Variant identification underlying inherited dysfibrinogenemia quite exceptionally fails. We report on two dysfibrinogenemia cases whose underlying DNA variant could not be identified by Sanger ...analysis. These failures result from two distinct mechanisms. The first case involved raw signal overcorrection by a built-in software, and the second constituted the first description of mosaicism for one of the fibrinogen genes. This mosaicism was subsequently identified by next-generation sequencing reanalysis of the sample.
Objectives
Venetoclax combined with hypomethylating agents is a new therapeutic strategy frequently used for treating AML patients who are not eligible for conventional chemotherapy. However, high ...response rates are heterogeneous due to different mechanisms mediating resistance to venetoclax such as up‐regulation of MCL‐1 expression. We thus tested the anti‐leukemic activity of S63845, a specific MCL‐1 inhibitor.
Methods
Apoptosis induces by S63845 with or without venetoclax was evaluated in primary AML samples and in AML cell lines co‐cultured or not with bone marrow (BM) mesenchymal stromal cells. Sensitivity of leukemic cells to S63845 was correlated to the expression level of BCL‐2, MCL‐1, and BCL‐XL determined by Western Blot and mass spectrometry‐based proteomics.
Results
We observed that even if MCL‐1 expression is weak compared to BCL‐2, S63845 induces apoptosis of AML cells and strongly synergizes with venetoclax. Furthermore, AML cells resistant to venetoclax are highly sensitive to S63845. Interestingly, the synergistic effect of S63845 toward venetoclax‐mediated apoptosis of AML cells is still observed in a context of interaction with the BM microenvironment that intrinsically mediates resistance to BCL2 inhibition.
Conclusion
These results are therefore of great relevance for clinicians as they provide the rational for combining BCL‐2 and MCL‐1 inhibition in AML.
TP53 aberrations are a major predictive factor of resistance to chemoimmunotherapy in chronic lymphocytic leukemia (CLL), and an assessment of them before each line of treatment is required for ...theranostic stratification. Acquisition of subclonal TP53 abnormalities underlies the evolution of CLL. To better characterize the distribution, combination, and impact of TP53 variants in CLL, 1,056 TP53 variants collected from 683 patients included in a multicenter collaborative study in France were analyzed and compared to UMD_CLL, a dataset built from published articles collectively providing 5,173 TP53 variants detected in 3,808 patients. Our analysis confirmed the presence of several CLL-specific hotspot mutations, including a two-base pair deletion in codon 209 and a missense variant at codon 234, the latter being associated with alkylating treatment. Our analysis also identified a novel CLL-specific variant in the splice acceptor signal of intron 6 leading to the use of a cryptic splice site, similarly utilized by TP53 to generate p53psi, a naturally truncated p53 isoform localized in the mitochondria. Examination of both UMD_CLL and several recently released large-scale genomic analyses of CLL patients confirmed that this splice variant is highly enriched in this disease when compared to other cancer types. Using a TP53-specific single-nucleotide polymorphism, we also confirmed that copy-neutral loss of heterozygosity is frequent in CLL. This event can lead to misinterpretation of TP53 status. Unlike other cancers, CLL displayed a high proportion of patients harboring multiple TP53 variants. Using both in silico analysis and single molecule smart sequencing, we demonstrated the coexistence of distinct subclones harboring mutations on distinct alleles. In summary, our study provides a detailed TP53 mutational architecture in CLL and gives insights into how treatments may shape the genetic landscape of CLL patients.
Data regarding the use of QuantiFERON to assist the diagnosis of active tuberculosis (TB) in HIV-infected children are limited, especially in countries with low incidence of TB/HIV coinfection.
...QuantiFERON results were analyzed in 63 HIV-infected children who presented to our hospital in Paris, France. Seventeen HIV-uninfected children with active TB (4 culture-confirmed) were included for comparison.
The 63 HIV-infected children (median age: 11 yr) had 113 QuantiFERON tests. Thirty-four (54%) were born in sub-Saharan Africa. Vertical HIV transmission was documented for 50 of 52 (96%) and stage III HIV-infection for 30 of 50 children (60%). Over the study period, active TB was diagnosed in 7 of 63 HIV-infected children (3 culture-confirmed). Additional ongoing or previous opportunistic infections were present in 4 of 7. QuantiFERON results were positive in 2 of 7 HIV-infected children with active TB (sensitivity: 29%) and 16 of 17 HIV-uninfected children with active TB (sensitivity: 94%). At initial QuantiFERON testing of the 63 HIV-infected children, 8 (13%) had positive results (1, active TB; 5, latent TB; 2, previous TB) and 51 (81%) had negative results. Of 33 children with repeat testing after an initially positive or negative result, the only change was one conversion from a negative to a positive result at the onset of active TB. The 4 children (6%) with indeterminate quantiFERON results had a concomitant opportunistic infection. Results of repeat testing after clinical stabilization were negative in all 4.
