Within the past 2 years, transcription activator-like effector (TALE) DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. ...However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural, and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free, and universal method to overcome it.
Background: TALE-based technologies are poised to revolutionize the field of biotechnology; however, their sensitivity to cytosine methylation may drastically restrict their ranges of applications.
Results: TALE repeat N* proficiently accommodates 5-methylated cytosine.
Conclusion: Sensitivity of TALE to cytosine methylation can be overcome by using TALE repeat N*.
Significance: Utilization of TALE repeat N* enables broadening the scope of TALE-based technologies.
ALK-negative anaplastic large cell lymphoma (ALCL) comprises subgroups harboring rearrangements of DUSP22 (DUSP22- R) or TP63 (TP63-R). Two studies reported 90% and 40% 5-year overall survival (OS) ...rates in 21 and 12 DUSP22-R/TP63- not rearranged (NR) patients, respectively, making the prognostic impact of DUSP22-R unclear. Here, 104 newly diagnosed ALK-negative ALCL patients (including 37 from first-line clinical trials) from the LYSA TENOMIC database were analyzed by break-apart fluorescence in situ hybridization assays for DUSP22-R and TP63-R. There were 47/104 (45%) DUSP22-R and 2/93 (2%) TP63-R cases, including one DUSP22-R/TP63-R case. DUSP22-R tumors more frequently showed CD3 expression (62% vs. 35%, P=0.01), and less commonly a cytotoxic phenotype (27% vs. 82%; P<0.001). At diagnosis, DUSP22- R ALCL patients more frequently had bone involvement (32% vs. 13%, P=0.03). The patient with DUSP22-R/TP63-R ALCL had a rapidly fatal outcome. After a median follow-up of 4.9 years, 5-year progression-free survival (PFS) and OS rates of 84 patients without TP63-R treated with curative-intent anthracycline-based chemotherapy were 41% and 53%, respectively. According to DUSP22 status, 5-year PFS was 57% for 39 DUSP22-R versus 26% for 45 triple-negative (DUSP22-NR/TP63-NR/ALK-negative) patients (P=0.001). The corresponding 5-year OS rates were 65% and 41%, respectively (P=0.07). In multivariate analysis, performance status and DUSP22 status significantly affected PFS, and distinguished four risk groups, with 4-year PFS and OS ranging from 17% to 73% and 21% to 77%, respectively. Performance status but not DUSP22 status influenced OS. The use of brentuximab vedotin in relapsed/refractory patients improved OS independently of DUSP22 status. Our findings support the biological and clinical distinctiveness of DUSP22- R ALK-negative ALCL. Its relevance to outcome in patients receiving frontline brentuximab vedotin remains to be determined.
Peripheral T-cell lymphoma comprises a heterogeneous group of mature non-Hodgkin lymphomas. Their diagnosis is challenging, with up to 30% of cases remaining unclassifiable and referred to as "not ...otherwise specified". We developed a reverse transcriptase-multiplex ligation-dependent probe amplification gene expression profiling assay to differentiate the main T-cell lymphoma entities and to study the heterogeneity of the "not specified" category. The test evaluates the expression of 20 genes, including 17 markers relevant to T-cell immunology and lymphoma biopathology, one Epstein-Barr virus-related transcript, and variants of
(G17V) and
(R172K/T). By unsupervised hierarchical clustering, our assay accurately identified 21 of 21 ALK-positive anaplastic large cell lymphomas, 16 of 16 extranodal natural killer (NK)/T-cell lymphomas, 6 of 6 hepatosplenic T-cell lymphomas, and 13 of 13 adult T-cell leukemia/lymphomas. ALK-negative anaplastic lymphomas (n=34) segregated into one cytotoxic cluster (n=10) and one non-cytotoxic cluster expressing Th2 markers (n=24) and enriched in
-rearranged cases. The 63 T
-derived lymphomas divided into two subgroups according to a predominant T
(n=50) or an enrichment in Th2 (n=13) signatures. We next developed a support vector machine predictor which attributed a molecular class to 27 of 77 not specified T-cell lymphomas: 17 T
, five cytotoxic ALK-negative anaplastic and five NK/T-cell lymphomas. Among the remaining cases, we identified two cell-of-origin subgroups corresponding to cytotoxic/Th1 (n=19) and Th2 (n=24) signatures. A reproducibility test on 40 cases yielded a 90% concordance between three independent laboratories. This study demonstrates the applicability of a simple gene expression assay for the classification of peripheral T-cell lymphomas. Its applicability to routinely-fixed samples makes it an attractive adjunct in diagnostic practice.
