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Here, we describe the immunoglobulin and T cell receptor (Ig/TCR) molecular rearrangements identified as a leukemic clone hallmark for minimal residual disease assessment in relation to TP53 mutational status in 171 Ph-negative Acute Lymphoblastic Leukemia (ALL) adult patients at diagnosis. The presence of a TP53 alterations, which represents a marker of poor prognosis, was strictly correlated with an immature DH/JH rearrangement of the immunoglobulin receptor (p < 0.0001). Furthermore, TP53-mutated patients were classified as pro-B ALL more frequently than their wild-type counterpart (46% vs. 25%, p = 0.05). Although the reasons for the co-presence of immature Ig rearrangements and TP53 mutation need to be clarified, this can suggest that the alteration in TP53 is acquired at an early stage of B-cell maturation or even at the level of pre-leukemic transformation.

Here, we describe the immunoglobulin and T cell receptor (Ig/TCR) molecular rearrangements identified as a leukemic clone hallmark for minimal residual disease assessment in relation to TP53 mutational status in 171 Ph-negative Acute Lymphoblastic Leukemia (ALL) adult patients at diagnosis. The presence of a TP53 alterations, which represents a marker of poor prognosis, was strictly correlated with an immature DH/JH rearrangement of the immunoglobulin receptor (p < 0.0001). Furthermore, TP53-mutated patients were classified as pro-B ALL more frequently than their wild-type counterpart (46% vs. 25%, p = 0.05). Although the reasons for the co-presence of immature Ig rearrangements and TP53 mutation need to be clarified, this can suggest that the alteration in TP53 is acquired at an early stage of B-cell maturation or even at the level of pre-leukemic transformation.

Background Chromothripsis, which is the local massive shattering of one or more chromosomes and their reassembly in a disordered array with frequent loss of some fragments, has been mainly reported in association with abnormal phenotypes. We report three unrelated healthy persons, two of which parenting a child with some degree of intellectual disability, carrying a chromothripsis involving respectively one, two, and three chromosomes, which was detected only after whole‐genome sequencing. Unexpectedly, in all three cases a fragment from one of the chromothripsed chromosomes resulted to be inserted within a nonchromothripsed one. Methods Conventional cytogenetic techniques, paired‐end whole‐genome sequencing, polymerase chain reaction, and Sanger sequencing were used to characterize complex rearrangements, copy‐number variations, and breakpoint sequences in all three families. Results In two families, one parent was carrier of a balanced chromothripsis causing in the index case a deletion and a noncontiguous duplication at 3q in case 1, and a t(6;14) translocation associated with interstitial 14q deletion in case 2. In the third family, an unbalanced chromothripsis involving chromosomes 6, 7, and 15 was inherited to the proband by the mosaic parent. In all three parents, the chromothripsis was concurrent with an insertional translocation of a portion of one of the chromothriptic chromosomes within a further chromosome that was not involved in the chromothripsis event. Conclusion Our findings show that (a) both simple and complex unbalanced rearrangements may result by the recombination of a cryptic parental balanced chromothripsis and that (b) insertional translocations are the spy of more complex rearrangements and not simply a three‐breakpoint event.

In-frame missense and splicing mutations (resulting in a 2 amino acid insertion or a 34 amino acid deletion) dispersed through the MAP3K1 gene tilt the balance from the male to female sex-determining pathway, resulting in 46,XY disorder of sex development. These MAP3K1 mutations mediate this balance by enhancing WNT/β-catenin/FOXL2 expression and β-catenin activity and by reducing SOX9/FGF9/FGFR2/SRY expression. These effects are mediated at multiple levels involving MAP3K1 interaction with protein co-factors and phosphorylation of downstream targets. In transformed B-lymphoblastoid cell lines and NT2/D1 cells transfected with wild-type or mutant MAP3K1 cDNAs under control of the constitutive CMV promoter, these mutations increased binding of RHOA, MAP3K4, FRAT1 and AXIN1 and increased phosphorylation of p38 and ERK1/2. Overexpressing RHOA or reducing expression of MAP3K4 in NT2/D1 cells produced phenocopies of the MAP3K1 mutations. Using siRNA knockdown of RHOA or overexpressing MAP3K4 in NT2/D1 cells produced anti-phenocopies. Interestingly, the effects of the MAP3K1 mutations were rescued by co-transfection with wild-type MAP3K4. Although MAP3K1 is not usually required for testis determination, mutations in this gene can disrupt normal development through the gains of function demonstrated in this study.

