Genomic imbalances analysis provides new insight into prognostic factors in adult and pediatric T-ALL.

Given the poor outcome of refractory and relapsing T-ALL, identifying prognostic markers is still challenging. Using SNP-array analysis, we provide a comprehensive analysis of genomic imbalances in a cohort of 317 newly-diagnosed T-ALL patients including 135 children and 182 adults with respect to clinical and biological features and outcomes. SNP-array results identified at least one somatic genomic imbalance in virtually all T-ALL patients (~96%). Del(9)(p21) (~70%) and UPD(9)p21)/CDKN2A/B (~28%) were the most frequent genomic imbalances. Unexpectedly del(13q14)/RB1/DLEU1 (~14%) was the second more frequent CNV followed by del(6)(q15)/CASP8AP2 (~11%), del(1)(p33)/SIL-TAL1 (~11%), del(12)(p13)ETV6/CDKN1B (~9%), del(18)(p11)/PTPN2 (~9%), del(1)(p36)/RPL22 (~9%), and del(17)(q11)/NF1/SUZ12 (~8%). SNP-array also revealed distinct profiles of genomic imbalances according to age, immunophenotype, and oncogenetic subgroups. In particular, adult T-ALL patients demonstrated a significantly higher incidence of del(1)(p36)/RPL22, and del(13)(q14)/RB1/DLEU1, and lower incidence of del(9)(p21) and UPD(9p21)/CDKN2A/B. We determined a threshold of 15 genomic imbalances to stratify patients into high- and low-risk groups of relapse. Survival analysis also revealed the poor outcome, despite the low number of affected cases, conferred by the presence of chromothripsis (n=6, ~2%), del(16)(p13)/CREBBP (n=15, ~5%) as well as the newly identified recurrent gain at 6q27 involving MLLT4 (n=10, ~3%). Genomic complexity, del(16)(p13)/CREBBP and gain at 6q27 involving MLLT4 maintained their significance in multivariate analysis for survival outcome. Our study thus demonstrated that whole genome analysis of imbalances provides new insights to refine risk stratification in T-ALL.

NGS-based stratification refines the risk stratification in T-ALL and identifies a Very High-Risk subgroup of patients.

We previously reported a better outcome in adult and pediatric T-cell acute lymphoblastic leukemia (T-ALL) harboring NOTCH1 and/or FBXW7 mutations without alterations of K-N-RAS and PTEN genes. Availability of high-throughput next-generation sequencing strategies (NGS) led us to refine the outcome prediction in T-ALL. Targeted whole-exome sequencing of 72 T-ALL related oncogenes was performed in 198 adult T-ALLs in first remission (CR1) from the GRAALL-2003/2005 protocols (ClinicalTrial.gov, NCT00222027, NCT00327678) and 242 pediatric T-ALLs from the FRALLE2000T. This approach enabled the identification of the first NGS-based classifier in T-ALL categorizing low-risk patients as those with N/F, PHF6, or EP300 mutations, excluding N-K-RAS, PI3K pathway (PTEN, PIK3CA, and PIK3R1), TP53, DNMT3A, IDH1/2, and IKZF1 alterations, with a 5-year cumulative incidence of relapse (CIR) estimated at 21%. Conversely, the remaining patients were classified as high-risk, exhibiting a 5-year CIR estimated at 47%. We externally validated this stratification in the pediatric cohort. NGS-based classifier was highly prognostic, independently of minimal residual disease (MRD) and white blood cells counts (WBC), in both adult and pediatric cohorts. Integration of the NGS-based classifier into a comprehensive risk stratification model, including WBC count at diagnosis and MRD at the end of induction, enabled the identification of an adverse risk subgroup (25%) with a 5-year CIR estimated at 51%, and a favorable risk group (32%) with a 5-year CIR estimated at 12%. NGS-based stratification combined with WBC and MRD sharpens the prognostic classification in T-ALL and identifies a new subgroup of patients who may benefit from innovative therapeutic approaches.

Combined germline and somatic human FADD mutations cause autoimmune lymphoproliferative syndrome.

