Revisiting the initial diagnosis and blood staging of mycosis fungoides and Sézary syndrome with the KIR3DL2 marker.

BACKGROUND: The early diagnosis of Sézary syndrome (SS) is challenging. Loss of CD7 and CD26 expression on CD4+ T cells is the currently used criterion in the initial diagnosis and staging of patients with SS. OBJECTIVES: Our aim was to evaluate the respective value of CD26, CD7 and KIR3DL2 expression on CD4+ T cells and total lymphocytes at initial diagnosis of SS. METHODS: This prospective study included 254 patients with clinical features consistent with cutaneous T-cell lymphoma seen at our institution between March 2014 and February 2019. Peripheral blood analysis by flow cytometry was performed for each patient at the time of diagnosis and during follow-up. The diagnosis of SS was based on ISCL/EORTC criteria. RESULTS: The presence of KIR3DL2+ Sézary cells (SCs) ≥ 200 µL-1 correlated with the diagnosis of SS, with sensitivity of 88·6% and specificity of 96·3%. All 154 patients with either inflammatory skin disease or other haematological disease had KIR3DL2+ cells < 200 µL-1 , while eight of them had CD4+ CD26- T cells ≥ 1000 µL-1 . Of five patients with SS and lymphopenia, four had CD4+ CD7- T cells < 1000 µL-1 and three had CD4+ CD26- T cells < 1000 µL-1 . However, all of them had KIR3DL2+ CD4+ T cells ≥ 200 µL-1 . Among patients with available samples during evolution, all B1-staged patients with ≥ 200 µL-1 KIR3DL2+ SCs at diagnosis evolved to B2 stage within 7 months. CONCLUSIONS: KIR3DL2 expression on T cells is highly specific and helps the early diagnosis of SS, especially in those patients with lymphopenia. What's already known about this topic? In the ISCL/EORTC cutaneous T-cell lymphoma (CTCL) categorization of blood involvement (B0-B2), B2 is defined as a T-cell receptor clonal rearrangement in blood, associated with high blood-smear Sézary cell (SC) count. Flow cytometry was developed to circumvent interobserver variability of SC manual counts; however, it mostly relies on detection of cells lacking CD7 and/or CD26 expression. We previously reported the reliability of KIR3DL2 as the first positive SC marker. What does this study add? Based on our analysis of 254 patients, we propose that KIR3DL2 be added to the ISCL/EORTC criteria for initial diagnosis of Sézary syndrome (SS) and B2 staging. This marker improved sensitivity of SS B2-stage CTCL diagnosis with a specificity > 95%, especially for patients with lymphopenia. We found KIR3DL2 helped early diagnosis of SS and was more reliable than CD26 in assessing blood tumour burden during therapy. What is the translational message? SC quantification is the major means of staging at initial diagnosis and monitoring blood tumour burden in a clinical trials setting. We recommend using a threshold value of KIR3DL2+ SCs ≥ 200 µL-1 or KIR3DL2+ SCs/lymphocytes ≥ 10% in the diagnostic criteria of SS and propose a novel algorithm for CTCL B2 blood staging.

Bioactivity of hydrolysates obtained from bovine casein using artichoke (Cynara scolymus L.) proteases.

The objective of this work was to obtain casein hydrolysates with aspartic proteinases present in extracts from the artichoke flower (Cynara scolymus L.) and evaluate their antioxidant, antimicrobial, and angiotensin-I converting enzyme (ACE) inhibitory activity in vitro. The casein hydrolysates produced by the action of C. scolymus had elevated antihypertensive and antioxidant activity due to their high hydrophobic peptide content (93.84, 96.58, and 90.54% at 2, 4, and 16 h of hydrolysis, respectively). Hydrolysis time and molecular weight (<3 kDa) had a significant influence on the hypertensive and antioxidant activity of the hydrolysates, which were greater at hydrolysis times of 4 and 16 h and corresponding to the <3 kDa fractions. The <3 kDa fraction of the 16 h hydrolysate had an ACE inhibitory activity with a half-maximal inhibitory concentration (IC50) of 71.77 µg peptides per mL; DPPH and ABTS•+ radical scavenging activities of 6.27 µM and 6.21 mM Trolox equivalents per mg of peptides, respectively; and iron (II) chelation activity with an IC50 of 221.49 µg of peptides per mL. Antimicrobial activity against Enterococcus faecalis was also observed in the hydrolysates. From the peptide sequences identified in the hydrolysates, we detected 22 peptides (from the BIOPEP database) that were already in their bioactive form (AMKPWIQPK, AMKPWIQPKTKVIPYVRYL, ARHPHPHLSFM, DAQSAPLRVY, FFVAPFPEVFGK, GPVRGPFPII, KVLPVPQK, LLYQEPVLGPVRGPFPIIV, MAIPPKKNQDK, NLHLPLPLL, PAAVRSPAQILQ, RELEELNVPGEIVESLSSSEESITR, RPKHPIKHQ, RPKHPIKHQGLPQEVLNENLLRF, SDIPNPIGSENSEK, TPVVVPPFLQP, VENLHLPLPLL, VKEAMAPK, VLNENLLR, VYPFPGPIH, VYQHQKAMKPWIQPKTKVIPYVRY, VYQHQKAMKPWIQPKTKVIPYVRYL) and are reported to display antioxidant, antimicrobial, and ACE inhibitory activity. We also identified 12,116, 14,513, and 25,169 peptide sequences in the hydrolysates at 2, 4, and 16 h, respectively, that were contained in the primary sequence, and these are reported to display ACE inhibitory, antioxidant, dipeptidyl peptidase IV inhibition, antithrombotic, opioid, immunomodulation, antiamnesic, anticancer, chelating, and hemolytic bioactivity.