Objective We surveyed the datasheets of 29 laboratories concerning prenatal diagnosis of de novo apparently balanced chromosome rearrangements to assess the involvement of specific chromosomes, the breakpoints distribution and the impact on the pregnancy outcome. Method By means of a questionnaire, data on 269.371 analyses performed from 1983 to 2006 on amniotic fluid, chorionic villus and fetal blood samples were collected. Results A total of 246 balanced anomalies were detected at frequencies of 72% for reciprocal translocations, 18% for Robertsonian translocations, 7% for inversions and 3% for complex chromosome rearrangements. The total frequencies of balanced rearrangements were 0.09%, 0.08% and 0.05% on amniotic fluid, chorionic villus and fetal blood samples. Conclusion A preferential involvement of chromosomes 22, 7, 21, 3, 9 and 11 and a less involvement of chromosomes X, 19, 12, 6 and 1 was observed. A nonrandom distribution of the breakpoints across chromosomes was noticed. Association in the location of recurrent breakpoints and fragile sites was observed for chromosomes 11, 7, 10 and 22, while it was not recorded for chromosome 3. The rate of pregnancy termination was about 20%, with frequencies decreasing from complex chromosomal rearrangements (33%), reciprocal translocations (24%) to inversions (11%) and Robertsonian translocations (3%). Copyright © 2009 John Wiley & Sons, Ltd.

The diagnosis of VACTERL syndrome can be elusive, especially in the prenatal life, due to the presence of malformations that overlap those present in other genetic conditions, including the Fanconi anemia (FA). We report on three VACTERL cases within two families, where the two who arrived to be born died shortly after birth due to severe organs' malformations. The suspicion of VACTERL association was based on prenatal ultrasound assessment and postnatal features. Subsequent chromosome breakage analysis suggested the diagnosis of FA. Finally, by next-generation sequencing based on the analysis of the exome in one family and of a panel of Fanconi genes in the second one, we identified novel FANCL truncating mutations in both families. We used ectopic expression of wild-type FANCL to functionally correct the cellular FA phenotype for both mutations. Our study emphasizes that the diagnosis of FA should be considered when VACTERL association is suspected. Furthermore, we show that loss-of-function mutations in FANCL result in a severe clinical phenotype characterized by early postnatal death.

Human bone marrow mesenchymal stromal cells (BM-MSC) represent one of the most investigated “advanced therapeutic medicinal products”.[1][1] Recent safety concerns have focused attention on the possible malignant transformation due to mutations acquired during their large-scale in vitro

Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells. The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician's decision to vary or to STOP IM therapy.

Background The identification of the JAK2 V617F mutation is mandatory in the diagnostic work-up of Philadelphia chromosome-negative myeloproliferative neoplasms. Several molecular techniques to detect this mutation are currently available, but each of them has some limits. Design and Methods We set up a novel molecular method for the identification of the JAK2 V617F mutation based on an allele-specific loop-mediated amplification, not polymerase chain reaction analysis. This innovative technique amplifies DNA targets under isothermal conditions with high specificity, efficiency and rapidity. The method does not require either a thermal cycler or gel separation and the DNA amplification reaction is visible to the naked eye and can be monitored by turbidimetry. This method was validated on DNA from cell lines as well as from patients with myeloproliferative neoplasms. The results were compared with those obtained by conventional polymerase chain reaction methods. Results This assay detects, within 1 hour, the JAK2 V617F mutation down to an allele burden of 0.1–0.01%. All samples positive by polymerase chain reaction (n=146) proved positive when tested by allele-specific loop-mediated amplification and none of the 80 negative controls gave false positive results. In addition, six patients with essential thrombocythemia previously diagnosed as being JAK2 V617F negative by polymerase chain reaction analysis were found to be positive (at a low level) by allele-specific loop-mediated amplification. Furthermore, this assay discriminated the amount of JAK2 V617F tumor allele within intervals of positivity, above 50%, between 50% and 10% and below 10%. Conclusions Allele-specific loop-mediated amplification is a simple, robust and easily applicable method for the molecular diagnosis and monitoring of JAK2 V617F mutation in patients with chronic myeloproliferative neoplasms.