BACKGROUND: The autoimmune lymphoproliferative syndrome (ALPS) is a noninfectious and nonmalignant lymphoproliferative disease frequently associated with autoimmune cytopenia resulting from defective FAS signaling. We previously described germline monoallelic FAS (TNFRSF6) haploinsufficient mutations associated with somatic events, such as loss of heterozygosity on the second allele of FAS, as a cause of ALPS-FAS. These somatic events were identified by sequencing FAS in DNA from double-negative (DN) T cells, the pathognomonic T-cell subset in ALPS, in which the somatic events accumulated. OBJECTIVE: We sought to identify whether a somatic event affecting the FAS-associated death domain (FADD) gene could be related to the disease onset in 4 unrelated patients with ALPS carrying a germline monoallelic mutation of the FADD protein inherited from a healthy parent. METHODS: We sequenced FADD and performed array-based comparative genomic hybridization using DNA from sorted CD4+ or DN T cells. RESULTS: We found homozygous FADD mutations in the DN T cells from all 4 patients, which resulted from uniparental disomy. FADD deficiency caused by germline heterozygous FADD mutations associated with a somatic loss of heterozygosity was a phenocopy of ALPS-FAS without the more complex symptoms reported in patients with germline biallelic FADD mutations. CONCLUSIONS: The association of germline and somatic events affecting the FADD gene is a new genetic cause of ALPS.

KIR3DL2 contributes to the typing of acute adult T-cell leukemia and is a potential therapeutic target.

Adult T-cell leukemia (ATL) is a lymphoid neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1), which encodes the transcriptional activator Tax, which participates in the immortalization of infected T cells. ATL is classified into 4 subtypes: smoldering, chronic, acute, and lymphoma. We determined whether natural killer receptors (NKRs) were expressed in ATL. NKR expression (KIR2DL1/2DS1, KIR2DL2/2DL3/2DS2, KIR3DL2, NKG2A, NKG2C, and NKp46) was assessed in a discovery cohort of 21 ATL, and KIR3DL2 was then assessed in 71 patients with ATL. KIR3DL2 was the only NKR among those studied frequently expressed by acute-type vs lymphoma- and chronic/smoldering-type ATL (36 of 40, 4 of 16, and 1 of 15, respectively; P = .001), although acute- and lymphoma-type ATL had similar mutation profiles by targeted exome sequencing. The correlation of KIR3DL2 expression with promoter demethylation was determined by microarray-based DNA methylation profiling. To explore the role of HTLV-1, KIR3DL2 and TAX messenger RNA (mRNA) expression levels were assessed by PrimeFlow RNA in primary ATL and in CD4+ T cells infected with HTLV-1 in vitro. TAX mRNA and KIR3DL2 protein expressions were correlated on ATL cells. HTLV-1 infection triggered KIR3DL2 by CD4+ cells but Tax alone did not induce KIR3DL2 expression. Ex vivo, autologous, antibody-dependent cell cytotoxicity using lacutamab, a first-in-class anti-KIR3DL2 humanized antibody, selectively killed KIR3DL2+ primary ATL cells ex vivo. To conclude, KIR3DL2 expression is associated with acute-type ATL. Transcription of KIR3DL2 may be triggered by HTLV-1 infection and correlates with hypomethylation of the promoter. The benefit of targeting KIR3DL2 with lacutamab is being further explored in a randomized phase 2 study in peripheral T-cell lymphoma, including ATL (registered on https://clinicaltrials.gov as #NCT04984837).

Harnessing the MYB-dependent TAL1 5'super-enhancer for targeted therapy in T-ALL.

The acquisition of genetic abnormalities engendering oncogene dysregulation underpins cancer development. Certain proto-oncogenes possess several dysregulation mechanisms, yet how each mechanism impacts clinical outcome is unclear. Using T-cell acute lymphoblastic leukemia (T-ALL) as an example, we show that patients harboring 5'super-enhancer (5'SE) mutations of the TAL1 oncogene identifies a specific patient subgroup with poor prognosis irrespective of the level of oncogene dysregulation. Remarkably, the MYB dependent oncogenic 5'SE can be targeted using Mebendazole to induce MYB protein degradation and T-ALL cell death. Of note Mebendazole treatment demonstrated efficacy in vivo in T-ALL preclinical models. Our work provides proof of concept that within a specific oncogene driven cancer, the mechanism of oncogene dysregulation rather than the oncogene itself can identify clinically distinct patient subgroups and pave the way for future super-enhancer targeting therapy.

