Claudia Brunetti

Alterations in the antigen processing-presenting machinery of transformed plasma cells are associated with reduced recognition by CD8+ T cells and characterize the progression of MGUS to multiple myeloma.

We hypothesized that progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) reflects the escape of transformed plasma cells from T-cell recognition because of impaired antigen processing-presenting machinery (APM). We studied plasma cells and CD8(+) T cells from bone marrow of 20 MGUS patients, 20 MM patients, and 10 control patients. Immunofluorescence and flow cytometry revealed significantly different patterns of APM component expression in plasma cells from the 3 groups. Compared with control patients, MM samples had lower expression of proteasome subunits and peptide transporters and greater expression of chaperones, considering both percentages of stained cells and molecular equivalents of soluble fluorochrome. MGUS samples had intermediate percentages of stained cells but molecular equivalents of soluble fluorochrome similar to control patients. Real-time polymerase chain reaction documented that APM changes occurred at the transcriptional level. Cytotoxicity assays demonstrated that MGUS CD8(+) T cells lysed autologous transformed plasma cells more than MM CD8(+) T cells did. MGUS progression correlated directly with calnexin, calreticulin, and tapasin and indirectly with delta, LMP2, and LMP10 expression levels; MM disease status did not correlate with APM levels. APM changes may allow transformed plasma cells to elude immunesurveillance in the MGUS-MM pathogenetic sequence.

Antibody Vh Repertoire Differences between Resolving and Chronically Evolving Hepatitis C Virus Infections

Despite the production of neutralizing antibodies to hepatitis C virus (HCV), many patients fail to clear the virus and instead develop chronic infection and long-term complications. To understand how HCV infection perturbs the antibody repertoire and to identify molecular features of antibody genes associated with either viral clearance or chronic infection, we sequenced the V(D)J region of naïve and memory B cells of 6 persons who spontaneously resolved an HCV infection (SR), 9 patients with a newly diagnosed chronically evolving infection (CE), and 7 healthy donors. In both naïve and memory B cells, the frequency of use of particular antibody gene subfamilies and segments varied among the three clinical groups, especially between SR and CE. Compared to CE, SR antibody genes used fewer VH, D and JH gene segments in naïve B cells and fewer VH segments in memory B cells. SR and CE groups significantly differed in the frequency of use of 7 gene segments in naïve B cell clones and 3 gene segments in memory clones. The nucleotide mutation rates were similar among groups, but the pattern of replacement and silent mutations in memory B cell clones indicated greater antigen selection in SR than CE. Greater clonal evolution of SR than CE memory B cells was revealed by analysis of phylogenetic trees and CDR3 lengths. Pauciclonality of the peripheral memory B cell population is a distinguishing feature of persons who spontaneously resolved an HCV infection. This finding, previously considered characteristic only of patients with HCV-associated lymphoproliferative disorders, suggests that the B cell clones potentially involved in clearance of the virus may also be those susceptible to abnormal expansion.

Bone Marrow of Persistently Hepatitis C Virus-Infected Individuals Accumulates Memory CD8+ T Cells Specific for Current and Historical Viral Antigens: A Study in Patients with Benign Hematological Disorders

The role of virus-specific T cells in hepatitis C virus (HCV) pathogenesis is not clear. Existing knowledge on the frequency, phenotype, and behavior of these cells comes from analyses of blood and liver, but other lymphoid compartments that may be important sites for functionally mature T cells have not yet been analyzed. We studied HCV-specific T cells from bone marrow, in comparison to those from peripheral blood and liver biopsy tissue, from 20 persistently HCV-infected patients with benign hematological disorders. Bone marrow contained a sizeable pool of CD8+ T cells specific for epitopes from structural and nonstructural HCV proteins. These cells displayed the same effector memory phenotype as liver-derived equivalents and the same proliferative potential as blood-derived equivalents but had greater antiviral effector functions such as Ag-specific cytotoxicity and IFN-γ production. These features were not shared by influenza virus-specific CD8+ T cells in the same bone marrow samples. Despite their highly differentiated phenotype and activated status, some bone marrow-resident HCV-specific CD8+ T cells were not directed against the infecting virus but, instead, against historical HCV Ags (i.e., viral species of a previous infection or minor viral species of the current infection). These findings provide a snapshot view of the distribution, differentiation, and functioning of virus-specific memory T cells in patients with persistent HCV infection.