Xeroderma pigmentosum group C (XP-C) is a rare human syndrome characterized by hypersensitivity to UV light and a dramatic predisposition to skin neoplasms. XP-C cells are deficient in the nucleotide ...excision repair (NER) pathway, a complex process involved in the recognition and removal of DNA lesions. Several XPC mutations have been described, including a founder mutation in North African patients involving the deletion of a TG dinucleotide (ΔTG) located in the middle of exon 9. This deletion leads to the expression of an inactive truncated XPC protein, normally involved in the first step of NER. New approaches used for gene correction are based on the ability of engineered nucleases such as Meganucleases, Zinc-Finger nucleases or TALE nucleases to accurately generate a double strand break at a specific locus and promote correction by homologous recombination through the insertion of an exogenous DNA repair matrix. Here, we describe the targeted correction of the ΔTG mutation in XP-C cells using engineered meganuclease and TALEN™. The methylated status of the XPC locus, known to inhibit both of these nuclease activities, led us to adapt our experimental design to optimize their in vivo efficacies. We show that demethylating treatment as well as the use of TALEN™ insensitive to CpG methylation enable successful correction of the ΔTG mutation. Such genetic correction leads to re-expression of the full-length XPC protein and to the recovery of NER capacity, attested by UV-C resistance of the corrected cells. Overall, we demonstrate that nuclease-based targeted approaches offer reliable and efficient strategies for gene correction.
Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by ...germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.
IDH1 and IDH2 somatic mutations have been identified in solid tumors and blood malignancies. The development of inhibitors of mutant IDH1 and IDH2 in the past few years has prompted the development ...of a fast and sensitive assay to detect IDH1R132, IDH2R140 and IDH2R172 mutations to identify patients eligible for these targeted therapies. This study aimed to compare two new multiplexed PCR assays – an automated quantitative PCR (qPCR) on the PGX platform and a droplet digital PCR (ddPCR) with next‐generation sequencing (NGS) for IDH1/2 mutation detection. These assays were evaluated on 102 DNA extracted from patient peripheral blood, bone marrow and formalin‐fixed paraffin‐embedded tissue samples with mutation allelic frequency ranging from 0.6% to 45.6%. The ddPCR assay had better analytical performances than the PGX assay with 100% specificity, 100% sensitivity and a detection limit down to 0.5% on IDH1R132, IDH2R140 and IDH2R172 codons, and a high correlation with NGS results. Therefore, the new highly multiplexed ddPCR is a fast and cost‐effective assay that meets most clinical needs to identify and follow cancer patients in the era of anti‐IDH1/2‐targeted therapies.
This study presents the first evaluation of two new highly multiplexed PCR assays as compared with next‐generation sequencing (NGS): an automated qPCR on the PGX platform and an allele‐specific droplet digital PCR (ddPCR) assay, both designed to detect most IDH1 and IDH2 hotspot mutations. The ddPCR assay was the best assay in terms of sensitivity, specificity, robustness, cost and timeliness, regardless of patient sample processing. It is, therefore, an appealing alternative to NGS to screen patients eligible for IDH1/2 targeted therapies.