TREC mediated oncogenesis in human immature T lymphoid malignancies preferentially involves ZFP36L2.

The reintegration of excised signal joints resulting from human V(D)J recombination was described as a potent source of genomic instability in human lymphoid cancers. However, such molecular events have not been recurrently reported in clinical patient lymphoma/leukemia samples. Using a specifically designed NGS-capture pipeline, we here demonstrated the reintegration of T-cell receptor excision circles (TRECs) in 20/1533 (1.3%) patients with T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL). Remarkably, the reintegration of TREC recurrently targeted the tumor suppressor gene, ZFP36L2, in 17/20 samples. Thus, our data identified a new and hardly detectable mechanism of gene deregulation in lymphoid cancers providing new insights in human oncogenesis.

Insights into the expanding intestinal phenotypic spectrum of SOCS1 haploinsufficiency and therapeutic options.

PURPOSE: Hyper activation of the JAK-STAT signaling underlies the pathophysiology of many human immune-mediated diseases. Herein, the study of 2 adult patients with SOCS1 haploinsufficiency illustrates the severe and pleomorphic consequences of its impaired regulation in the intestinal tract. METHODS: Two unrelated adult patients presented with gastrointestinal manifestations, one with Crohn's disease-like ileo-colic inflammation refractory to anti-TNF and the other with lymphocytic leiomyositis causing severe chronic intestinal pseudo-occlusion. Next-generation sequencing was used to identify the underlying monogenic defect. One patient received anti-IL-12/IL-23 treatment while the other received the JAK1 inhibitor, ruxolitinib. Peripheral blood, intestinal tissues, and serum samples were analyzed before-and-after JAK1 inhibitor therapy using mass cytometry, histology, transcriptomic, and Olink assay. RESULTS: Novel germline loss-of-function variants in SOCS1 were identified in both patients. The patient with Crohn-like disease achieved clinical remission with anti-IL-12/IL-23 treatment. In the second patient with lymphocytic leiomyositis, ruxolitinib induced rapid resolution of the obstructive symptoms, significant decrease of the CD8+ T lymphocyte muscular infiltrate, and normalization of serum and intestinal cytokines. Decreased frequencies of circulating Treg cells, MAIT cells, and NK cells, with altered CD56bright:CD16lo:CD16hi NK subtype ratios were not modified by ruxolitinib. CONCLUSION: SOCS1 haploinsufficiency can result in a broad spectrum of intestinal manifestations and need to be considered as differential diagnosis in cases of severe treatment-refractory enteropathies, including the rare condition of lymphocytic leiomyositis. This provides the rationale for genetic screening and considering JAK inhibitors in such cases.

Human Autosomal Recessive DNA Polymerase Delta 3 Deficiency Presenting as Omenn Syndrome.

The DNA polymerase δ complex (PolD), comprising catalytic subunit POLD1 and accessory subunits POLD2, POLD3, and POLD4, is essential for DNA synthesis and is central to genome integrity. We identified, by whole exome sequencing, a homozygous missense mutation (c.1118A > C; p.K373T) in POLD3 in a patient with Omenn syndrome. The patient exhibited severely decreased numbers of naïve T cells associated with a restricted T-cell receptor repertoire and a defect in the early stages of TCR recombination. The patient received hematopoietic stem cell transplantation at age 6 months. He manifested progressive neurological regression and ultimately died at age 4 years. We performed molecular and functional analysis of the mutant POLD3 and assessed cell cycle progression as well as replication-associated DNA damage. Patient fibroblasts showed a marked defect in S-phase entry and an enhanced number of double-stranded DNA break-associated foci despite normal expression levels of PolD components. The cell cycle defect was rescued by transduction with WT POLD3. This study validates autosomal recessive POLD3 deficiency as a novel cause of profound T-cell deficiency and Omenn syndrome.

TAL1 activation in T-cell acute lymphoblastic leukemia: a novel oncogenic 3' neo-enhancer.