Droplet digital PCR analysis of NOTCH1 gene mutations in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), NOTCH1 gene mutations (NOTCH1mut) have been associated with adverse prognostic features but the independence of these as a prognostic factor is still controversial. In our study we validated a c.7541-7542delCT NOTCH1 mutation assay based on droplet digital PCR (ddPCR); we also analyzed the NOTCH1mut allelic burden, expressed as fractional abundance (FA), in 88 CLL patients at diagnosis to assess its prognostic role and made a longitudinal ddPCR analysis in 10 cases harboring NOTCH1mut to verify the FA variation over time. Our data revealed that with the ddPCR approach the incidence of NOTCH1mut in CLL was much higher (53.4%) than expected. However, longitudinal ddPCR analysis of CLL cases showed a statistically significant reduction of the NOTCH1mut FA detected at diagnosis after treatment (median FA 11.67 % vs 0.09 %, respectively, p = 0.01); the same difference, in terms of NOTCH1mut FA, was observed in the relapsed cases compared to the NOTCH1mut allelic fraction observed in patients in complete or partial remission (median FA 4.75% vs 0.43%, respectively, p = 0.007). Our study demonstrated a much higher incidence of NOTCH1mut in CLL than has previously been reported, and showed that the NOTCH1mut allelic burden evaluation by ddPCR might identify patients in need of a closer clinical follow-up during the “watch and wait” interval and after standard chemotherapy.

Droplet digital PCR assay for quantifying of CALR mutant allelic burden in myeloproliferative neoplasms

Dear Editor, Calreticulin (CALR) gene mutations (CALR) have recently been discovered in about 20–35 % of patients affected by essential thrombocythemia (ET) and primarymyelofibrosis (PMF) [1, 2]. Several molecular assays have been developed to detect the most frequentCALR (type 1 consisting of a 52bp deletion, and type 2 of a 5-bp insertion) [3, 4]. All these techniques are useful for identifyingCALR at the diagnosis, but they are not suitable for minimal residual disease (MRD) monitoring, since the maximum sensitivity is 1 %. The droplet digital PCR (ddPCR) technology is a third-generation PCR method that started to be used in hematological malignancies [5–7].We describe a ddPCR assay with a sensitivity of 0.01% developed for the absolute quantification of CALR type 1 and 2 mutations and analyze a cohort of 57 JAK2V617F-negative myeloproliferative neoplasm patients. ddPCR experiments were performed using the QX-200 instrument (BioRad) and specific primers and probes were designed for both type 1 and type 2 mutations (see Supplementary Files). CALR load in each sample was expressed as fractional abundance (FA, mutant allele/mutant allele + wild-type allele). The CALR allelic burden resulted heterogeneous in both ET (min.13.8 %– max. 51%) and PMF (min. 34.5%–max. 51.3%) patients.We show that the medianCALR allelic burden at diagnosis was significantly higher in PMF patients as compared to ET case (47.9 vs 43.8 %, p = 0.008) whereas no significant difference was observed between the type 1 and 2 mutations (Fig. 1a). Moreover, no relationship between the genemutation type and the CALR amount was observed within each group of ET and PMF patients. In our ET series, there were 14 (29.7 %) patients with a very low FA, <30 %; this group was not statistically different in terms of hemoglobin, white blood cells and platelet counts, age, sex, thrombosis and/or hemorrhage, and CALR type compared to those with FA >30 %. The PMF group included ten patients, too few for any type of statistical considerations. Sequential evaluations by ddPCR experiments were performed in three patients to monitor the CALR load during treatment. CALR load at diagnosis was 15.8 and 48 % in two ET patients. The former patient was treated with interferon-α (IFN-α) and after 5 years from diagnosis the FA was 7.7 %. The latter was also treated with IFN-α and after 2 years from diagnosis, the CALR load was 14.7 %. Both patients had stable disease and a well-controlled platelet count. A 44-year-old man at PMF diagnosis showed a FA of 49.7 %; 8 years later, we observed a leukemic transformation. At the time of the AML evolution, theCALR load was 0 %; this finding was also confirmed by PCR qualitative analysis. The patient underwent induction chemotherapy, achieving complete remission, then allogeneic bone marrow transplantation (ABMT) from a HLA-matched related donor. Two months later, the FA observed by ddPCR analysis was 0.01 %; 7 months after, the ABMT and AML relapsed and at this time, the CALR load was 13.5 % (Fig. 1b, c). Although the importance of the CALR allelic burden determination has not yet been defined at the disease onset, the utility of and need for a sensitive method, like our ddPCR assay, are unquestionable for the purposes of MRD monitoring [8–10]. Electronic supplementary material The online version of this article (doi:10.1007/s00277-016-2739-2) contains supplementary material, which is available to authorized users.

Overexpression of the LSAMP and TUSC7 genes in acute myeloid leukemia following microdeletion/duplication of chromosome 3

The 3q13.31 microdeletion syndrome is characterized by developmental delay, postnatal growth above the mean, characteristic facial features, and abnormal male genitalia. Moreover, a frequent deletion in the 3q13.31 chromosome region has been identified in patients who are affected by osteosarcomas. Among the genes located within the deleted region, the involvement of the limbic system-associated membrane protein gene (LSAMP), together with a non-coding RNA tumor suppressor candidate 7 gene (TUSC7), has been suggested. We describe the case of an adult acute myeloid leukemia (AML) patient with a novel chromosomal rearrangement characterized by a 3q13.31 microdeletion and an extra copy of the 3q13.31-q29 chromosomal region translocated to the long arm of the Y chromosome. This karyotypic aberration seems to cause LSAMP and TUSC7 gene expression dysregulation. In conclusion, we report the first case of LSAMP and TUSC7 gene overexpression, possibly due to a position effect in an AML patient bearing a 3q13.31 cryptic deletion.

