SummaryThis study aimed to determine the incidence/prognostic impact of TP53 mutation in 318 myelodysplastic syndrome (MDS) patients, and to correlate the changes to cytogenetics, single nucleotide polymorphism array karyotyping and clinical outcome. The median age was 65 years (17-89 years) and median follow-up was 45 months [95% confidence interval (CI) 27-62 months]. TP53 mutations occurred in 30 (9Á4%) patients, exclusively in isolated del5q (19%) and complex karyotype (CK) with -5/5q-(72%), correlated with International Prognostic Scoring System intermediate-2/high, TP53 protein expression, higher blast count and leukaemic progression. Patients with mutant TP53 had a paucity of mutations in other genes implicated in myeloid malignancies. Median overall survival of patients with TP53 mutation was shorter than wild-type (9 versus 66 months, P < 0Á001) and it retained significance in multivariable model (Hazard Ratio 3Á8, 95% CI 2Á3-6Á3,P < 0Á001). None of the sequentially analysed samples showed a disappearance of the mutant clone or emergence of new clones, suggesting an early occurrence of TP53 mutations. A reduction in mutant clone correlated with response to 5-azacitidine, however clones increased in nonresponders and persisted at relapse. The adverse impact of TP53 persists after adjustment for cytogenetic risk and is of practical importance in evaluating prognosis. The relatively common occurrence of these mutations in two different prognostic spectrums of MDS, i.e. isolated 5q-and CK with -5/5q-, possibly implies two different mechanistic roles for TP53 protein.

Key Points• Acquired mutations of myeloid-related genes are present in a proportion of AA patients.• Somatic mutations in AA predict higher risk of transformation to MDS.The distinction between acquired aplastic anemia (AA) and hypocellular myelodysplastic syndrome (hMDS) is often difficult, especially nonsevere AA. We postulated that somatic mutations are present in a subset of AA, and predict malignant transformation. From our database, we identified 150 AA patients with no morphological evidence of MDS, who had stored bone marrow (BM) and constitutional DNA. We excluded Fanconi anemia, mutations of telomere maintenance, and a family history of BM failure (BMF) or cancer. The initial cohort of 57 patients was screened for 835 known genes associated with BMF and myeloid cancer; a second cohort of 93 patients was screened for mutations in ASXL1, DNMT3A, BCOR, TET2, and MPL. Somatic mutations were detected in 19% of AA, and included ASXL1 (n 5 12), DNMT3A (n 5 8) and BCOR (n 5 6). Patients with somatic mutations had a longer disease duration (37 vs 8 months, P < .04), and shorter telomere lengths (median length, 0.9 vs 1.1, P < .001), compared with patients without mutations. Somatic mutations in AA patients with a disease duration of >6 months were associated with a 40% risk of transformation to MDS (P < .0002). Nearly one-fifth of AA patients harbor mutations in genes typically seen in myeloid malignancies that predicted for later transformation to MDS. (Blood. 2014;124(17):2698-2704

These findings provide a comprehensive assessment of genetic abnormalities in CTCL and a rational approach for further studies.

Sézary syndrome (SS) is a rare form of erythrodermic cutaneous T-cell lymphoma with hematological involvement and a poor prognosis. At present little is known about the molecular pathogenesis of this malignancy. To address this issue, we analyzed 28 SS cases through the use of molecular cytogenetic techniques. Conventional cytogenetic analysis showed 12 of 28 cases with clonal chromosome abnormalities (43%). Seven cases had aberrations affecting chromosomes 1 and 17; five demonstrated rearrangement of chromosomes 10 and 14; four presented with an abnormality of 6q. Multiplex-fluorescence in situ hybridization (M-FISH) revealed complex karyotypes in 6 of 17 cases (35%), and recurrent der(1)t(1;10)(p2;q2) and der(14)t(14;15)(q;q?) translocations were each identified in two cases, and confirmed by dual-color FISH. There was an overall difference in the incidence of clonal abnormalities detected by G-banded karyotyping and M-FISH. In addition, comparative genomic hybridization studies revealed chromosome imbalances (CIs) in 9 of 20 cases (45%), with a mean DNA copy number change per sample of 1.95 +/- 2.74, and losses (mean: 1.25 +/- 1.77) more frequent than gains (mean: 0.7 +/- 1.26). The most common CIs noted were loss of 1p, followed by losses of 10/10q, 17p, and 19, and gains of 17q and 18. Furthermore, in conjunction with this study a systematic literature review was conducted, which showed a high frequency and consistent pattern of chromosome changes in SS. These findings suggest that chromosomal instability is common in SS, although there are specific chromosomal abnormalities that appear to be characteristic, and the identification of two different recurrent chromosome translocations provides the basis for further studies.