Long-term efficacy and safety of alemtuzumab in advanced primary cutaneous T-cell lymphomas.

BACKGROUND: Alemtuzumab has been proposed as salvage therapy for refractory cutaneous T-cell lymphomas (CTCLs). Long-term follow-up data are scarce. OBJECTIVES: To assess the efficacy and safety of alemtuzumab in the treatment of advanced CTCL. METHODS: A multicentre retrospective analysis was carried out of 39 patients with advanced CTCL treated with alemtuzumab between 2003 and 2013. RESULTS: Thirty-nine patients (median age 62 years, range 20-83) with Sézary syndrome (SS, n = 23) or advanced mycosis fungoides (MF, n = 16) received alemtuzumab 30 mg two to three times per week for a median duration of 12 weeks (range 1-35). Fifteen patients received maintenance therapy for a median duration of 24 weeks (range 6-277). Eleven patients (28%) had transformed disease (MF, n = 10; SS, n = 1). After a median follow-up of 24 months (range 0.3-124), eight patients (21%) were still alive. The overall response rate was 51% in the whole study group (partial response, n = 13; complete response, n = 7); 70% in patients with SS and 25% in patients with MF (P = 0.009). The median time to progression was 3.4 months (range 0.4-42). Six patients (15%; SS, n = 5; MF, n = 1) remained progression free for > 2 years (median 56 months, range 28-117). Five patients experienced cutaneous large T-cell transformation during alemtuzumab treatment and one patient developed primary cutaneous large B-cell lymphoma. Twenty-four patients (62%) had a grade three or higher infectious adverse event and 10 (26%) a haematological toxicity, which led to treatment discontinuation in 17 cases (44%) and death in two (5%). CONCLUSIONS: Alemtuzumab may induce long-term remission in SS but seems ineffective in MF and transformed CTCL.

Outcomes, infections, and immune reconstitution after double cord blood transplantation in patients with high-risk hematological diseases.

Double unrelated cord blood transplant (dUCBT) has been used to circumvent cell dose limitation of single UCBT; however, few data are available describing outcomes, infectious disease, and immune recovery. We analyzed 35 consecutive dUCBT recipients with high-risk malignant disorders (n=21) and bone marrow failure syndromes (n=14). Median follow-up was 32 months. Conditioning regimen was myeloablative in 14 and reduced intensity in 21 patients. Median infused nucleated cell dose was 4 × 10(7) /kg. Median time to absolute neutrophil count >0.5 × 10(9) /L was 25 days. Cumulative incidence (CI) of acute grade II-IV graft-versus-host disease was 47%. Estimated overall survival at 2 years was 48%. CI of first viral infections at 1 year was 92%. We observed 49 viral infections in 30 patients, 34 bacterial infections in 19 patients, and 16 fungal or parasitic infections in 12 patients. Lymphocyte subset analyses were performed at 3, 6, 9, and >12 months after dUCBT. Decreased T-cell and B-cell counts with expansion of natural killer cells were observed until 9 months post transplantation. Recovery of thymopoiesis measured by T-cell receptor excision circles was impaired until 9 months after dUCBT, when the appearance of new thymic precursors was observed. Delayed immune recovery and high incidence of infectious complications were observed after dUCBT in patients with high-risk hematological diseases.

Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.

Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

The MLL recombinome of acute leukemias in 2017.

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

Cyclin D2 dysregulation by chromosomal translocations to TCR loci in T-cell acute lymphoblastic leukemias.

