT-PCR). Endomyocardial biopsies (EMB) were obtained at 1 and 9 months post-transplant from cardiac allograft recipients with (n = 11) and without (n = 11) angiographic evidence of GVD at 1 year. In 1-month EMB, comparable TGF-beta1 mRNA levels were found in patients with and without GVD at 1 year (p = 0.84, Mann-Whitney U-test). In contrast, in 9-month EMB during the development of GVD, intragraft mRNA levels of both PDGF-alpha (p = 0.08) and TGF-beta1 (p = 0.03) were higher in patients with GVD after the first year compared to patients without GVD. These results suggest that intragraft PDGF-alpha and TGF-beta1 play a role in the pathogenesis of accelerated GVD after clinical heart transplantation.