We performed flow cytometric analysis of CD34+ cell apoptosis in 59 patients with myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML) secondary to MDS (MDS‐AML) using annexin V‐FITC, which binds to exposed phosphatidylserine on apoptotic cells. Apoptosis was significantly increased in FAB subtypes RA, RARS and RAEB (<10% blasts) (56.5% (15.1–86.5%)) compared to normal controls (18.5% (3.4–33.4%), P < 0.0001) and RAEB‐t/MDS‐AML (16% (2.1–43.2%), P < 0.0001). There was no correlation between % apoptosis, Full blood count or cytogenetics in any disease category. Two‐colour cytometric analysis of permeabilized CD34+ cells stained with antibodies to Bcl‐2, Bcl‐X (anti‐apoptotic), Bax and Bad (pro‐apoptotic), demonstrated significantly higher ratios of pro‐ v anti‐apoptotic proteins in early MDS (2.47 (1.19–9.42) compared to advanced disease (1.14 (0.06–3.32), P = 0.0001). Moreover, using repeated measures of variants (ANOVA), we found that variations between individual Bcl‐2‐related proteins differed significantly according to disease subtype (P < 0.0005). Our results confirm that CD34+ cell apoptosis was significantly increased in MDS subtypes RA and RARS and fell with disease progression. Early MDS was also associated with a significantly higher CD34+ cell pro‐ v anti‐apoptotic Bcl‐2‐family‐protein ratio than advanced disease. Furthermore, patterns of expression of individual Bcl‐2 related proteins differed significantly between different disease categories. However, no correlation between pro‐ v anti‐apoptotic Bcl‐2‐family‐protein ratios and the degree of apoptosis was observed.

Nineteen patients with high‐risk myelodysplastic syndrome (MDS)/acute myeloid leukaemia (AML) received fludarabine, cytarabine, granulocyte‐colony stimulating factor (G‐CSF), and idarubicin chemotherapy (de novo MDS/MDS‐AML, nine; relapsed/refractory MDS/AML, seven; therapy‐related MDS, three). Median age was 44 years and median disease duration 10 months. 16/19 (84%) patients had abnormal cytogenetics with seven (37%) harbouring abnormalities of chromosome 7. 18/19 (94.7%) patients responded to FLAG‐idarubicin with 12 (63%) achieving complete remission (CR) (<5% blasts and normal cytogenetics). 7/9 (78%) patients with de novo MDS/MDS‐AML achieved CR compared to 5/10 (50%) with alternative diagnoses. Response was associated with age < 50 years, disease duration < 3 months, and cytogenetics other than abnormalities of chromosome 7. Haemopoietic regeneration was rapid in most patients and there were no toxic deaths. Nine patients received a second course of chemotherapy, three have proceeded to allogeneic bone marrow transplant and three to autologous blood stem cell/bone marrow transplantation. Follow‐up is short (median 10 months). 12/19 (63%) patients remain alive and 5/12 (42%) have relapsed at a median 5 months following CR achievement. FLAG‐idarubicin was well tolerated. High rates of morphological and cytogenetic remission, especially in de novo MDS, offer a window of opportunity for assessment of autologous BMT in this group of diseases where no treatment except alloBMT has led to prolongation of survival.

References1 Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the Bcl2 family and cell death.

Cancer testis (CT) antigens provide attractive targets for cancer-specific immunotherapy. Although CT genes are expressed in some normal tissues, such as the testis and in some cases placenta, these immunologically protected sites lack MHC I expression and as such, do not present 'self' antigens to T cells. To date, CT genes have been shown to be expressed in a range of solid tumours, but rarely in haematological malignancies. We have extended previous studies to investigate the expression of a comprehensive range of CT genes (MAGE-A1, -A3, -A6, -A12, BAGE, GAGE, HAGE, LAGE-1, NY-ESO-1 and RAGE) for their expression in a cohort of acute and chronic myeloid leukaemia patient samples. CT expression was not detected in 20 normal bone marrow or peripheral blood stem cell samples. In acute myeloid leukaemia (AML) nine of the 26 (35%) samples analysed expressed one or more of the CT genes with six of the samples (23%) expressing HAGE. In chronic myeloid leukaemia (CML) 24 of 42 (57%) presentation chronic myeloid leukaemia (CML) patient samples expressed one or more CT antigen with 23 expressing HAGE. We have shown that HAGE is frequently expressed in CML, and to a lesser extent in AML patient samples. This is the first demonstration of HAGE gene expression in myeloid leukaemia patients and the frequent expression of HAGE at disease presentation opens up the possibility of early immunotherapeutic treatments.