QuantiFERON testing performed poorly for active TB diagnosis in this series of children with advanced HIV infection.
We report a case of acquired von Willebrand syndrome relapse in association with Crohn's disease, in a context of non-compliance in a 85-year-old woman suffering from epistaxis and melena. The ...acquired von Willebrand syndrome is a rare bleeding disorder. This case underlines the importance of maintaining the corticosteroid therapy in order to prevent the reappearance of autoantibodies and the recurrence of this syndrome.
TP53 aberrations, including somatic mutations of TP53 gene or 17p deletion leading to the loss of the TP53 locus, are a major predictive factor of resistance to fludarabin based chemotherapy in ...chronic lymphocytic leukemia (CLL) and remain an adverse prognostic factor in the chemofree era. Therefore, detection of TP53 alteration before each new line of treatment is required for theranostic stratification.
In order to better characterize the distribution and combination of the TP53 variants in CLL, we collected the TP53 sequencing data of 343 patients harboring TP53 mutations from centers of the French Innovative Leukemia Organization-CLL (FILO) and established a large data base of 573 TP53 mutations. Mutations were identified through NGS sequencing (covering exon 2 to 11) allowing the detection of low frequency variants down to 1% VAF. Several distinct low VAF mutations were orthogonally confirmed by digital PCR. TP53 variants were analyzed through UMD_TP53 data gathering 90 000 TP53 mutations from all type of cancers. IGHV mutational status and FISH analysis were available for 224 and 176 patients respectively.
Using ACMG criteria from the UMD_TP53 database, we confirmed that 523 could be classified as pathogenic, 42 were likely pathogenic and 8 were VUS (Variants of Unknown Significance). As expected, the mutation distribution along the p53 protein exhibited a clustering of variants in the DNA binding domain of the protein. We also confirmed the presence of a specific hotspot at codon 234 (6%) which is noticeable in other CLL cohorts but absent in solid tumors. 431 TP53 variants led to the expression of a mutant protein whereas the remaining 142 led a TP53 null phenotype. For 8 patients without 17p deletion and a mutation VAF larger than 50%, SNP analysis indicate that these tumors had a copy number neutral loss of heterozygosis at 17p with a duplication of the mutant allele leading to homozygous mutations of TP53.
When focusing on the allele burden of TP53 mutations, 264/573 (46%) variants had an allele frequency <10%. Even if they were predominantly found in polymutated cases, presence of only low VAF (<10%) mutations was evidenced in 74 (21%) patients (50 patients with a single TP53 mutation and 24 patients with more than one). All these cases would have been missed by conventional sequencing.
Among the 343 patients, 113 (33%) were poly-mutated and harbored more than one pathogenic TP53 variants (2 to 11 variants per patient): 57 (16,7 %) had 2 variants, 32 (9,3%) had 3, 10 had 4 (3%) and 14 patients (4%) had 5 to 11 variants. Using both long range sequencing and in silico analysis, we could show that all these variants were distributed in different alleles supporting an important intratumoral heterogeneity and a strong selection for TP53 loss of function during tumor progression in these patients.
Null variants were rarely found as single alteration: only 46 patients (13,4%) patients harbored a single null mutation. Null mutations were predominantly found in patients with multiclonal mutations (87% with 4 or more).
Median size of variants significantly decreased with the number of mutations and most of low VAF (less than 10%) variants were found in multiclonal combinations. Multiclonal mutations were predominantly found in previously treated patients (41% treated versus 10 % untreated) but whether all these variants preceded treatment and were further selected is currently unknown. We observed that 71,5 % of patients were IGHV unmutated and multiclonal mutations were surprisingly more frequent in mutated IGHV cases than in unmutated ones. Only 50% of cases carried a 17p deletion, highlighting again the importance of testing for TP53 mutations in addition to FISH analysis. Presence or absence of 17p deletion was unrelated to the number of TP53 mutations.
Taken together these observations suggest that the TP53 mutational landscape in CLL is very complex and can involve multiple mechanisms, converging to a total loss of TP53 function and tumor progression. NGS provides a powerful tool for detecting all these alterations including variants with low VAF and should become a standard for CLL screening prior to each line of treatment.
Leblond:Amgen: Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Letestu:Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Roche: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Alexion: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts. Cymbalista:Abbvie: Honoraria; Roche: Research Funding; Sunesis: Research Funding; Gilead: Honoraria; Janssen: Honoraria; AstraZeneca: Honoraria.