Strong expression of at least one of the three D-type cyclins is common in human cancers. While the cyclin D1 and D3 genes (CCND1 and CCND3) are recurrently involved in genomic rearrangements, especially in B-cell lymphoid neoplasias, no clear involvement of the cyclin D2 gene (CCND2) has been reported to date. Here, we identified chromosomal translocations targeting the CCND2 locus at 12p13, and the T-cell receptor beta (TCRB) or the TCRA/D loci in T-cell acute lymphoblastic leukemias (T-ALLs). Expression analysis demonstrated dramatic cyclin D2 overexpression in the translocated cases (n=3) compared to other T-ALLs (total, n=89). In order to evaluate dysregulation in T-ALL with respect to normal T-cell differentiation, we analyzed CCND2 expression in normal purified human thymic subpopulations. CCND2 levels were downregulated through progression from the early stages of human T-cell differentiation, further suggesting that the massive and sustained expression in the CCND2-rearranged T-ALL cases was oncogenic. Association with other oncogene expression (TAL1, HOXAs, or TLX3/HOX11L2), NOTCH1 activating mutations, and/or CDKN2A/p16/ARF deletion, showed that cyclin D2 dysregulation could contribute to multi-event oncogenesis in various T-ALL groups. This report is the first clear evidence of a direct involvement of cyclin D2 in human cancer due to recurrent somatic genetic alterations.

Mutation status and clinical outcome of 89 imatinib mesylate-resistant chronic myelogenous leukemia patients: a retrospective analysis from the French intergroup of CML (Fi(phi)-LMC GROUP).

The emergence of ABL point mutations is the most frequent cause for imatinib resistance in chronic myelogenous leukemia (CML) patients and can occur during any phase of the disease; however, their clinical impact remains controversial. In this study, we retrospectively analyzed the predictive impact of 94 BCR-ABL kinase domain mutations (18 T315I, 26 P-loop, 50 in other sites) found in 89 imatinib-resistant CML patients. At imatinib onset, 64% of patients (57/89) were in chronic phase (CP), 24% (21/89) in accelerated phase (AP) and 12% (11/89) in blastic phase (BP). T315I and P-loop mutations were preferentially discovered in accelerated phase of BP CML, and other types of mutations in CP (P=0.003). With a median follow-up of 39.2 months (6.3-67.2), since imatinib initiation, overall survival (OS) was significantly worse for P-loop (28.3 months) and for T315I (12.6 months), and not reached for other mutations (P=0.0004). For CP only, multivariate analysis demonstrated a worse OS for P-loop mutations (P=0.014), and a worse progression-free survival (PFS) for T315I mutations (P=0.014). Therefore, P-loop and T315I mutations selectively impair the outcome of imatinib-resistant CML patients, in contrast to other mutations, which may benefit from dose escalation of imatinib, able to improve or stabilize disease response.

Allogeneic stem cell transplantation improves the outcome of adults with t(1;19)/E2A-PBX1 and t(4;11)/MLL-AF4 positive B-cell acute lymphoblastic leukemia: results of the prospective multicenter LALA-94 study.

Adult patients with acute lymphoblastic leukemia (ALL) and t(1;19)/E2A-PBX1 or t(4;11)/MLL-AF4 have a poor outcome. We have evaluated the impact of an intensified post-remission therapy using a high-dose chemotherapy course followed by allogeneic or autologous SCT on the outcome of 58 patients with t(1;19)/E2A-PBX1 (E2A group, n=24) or t(4;11)/MLL-AF4 (MLL group, n=34) treated in the LALA-94 multicenter prospective study. Patients in the MLL group had higher WBC counts and more frequent DIC. CR rates achieved by MLL and E2A groups were similar to other B-cell ALL (87, 82 and 86% respectively). While in CR, patients with a donor were assigned to alloSCT (n=22), the remaining patients with were randomized between autoSCT (n=15) or chemotherapy (n=8). Five-year overall survival was 31 and 45% for E2A and MLL groups, respectively. In both groups, DFS was higher in the alloSCT arm as compared to autoSCT and chemotherapy arms. The results of this study show that chemotherapy intensification did not overcome the poor prognosis of adults with t(1;19)/E2A-PBX1. Allogeneic SCT should thus be offered in first CR to patients with t(1;19)/E2A-PBX1 or t(4;11)/MLL-AF4. New therapeutic approaches are needed for patients without donor.

Prognostic implication of FLT3 and Ras gene mutations in patients with acute promyelocytic leukemia (APL): a retrospective study from the European APL Group.