T-cell acute lymphocytic leukemia protein 1 (TAL1) is one of the most frequently deregulated oncogenes in T-cell acute lymphoblastic leukemia (T-ALL). Its deregulation can occur through diverse cis-alterations, including SIL-TAL1 microdeletions, translocations with T-cell Receptor loci, and more recently described upstream intergenic non-coding mutations. These mutations consist of recurrent focal microinsertions that create an oncogenic neo-enhancer accompanied by activating epigenetic marks. This observation laid the groundwork for an innovative paradigm concerning the activation of proto-oncogenes via genomic alterations of non-coding intergenic regions. However, for the majority of T-ALL expressing TAL1 (TAL1+), the deregulation mechanism remains 'unresolved'. We took advantage of H3K27ac and H3K4me3 chromatin immunoprecipitation sequencing data of eight cases of T-ALL, including five TAL1+ cases. We identified a putative novel oncogenic neo-enhancer downstream of TAL1 in an unresolved monoallelic TAL1+ case. A rare but recurrent somatic heterozygous microinsertion within this region creates a de novo binding site for MYB transcription factor. Here we demonstrate that this mutation leads to increased enhancer activity, gain of active epigenetic marks, and TAL1 activation via recruitment of MYB. These results highlight the diversity of non-coding mutations that can drive oncogene activation.

Oncogenetic landscape of T-cell lymphoblastic lymphomas compared to T-cell acute lymphoblastic leukemia.

In the latest 2016 World Health Organization classification of hematological malignancies, T-cell lymphoblastic lymphoma (T-LBL) and lymphoblastic leukemia (T-ALL) are grouped together into one entity called T-cell lymphoblastic leukemia/lymphoma (T-LBLL). However, the question of whether these entities represent one or two diseases remains. Multiple studies on driver alterations in T-ALL have led to a better understanding of the disease while, so far, little data on genetic profiles in T-LBL is available. We sought to define recurrent genetic alterations in T-LBL and provide a comprehensive comparison with T-ALL. Targeted whole-exome next-generation sequencing of 105 genes, multiplex ligation-dependent probe amplification, and quantitative PCR allowed comprehensive genotype assessment in 818, consecutive, unselected, newly diagnosed patients (342 T-LBL vs. 476 T-ALL). The median age at diagnosis was similar in T-LBL and T-ALL (17 vs. 15 years old, respectively; p = 0.2). Although we found commonly altered signaling pathways and co-occurring mutations, we identified recurrent dissimilarities in actionable gene alterations in T-LBL as compared to T-ALL. HOX abnormalities (TLX1 and TLX3 overexpression) were more frequent in T-ALL (5% of T-LBL vs 13% of T-ALL had TLX1 overexpression; p = 0.04 and 6% of T-LBL vs 17% of T-ALL had TLX3 overexpression; p = 0.006). The PI3K signaling pathway was significantly more frequently altered in T-LBL as compared to T-ALL (33% vs 19%; p < 0.001), especially through PIK3CA alterations (9% vs 2%; p < 0.001) with PIK3CAH1047 as the most common hotspot. Similarly, T-LBL genotypes were significantly enriched in alterations in genes coding for the EZH2 epigenetic regulator and in TP53 mutations (respectively, 13% vs 8%; p = 0.016 and 7% vs 2%; p < 0.001). This genetic landscape of T-LBLL identifies differential involvement of recurrent alterations in T-LBL as compared to T-ALL, thus contributing to better understanding and management of this rare disease.

Oncogenetic mutations combined with MRD improve outcome prediction in pediatric T-cell acute lymphoblastic leukemia.