Summary
Background
Primary cutaneous peripheral T‐cell lymphomas with a T‐follicular helper phenotype (pcTFH‐PTCL) are poorly characterized, and often compared to, but not corresponding with, mycosis ...fungoides (MF), Sézary syndrome, primary cutaneous CD4+ lymphoproliferative disorder, and skin manifestations of angioimmunoblastic T‐cell lymphomas (AITL).
Objectives
We describe the clinicopathological features of pcTFH‐PTCL in this original series of 23 patients, and also characterize these cases molecularly.
Methods
Clinical and histopathological data of the selected patients were reviewed. Patient biopsy samples were also analysed by targeted next‐generation sequencing.
Results
All patients (15 men, eight women; median age 66 years) presented with skin lesions, without systemic disease. Most were stage T3b, with nodular (n = 16), papular (n = 6) or plaque (atypical for MF, n = 1) lesions. Three (13%) developed systemic disease and died of lymphoma. Nine (39%) patients received more than one line of chemotherapy. Histologically, the lymphomas were CD4+ T‐cell proliferations, usually dense and located in the deep dermis (n = 14, 61%), with the expression of at least two TFH markers (CD10, CXCL13, PD1, ICOS, BCL6), including three markers in 16 cases (70%). They were associated with a variable proportion of B cells. Eight patients were diagnosed with an associated B‐cell lymphoproliferative disorder (LPD) on biopsy, including Epstein–Barr virus (EBV)‐positive diffuse large B‐cell lymphoma (n = 3), EBV+ LPD (n = 1) and monotypic plasma cell LPD (n = 4). Targeted sequencing showed four patients to have a mutated TET2–RHOAG17V association (as frequently seen in AITL) and another a TET2/DNMT3A/PLCG1/SETD2 mutational profile. The latter patient, one with a TET2–RHOA association, and one with no detected mutations, developed systemic disease and died. Five other patients showed isolated mutations in TET2 (n = 1), PLCG1 (n = 2), SETD2 (n = 1) or STAT5B (n = 1).
Conclusions
Patients with pcTFH‐PTCL have pathological and genetic features that overlap with those of systemic lymphoma of TFH derivation. Clinically, most remained confined to the skin, with only three patients showing systemic spread and death. Whether pcTFH‐PTCL should be integrated as a new subgroup of TFH lymphomas in future classifications is still a matter of debate.
What is already known about this topic?
There is a group of cutaneous lymphomas that express T‐follicular helper (TFH) markers that do not appear to correspond to existing World Health Organization diagnostic entities.
These include mycosis fungoides, Sézary syndrome, or primary cutaneous CD4+ small/medium‐sized T‐cell lymphoproliferative disorder or cutaneous extensions of systemic peripheral T‐cell lymphomas (PTCL) with TFH phenotype.
What does this study add?
This is the first large original series of patients with a diagnosis of primary cutaneous PTCL with a TFH phenotype (pcTFH‐PTCL) to be molecularly characterized.
pcTFH‐PTCL may be a standalone group of cutaneous lymphomas with clinicopathological and molecular characteristics that overlap with those of systemic TFH lymphomas, such as angioimmunoblastic T‐cell lymphoma, and does not belong to known diagnostic groups of cutaneous lymphoma.
This has an impact on the treatment and follow‐up of patients; the clinical behaviour needs to be better clarified in further studies to tailor patient management.
There is a group of cutaneous lymphomas that express TFH markers that do not appear to correspond to existing WHO diagnostic entities. This is the first large original series of patients with a diagnosis of primary cutaneous peripheral T‐cell lymphomas with a T‐follicular helper phenotype (pcTFH‐PTCL) to be molecularly characterised. pcTFH‐PTCL may be a standalone group of cutaneous lymphomas with clinicopathological and molecular characteristics that overlap with those of systemic TFH lymphomas, and does not belong to known diagnostic groups of cutaneous lymphoma.
Linked Comment: W. Kempf. Br J Dermatol 2022; 187:841–842.
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