In Chronic Lymphocytic Leukemia (CLL), it is well established that 17p deletions are associated with adverse prognosis and chemotherapy resistance. 17p deletions are most often associated with TP53 ...mutations, but TP53 mutations can occur in the absence of 17p deletion in about half of the cases with a similar unfavorable prognostic influence. Some patients harbor several subclones with different TP53 mutations. Nonetheless, little is known on the functional effect of the various alterations and of their associations.
We have retrospectively collected 450 TP53 variants from a real life cohort of 337 patients treated in centers of the FILO group (French Innovative Leukemia Organization). Among those, 218 were evidenced by Sanger sequencing (exons 4 to 9) with a sensitivity around 15% and 232 by next generation sequencing (exons 2 to 11) with a VAF (variant allelic frequency) >1%. All TP53 missense variants were previously described in the UMD TP53 mutation database strengthening the specificity of the data of this cohort.
Full clinical data were available for 245 patients. FISH analysis was performed in 230 cases, 17p deletion was absent in 100/230 cases (43%) confirming the importance of looking for TP53 mutation in these cases. Only 76 patients (31%) had an initial TP53 mutation assessment. The other 169 patients received a median of 2 treatments (0-11) before TP53 mutation was searched. Treatments included Fludarabine or Bendamustine based in 144 cases, Chlorambucil based in 47 cases and miscellaneous other treatments in 57 cases. After detection of TP53 mutation, 113 patients received either Ibrutinib or Idelalisib whereas 89 did not because there were not yet available. Overall survival of patients who received a BCR inhibitor is significantly better than those who did not (p<0,0001).
We analysed the mutation profile of TP53 . Mutations distribution along the TP53 gene was in agreement with the 2500 chronic lymphoproliferative disorders included in the UMD_TP53 database. There were 340 missense mutations (76%) and 110 null TP53 variants (24%), either nonsense mutations (n= 23), or frameshift insertions or deletions (n=51) or splice site mutations (n=36), leading to the potential loss of TP53 expression. Eighty patients (35%) had only a single TP53 alteration while 150 (65%) carried more than one TP53 alteration. The type of mutation was unrelated to the presence of several clones, of 17p deletion or the IGHV mutational status. But among the 80 patients with a single TP53 alteration, 70 had a TP53 missense mutant (versus only 10 null TP53 mutations, p<10-6) suggesting that the dominant negative effect of this mutant alleviates the need of a second event such as loss of 17p. Interestingly, the 10 patients who harboured only one null TP53 mutation appeared to have a milder CLL course and no strong chemorefractoriness.
We then studied the association between IGHV mutational status, presence of 17p deletion or presence of multiclonal TP53 mutations. IGHV status was mutated in 35/ 157 (22%) patients in which it was available. As expected, more multiclonal mutations cases were detected by NGS rather than by Sanger. Thirty cases harbored only small clones (VAF<15%), that would have been missed by Sanger sequencing. Two or more mutations were identified in 71 patients (21%), and 7 of them had 4 to 8 detectable clones. Presence of several clones also appeared independent of IGHV status and of presence or absence of 17p deletion. Richter's transformation occured in 29 patients experienced. We did not identify any specific pattern of TP53 mutation among those (hotspot, variant type or presence of subclones). Only 2 of them harbored mutated IGHV . Survival after Richter's transformation was very poor.
In conclusion, detection of 17p deletion by FISH analysis is currently included in the workout before therapy, whereas TP53 mutations are still not explored enough in routine practice. This retrospective real life cohort highlights the need for early detection of TP53 mutation in order to elude inefficient treatment and further development of resistant clones. Moreover, this cohort evidences that P53 alteration is a very heterogeneous process with multiple combinations depending on the penetrance of the various alterations, with disparity between missense and null variants. Whether or not this is linked to the clinical course of the disease is currently under study.
Tournilhac:GILEAD: Honoraria, Other: Travel Funding, Research Funding; ROCHE: Honoraria, Other: Travel funding, Research Funding; AMGEN: Other: Travel funding, Research Funding; Janssen: Honoraria, Other: travel funding; Abbvie: Honoraria, Other: Travel funding. Leblond:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria; Novartis: Honoraria; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ysebaert:Janssen: Consultancy, Research Funding, Speakers Bureau. Dilhuydy:Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses. Dartigeas:Roche: Consultancy; Gilead: Other: travel grant; Mundipharma: Other: travel grant; Janssen: Consultancy. Troussard:ABBVIE: Honoraria; JANSSEN: Honoraria; GILEAD: Honoraria; ROCHE: Honoraria. Thieblemont:Bayer: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Letestu:Alexion: Consultancy, Honoraria. Cymbalista:Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Roche: Other: Travel, Accommodations, Expenses; AbbVie: Consultancy, Honoraria; Mundipharma: Honoraria.
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