Risk stratification in childhood T-cell acute lymphoblastic leukemia (T-ALL) is mainly based on minimal residual disease (MRD) quantification. Whether oncogenetic mutation profiles can improve the discrimination of MRD-defined risk categories was unknown. Two hundred and twenty FRALLE2000T-treated patients were tested retrospectively for NOTCH1/FBXW7/RAS and PTEN alterations. Patients with NOTCH1/FBXW7 (N/F) mutations and RAS/PTEN (R/P) germ line (GL) were classified as oncogenetic low risk (gLoR; n = 111), whereas those with N/F GL and R/P GL mutations or N/F and R/P mutations were classified as high risk (gHiR; n = 109). Day 35 MRD status was available for 191 patients. Five-year cumulative incidence of relapse (CIR) and disease-free survival were 36% and 60% for gHiR patients and 11% and 89% for gLoR patients, respectively. Importantly, among the 60% of patients with MRD <10-4, 5-year CIR was 29% for gHiR patients and 4% for gLoR patients. Based on multivariable Cox models and stepwise selection, the 3 most discriminating variables were the oncogenetic classifier, MRD, and white blood cell (WBC) count. Patients harboring a WBC count ≥200 × 109/L, gHiR classifier, and MRD ≥10-4 demonstrated a 5-year CIR of 46%, whereas the 58 patients (30%) with a WBC count <200 × 109/L, gLoR classifier, and MRD <10-4 had a very low risk of relapse, with a 5-year CIR of only 2%. In childhood T-ALL, the N/F/R/P mutation profile is an independent predictor of relapse. When combined with MRD and a WBC count ≥200 × 109/L, it identifies a significant subgroup of patients with a low risk of relapse.

Low level CpG island promoter methylation predicts a poor outcome in adult T-cell acute lymphoblastic leukemia.

Cancer cells undergo massive alterations in their DNA methylation patterns which result in aberrant gene expression and malignant phenotypes. Abnormal DNA methylation is a prognostic marker in several malignancies, but its potential prognostic significance in adult T-cell acute lymphoblastic leukemia (T-ALL) is poorly defined. Here, we performed methylated DNA immunoprecipitation to obtain a comprehensive genome-wide analysis of promoter methylation in adult T-ALL (n=24) compared to normal thymi (n=3). We identified a CpG hypermethylator phenotype that distinguishes two T-ALL subgroups and further validated it in an independent series of 17 T-lymphoblastic lymphoma. Next, we identified a methylation classifier based on nine promoters which accurately predict the methylation phenotype. This classifier was applied to an independent series of 168 primary adult T-ALL treated accordingly to the GRAALL03/05 trial using methylation-specific multiplex ligation-dependent probe amplification. Importantly hypomethylation correlated with specific oncogenic subtypes of T-ALL and identified patients associated with a poor clinical outcome. This methylation-specific multiplex ligation-dependent probe amplification based methylation profiling could be useful for therapeutic stratification of adult T-ALL in routine practice. The GRAALL-2003 and -2005 studies were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively.

DNMT3A mutation is associated with increased age and adverse outcome in adult T-cell acute lymphoblastic leukemia.

The prognostic implications of DNMT3A genotype in T-cell acute lymphoblastic leukemia are incompletely understood. We performed comprehensive genetic and clinico-biological analyses of T-cell acute lymphoblastic leukemia patients with DNMT3A mutations treated during the GRAALL-2003 and -2005 studies. Eighteen of 198 cases (9.1%) had DNMT3A alterations. Two patients also had DNMT3A mutations in non-leukemic cell DNA, providing the first potential evidence of age-related clonal hematopoiesis in T-cell acute lymphoblastic leukemia. DNMT3A mutation was associated with older age (median 43.9 years vs 29.4 years, P<0.001), immature T-cell receptor genotype (53.3% vs 24.4%, P=0.016) and lower remission rates (72.2% mutated vs 94.4% non-mutated, P=0.006). DNMT3A alterations were significantly associated with worse clinical outcome, with higher cumulative incidence of relapse (HR 2.33, 95% CI: 1.05-5.16, P=0.037) and markedly poorer event-free survival (HR 3.22, 95% CI: 1.81-5.72, P<0.001) and overall survival (HR 2.91, 95% CI: 1.56-5.43, P=0.001). Adjusting for age as a covariate, or restricting the analysis to patients over 40 years, who account for almost 90% of DNMT3A-mutated cases, did not modify these observations. In multivariate analysis using the risk factors that were used to stratify treatment during the GRAALL studies, DNMT3A mutation was significantly associated with shorter event-free survival (HR 2.33, 95% CI: 1.06 - 4.04, P=0.02). Altogether, these results identify DNMT3A genotype as a predictor of aggressive T-cell acute lymphoblastic leukemia biology. The GRAALL-2003 and -2005 studies were registered at http://www.ClinicalTrials.gov as #NCT00222027 and #NCT00327678, respectively.

SET-NUP214 is a recurrent γδ lineage-specific fusion transcript associated with corticosteroid/chemotherapy resistance in adult T-ALL.