Internal tandem duplications (ITDs) of the FLT3 gene have been observed in about 35% of APL cases. If FLT3-ITD is associated with a worse outcome in patients with acute myeloid leukemia (AML) in general, its prognostic value in acute promyelocytic leukemia (APL) is still a matter of debate. We investigated incidence, associated clinical features, and prognostic implication of FLT3-ITD, but also FLT3-D835 point mutation and N-Ras or K-Ras mutations in 119 APL patients, all prospectively enrolled in the two consecutive APL-93 and APL-2000 trials. Mutation incidences were 38, 20, and 4%, for FLT3-ITD, FLT3-D835, and Ras, respectively. The presence of FLT3-ITD was associated with high white blood cell count, high Sanz index, M3-variant subtype, and V/S PML-RAR alpha isoforms. Complete remission (CR), induction death, and death in CR rates were not affected by FLT3 or Ras mutations, as well as cumulative incidence of relapse. However, a trend for a shorter overall survival (P=0.09) was observed in FLT3-ITD patients, because of a very poor postrelapse survival (P=0.02). This feature, which has been also reported in patients with AML in general, is suggestive of an underlying genetic instability in FLT3-ITD patients, leading to the acquisition of additional unknown bad-prognosis gene mutations at relapse.

Germ-line deletion involving the INK4 locus in familial proneness to melanoma and nervous system tumors.

Joint predisposition to malignant melanoma and nervous system tumors (NSTs) is a puzzle. Several melanoma susceptibility genes have been identified, including p16, a clustered tumor suppressor. However, the molecular bases of inherited proclivity to NSTs in the absence of a recognizable genetic syndrome are unknown. We analyzed two families with joint proneness to melanoma and NSTs in view of genetic linkage and identification of the causal molecular lesions. Highly informative linkage markers were used for segregation analyses of the predisposition alleles in the two pedigrees. Characterization of the molecular lesions required hemizygosity mapping based on microsatellite markers physically mapped to contigs of the 9p21 region and a Southern blot approach using several PCR-generated probes. Both families were found to be allelic and linked to p16 markers. In the family segregating the melanoma/NST syndrome, a large germ-line deletion ablated the whole p16, p19, and p15 gene cluster (or INK4 locus), whereas a more circumscribed molecular lesion disrupting p16 and p19 but leaving p15 unaltered segregated with the melanoma-astrocytoma syndrome (MIM 155755). Our results suggest that multiple cancer susceptibility in these two families ensues from contiguous tumor suppressor gene deletion. Indeed, known phenotypes associated with germ-line p16 mutations and an apparent correlation between the deletion span and tumor spectrum in the two families suggest a new model of cancer pathogenesis based on the inactivation of contiguous tumor suppressor genes, an alternative to the established pleiotropic effects of single-gene disruption.

Evaluation of minimal residual disease using reverse-transcription polymerase chain reaction in t(8;21) acute myeloid leukemia: a multicenter study of 51 patients.

PURPOSE: Most studies using various reverse-transcription polymerase chain reaction (RT-PCR) techniques reported that the detection of the AML1-ETO fusion transcript was a common finding in long-term complete remission (CR) in acute myeloid leukemia (AML) with t(8;21) translocation. However, larger prospective studies with interlaboratory quality control may be important to investigate more precisely the clinical usefulness of studying minimal residual disease with RT-PCR in t(8;21) AML. PATIENTS AND METHODS: We collected 223 marrow samples from 51 patients with t(8;21) AML diagnosed in five centers and tested all samples by two different RT-PCR techniques (a nested technique and a one-step technique, with a sensitivity of 10(-6) and 10(-5), respectively) in two different laboratories. RESULTS: Samples from 14 patients in long persistent CR (median follow-up duration, 112 months) were taken at least twice, and all were PCR-negative by both techniques. Samples were prospectively taken from 37 patients after achievement of first CR and/or second CR, before intensive consolidation treatment, and every 3 to 6 months after completion of therapy. Patients who converted to PCR negativity with the one-step technique (60%) or both techniques (48%) after CR achievement had a longer CR duration than those with persistently positive PCR results (two-sided log-rank test, P =.0001). Patients who became PCR-negative with the one-step technique before intensive consolidation (23%) had a lower relapse rate (11% v 72%) and a longer CR duration than those who remained persistently PCR-positive at that point (two-sided log-rank test, P =.0015). CONCLUSION: Patients with AML with t(8;21) in long-term remission were all PCR-negative. In prospectively studied patients, a good correlation was found between negative PCR results and absence of relapse. Early negative results with the one-step RT-PCR technique, before consolidation treatment, seemed to carry an especially good prognosis, suggesting that RT-PCR analysis could help in choosing the type of consolidation therapy in patients with t(8;21) AML.