Droplet Digital PCR Is a Reliable Tool for Monitoring Minimal Residual Disease in Acute Promyelocytic Leukemia

Nested RT-PCR (nPCR) and real-time quantitative PCR (qPCR) are well-established methods for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL). Despite their remarkable sensitivity and specificity, both methods have inherent limitations, such as qualitative MRD evaluation and relative quantification. Herein, we used droplet digital PCR (ddPCR) to monitor MRD in 21 APL patients and compared its performance with nPCR and qPCR. After assessing the limit of detection (LOD) for each technique on serial dilutions of PML-RARA bcr1 and bcr3 transcripts, a total of 48 follow-up samples were analyzed and the results compared. ddPCR showed good linearity and efficiency and reached an LOD comparable or even superior to nPCR and qPCR. When tested on primary samples, ddPCR exhibited a sensitivity and specificity of ≥95% and ≥91% for bcr1 and bcr3 transcripts and displayed a significant concordance with both techniques, particularly with nPCR. The peculiar advantage of ddPCR-based monitoring of MRD is represented by absolute quantification, which provides crucial information for the management of patients whose MRD fluctuates under the LOD of qPCR and is detectable, but not quantifiable, by nPCR. Our findings highlight ddPCR as a reliable complementary approach to monitor MRD in APL, and suggest its advantageous application, particularly for the molecular follow-up of patients at high risk of relapse.

5'RUNX1-3'USP42 chimeric gene in acute myeloid leukemia can occur through an insertion mechanism rather than translocation and may be mediated by genomic segmental duplications

BackgroundThe runt-related transcription factor 1 (RUNX1) gene is a transcription factor that acts as a master regulator of hematopoiesis and represents one of the most frequent targets of chromosomal rearrangements in human leukemias. The t(7;21)(p22;q22) rearrangement generating a 5‘RUNX1-3’USP42 fusion transcript has been reported in two cases of pediatric acute myeloid leukemia (AML) and further in eight adult cases of myeloid neoplasms. We describe the first case of adult AML with a 5‘RUNX1-3’USP42 fusion gene generated by an insertion event instead of chromosomal translocation.MethodsConventional and molecular cytogenetic analyses allowed the precise characterization of the chromosomal rearrangement and breakpoints identification. Gene expression analysis was performed by quantitative real-time PCR experiments, whereas bioinformatic studies were carried out for revealing structural genomic characteristics of breakpoint regions.ResultsWe identified an adult AML case bearing a ins(21;7)(q22;p15p22) generating a 5‘RUNX1-3’USP42 fusion gene on der(21) chromosome and causing USP42 gene over-expression. Bioinformatic analysis of the genomic regions involved in ins(21;7)/t(7;21) showed the presence of interchromosomal segmental duplications (SDs) next to the USP42 and RUNX1 genes, that may underlie a non-allelic homologous recombination between chromosome 7 and 21 in AML.ConclusionsWe report the first case of a 5‘RUNX1-3’USP42 chimeric gene generated by a chromosomal cryptic insertion in an adult AML patient. Our data revealed that there may be a pivotal role for SDs in this very rare but recurrent chromosomal rearrangement.

Genomic BCR-ABL1 breakpoint characterization by a multi-strategy approach for “personalized monitoring” of residual disease in chronic myeloid leukemia patients

For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology. Finally, after defining the BCR-ABL1 genomic junction, we performed the target CML patient–specific quantification, using droplet digital PCR (ddPCR). FISH and MinION steps, respectively, together with ddPCR analysis, greatly reduce the complexity that has impeded the use of “personalized monitoring” of CML in clinical practice. Our report suggests a feasible pipeline, in terms of costs and reproducibility, aimed at characterizing and quantifying the genomic BCR-ABL1 rearrangement during MRD monitoring in CML patients.

Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology

We report a third-generation sequencing assay on nanopore technology (MinION) for detecting BCR-ABL1 KD mutations and compare the results to a Sanger sequencing(SS)-based test in 24 Philadelphia-positive (Ph+) leukemia cases. Our data indicates that MinION is markedly superior to SS in terms of sensitivity, costs and timesaving, and has the added advantage of determining the clonal configuration of multiple mutations. We demonstrate that MinION is suitable for employment in the hematology laboratory for detecting BCR-ABL1 KD mutation in Ph+ leukemias.