The SET-NUP214 (TAF1/CAN) fusion gene is a rare genetic event in T-cell acute lymphoblastic leukemia (T-ALL). Eleven (6%) of 196 T-ALL patients enrolled in the French Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL) 2003 and 2005 trials harbored a SET-NUP214 transcript. SET-NUP214-positive patients were predominantly (10 [91%] of 11) T-cell receptor (TCR)-negative and strikingly associated with TCRγδ lineage T-ALLs, as defined by expression of TCRγδ, TCRδ and/or TCRγ rearrangements but no complete TCRß variable diversity joining rearrangement in surface CD3/TCR-negative cases. When compared with SET-NUP214-negative patients, SET-NUP214-positive patients showed a significantly higher rate of corticosteroid resistance (91% vs 44%; P = .003) and chemotherapy resistance (100% vs 44%; P = .0001). All SET-NUP214-positive patients but one achieved complete remission, and 9 were allografted. Despite the poor early-treatment sensitivity, the outcome of SET-NUP214-positive patients was similar to that of SET-NUP214-negative patients.

An early thymic precursor phenotype predicts outcome exclusively in HOXA-overexpressing adult T-cell acute lymphoblastic leukemia: a Group for Research in Adult Acute Lymphoblastic Leukemia study.

UNLABELLED: Gene expression studies have consistently identified a HOXA-overexpressing cluster of T-cell acute lymphoblastic leukemias, but it is unclear whether these constitute a homogeneous clinical entity, and the biological consequences of HOXA overexpression have not been systematically examined. We characterized the biology and outcome of 55 HOXA-positive cases among 209 patients with adult T-cell acute lymphoblastic leukemia uniformly treated during the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003 and -2005 studies. HOXA-positive patients had markedly higher rates of an early thymic precursor-like immunophenotype (40.8% versus 14.5%, P=0.0004), chemoresistance (59.3% versus 40.8%, P=0.026) and positivity for minimal residual disease (48.5% versus 23.5%, P=0.01) than the HOXA-negative group. These differences were due to particularly high frequencies of chemoresistant early thymic precursor-like acute lymphoblastic leukemia in HOXA-positive cases harboring fusion oncoproteins that transactivate HOXA Strikingly, the presence of an early thymic precursor-like immunophenotype was associated with marked outcome differences within the HOXA-positive group (5-year overall survival 31.2% in HOXA-positive early thymic precursor versus 66.7% in HOXA-positive non-early thymic precursor, P=0.03), but not in HOXA-negative cases (5-year overall survival 74.2% in HOXA-negative early thymic precursor versus 57.2% in HOXA-negative non-early thymic precursor, P=0.44). Multivariate analysis further revealed that HOXA positivity independently affected event-free survival (P=0.053) and relapse risk (P=0.039) of chemoresistant T-cell acute lymphoblastic leukemia. These results show that the underlying mechanism of HOXA deregulation dictates the clinico-biological phenotype, and that the negative prognosis of early thymic precursor acute lymphoblastic leukemia is exclusive to HOXA-positive patients, suggesting that early treatment intensification is currently suboptimal for therapeutic rescue of HOXA-positive chemoresistant adult early thymic precursor acute lymphoblastic leukemia. TRIAL REGISTRATION: The GRAALL-2003 and -2005 studies were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively.

Minimal residual disease monitoring by 8-color flow cytometry in mantle cell lymphoma: an EU-MCL and LYSA study.

Quantification of minimal residual disease may guide therapeutic strategies in mantle cell lymphoma. While multiparameter flow cytometry is used for diagnosis, the gold standard method for minimal residual disease analysis is real-time quantitative polymerase chain reaction (RQ-PCR). In this European Mantle Cell Lymphoma network (EU-MCL) pilot study, we compared flow cytometry with RQ-PCR for minimal residual disease detection. Of 113 patients with at least one minimal residual disease sample, RQ-PCR was applicable in 97 (86%). A total of 284 minimal residual disease samples from 61 patients were analyzed in parallel by flow cytometry and RQ-PCR. A single, 8-color, 10-antibody flow cytometry tube allowed specific minimal residual disease assessment in all patients, with a robust sensitivity of 0.01%. Using this cut-off level, the true-positive-rate of flow cytometry with respect to RQ-PCR was 80%, whereas the true-negative-rate was 92%. As expected, RQ-PCR frequently detected positivity below this 0.01% threshold, which is insufficiently sensitive for prognostic evaluation and would ideally be replaced with robust quantification down to a 0.001% (10-5) threshold. In 10 relapsing patients, the transition from negative to positive by RQ-PCR (median 22.5 months before relapse) nearly always preceded transition by flow cytometry (4.5 months), but transition to RQ-PCR positivity above 0.01% (5 months) was simultaneous. Pre-emptive rituximab treatment of 2 patients at minimal residual disease relapse allowed re-establishment of molecular and phenotypic complete remission. Flow cytometry minimal residual disease is a complementary approach to RQ-PCR and a promising tool in individual mantle cell lymphoma therapeutic management. (clinicaltrials identifiers: 00209209 and 00209222).

The kinetics of relapse in DEK-NUP214-positive acute myeloid leukemia patients.

Preemptive treatment of relapse of acute myeloid leukemia (AML) holds the promise to improve the prognosis of this currently highly lethal condition. Proposed treatment modalities applicable in preemptive cytoreduction (e.g., demethylating agents or standard chemotherapy) differ substantially in interval from administration to antileukemic effect. The t(6;9) balanced translocation, producing the DEK-NUP214 fusion protein, is seen in only 1% of patients with AML. We hypothesized that in these patients, who relapse with a very high frequency, a more detailed knowledge of leukemic relapse growth kinetics would improve the personalized decision-making regarding re-administration of chemotherapy. Based on standardized quantitative PCR data, we therefore delineated the relapse kinetics in a cohort of 27 relapsing DEK-NUP214-positive patients treated in four different European countries. The prerelapse leukemic burden increased with a median doubling time of 13 d (range: 5-51 d, median: 0.71 logs/month, range: 0.18-1.91 logs/month), with FLT3-ITD-positive patients relapsing significantly faster than FLT3-ITD-negative ones (median: 0.9 vs. 0.6 logs/month, Wilcoxon rank sum test, P = 0.041). Peripheral blood and bone marrow were equally useful for minimal residual disease (MRD) detection, and thus, we found that with sampling intervals of 2 months, 94% of relapses would be detected with a median time from MRD detection to hematological relapse of 64 d. In conclusion, this data provide algorithms for handling the rare patients with DEK-NUP214-positive AML allowing for planning of both MRD follow-up and, upon molecular relapse, the timing of cytoreduction or possibly transplant procedures.

PHF6-altered T-ALL Harbor Epigenetic Repressive Switch at Bivalent Promoters and Respond to 5-Azacitidine and Venetoclax.

PURPOSE: To assess the impact of PHF6 alterations on clinical outcome and therapeutical actionability in T-cell acute lymphoblastic leukemia (T-ALL). EXPERIMENTAL DESIGN: We described PHF6 alterations in an adult cohort of T-ALL from the French trial Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003/2005 and retrospectively analyzed clinical outcomes between PHF6-altered (PHF6ALT) and wild-type patients. We also used EPIC and chromatin immunoprecipitation sequencing data of patient samples to analyze the epigenetic landscape of PHF6ALT T-ALLs. We consecutively evaluated 5-azacitidine efficacy, alone or combined with venetoclax, in PHF6ALT T-ALL. RESULTS: We show that PHF6 alterations account for 47% of cases in our cohort and demonstrate that PHF6ALT T-ALL presented significantly better clinical outcomes. Integrative analysis of DNA methylation and histone marks shows that PHF6ALT are characterized by DNA hypermethylation and H3K27me3 loss at promoters physiologically bivalent in thymocytes. Using patient-derived xenografts, we show that PHF6ALT T-ALL respond to the 5-azacytidine alone. Finally, synergism with the BCL2-inhibitor venetoclax was demonstrated in refractory/relapsing (R/R) PHF6ALT T-ALL using fresh samples. Importantly, we report three cases of R/R PHF6ALT patients who were successfully treated with this combination. CONCLUSIONS: Overall, our study supports the use of PHF6 alterations as a biomarker of sensitivity to 5-azacytidine and venetoclax combination in R/R T-ALL.