In this study, we used deletions at 22q13, which represent a substantial source of human pathology (Phelan/McDermid syndrome), as a model for investigating the molecular mechanisms of terminal deletions that are currently poorly understood. We characterized at the molecular level the genomic rearrangement in 44 unrelated patients with 22q13 monosomy resulting from simple terminal deletions (72%), ring chromosomes (14%), and unbalanced translocations (7%). We also discovered interstitial deletions between 17–74 kb in 9% of the patients. Haploinsufficiency of the SHANK3 gene, confirmed in all rearrangements, is very likely the cause of the major neurological features associated with PMS. SHANK3 mutations can also result in language and/or social interaction disabilities. We determined the breakpoint junctions in 29 cases, providing a realistic snapshot of the variety of mechanisms driving non-recurrent deletion and repair at chromosome ends. De novo telomere synthesis and telomere capture are used to repair terminal deletions; non-homologous end-joining or microhomology-mediated break-induced replication is probably involved in ring 22 formation and translocations; non-homologous end-joining and fork stalling and template switching prevail in cases with interstitial 22q13.3. For the first time, we also demonstrated that distinct stabilizing events of the same terminal deletion can occur in different early embryonic cells, proving that terminal deletions can be repaired by multistep healing events and supporting the recent hypothesis that rare pathogenic germline rearrangements may have mitotic origin. Finally, the progressive clinical deterioration observed throughout the longitudinal medical history of three subjects over forty years supports the hypothesis of a role for SHANK3 haploinsufficiency in neurological deterioration, in addition to its involvement in the neurobehavioral phenotype of PMS., Author Summary Terminal chromosome deletions are among the most commonly observed rearrangements detected by cytogenetics and may result in several well-known genetic syndromes. We used 22q13 deletions to study how these types of chromosome abnormalities arise. Children with Phelan/McDermid syndrome, caused by deletion of the terminal portion of chromosome 22, experience developmental delay, absent or severely delayed speech, and frequent behavioral problems. Lack of one copy of SHANK3, a key gene for the correct development and organization of brain synapses, is very likely the basis of the syndrome's major neurological features. Deletion of additional genes probably causes more complex phenotypes in subjects with larger deletions. We also studied patients who only lack a portion of SHANK3 and demonstrated that small, hard-to-detect deletions of this gene may cause substantial clinical problems. Until now, the 22q distal deletion had been only diagnosed in very young people. We studied a large group of patients of different ages and discovered that all adult patients face progressive cognitive decline. Our study demonstrates that deletion of the terminal portion of chromosome 22, a prototype for terminal deletions in human chromosomes, can occur in several ways. Mosaic deletions of different size can also form in early embryogenesis.

In chronic myeloid leukemia, different methods are available to monitor the response to therapy: chromosome banding analysis (CBA), interphase fluorescence in situ hybridization (I-FISH), and real-time quantitative polymerase chain reaction (RT-Q-PCR). The GIMEMA CML WP (Gruppo Italiano Malattie Ematologiche Adulto Chronic Myeloid Leukemia Working Party) has performed a prospective study to compare CBA and I-FISH for the definition of complete cytogenetic response (CCgR). Samples (n = 664) were evaluated simultaneously by CBA and I-FISH. Of 537 cases in CCgR, the number of positive nuclei by I-FISH was less than 1% in 444 cases (82.7%). Of 451 cases with less than 1% positive nuclei by I-FISH, 444 (98.4%) were classified as CCgR by CBA. The major molecular response rate was significantly greater in cases with I-FISH less than 1% than in those with I-FISH 1% to 5% (66.8% vs 51.6%, P < .001) and in cases with CCgR and I-FISH less than 1% than in cases with CCgR and I-FISH 1% to 5% (66.1% vs 49.4%, P = .004). I-FISH is more sensitive than CBA and can be used to monitor CCgR. With appropriate probes, the cutoff value of I-FISH may be established at 1%. These trials are registered at http://www.clinicaltrials.gov as NCT00514488 and NCT00510926.

Clinical trials have shown that high dose chemotherapy (HDT) with peripheral stem cell autotransplantation is presently the best treatment for patients with symptomatic multiple myeloma (MM). In the context of an outcomes research project, we analyzed the feasibility of this strategy in clinical practice in a large cohort of consecutive, unselected patients with newly diagnosed MM and looked at the major determinants of response of patients enrolled in a HDT with tandem autotransplantation (Total Therapy I, TTI) program.Two hundred and fourteen patients were treated outside of a clinical trial and regularly followed-up at our Center for symptomatic MM. Ninety-seven patients (45%) received conventional chemo-radiotherapy regimens, 110 (51%) entered the TTI program and the remaining 7 patients (3.3%) were enrolled in other programs involving HDT with autotransplantation.Patients enrolled in HDT with tandem autotransplantation programs were 14 years younger and less likely to have co-morbidities than patients treated with conventional therapy. Median overall survivals of the two groups were 60 and 33 months, respectively. Thirteen percent of the patients enrolled in the TTI program did not receive the first HDT with autotransplantation, mostly because of disease progression, and another 16% did not proceed to the second HDT with autotransplantation mainly because of infections or drug-related complications. Most patients achieved complete remission after the second autotransplantation, with acceptable toxicity. However, only patients with a major reduction of the myeloma burden at the end of induction therapy enjoyed significantly prolonged event-free and overall survivals.Approximately one third of patients with newly diagnosed symptomatic MM completed the TTI program. These data suggest the need to improve the induction therapy in order to increase both the number of patients able to proceed to autotransplantation programs and to enhance the rate of early response.

INTRODUCTION An exploratory trial was conducted to evaluate toxicity and potential therapeutic role of all trans-retinoic acid (ATRA) given long-term together with chemotherapy and G-CSF to adult patients with acute myelogenous leukemia (AML). MATERIALS AND METHODS ATRA was administered orally at 45 mg/m(2)/day on days 1-14 and 25 mg/m(2)/day on days 15-28 of two standard cycles (idarubicin, etoposide, cytarabine, G-CSF) and of up to three high-dose courses (cytarabine, G-CSF). The results obtained in 19 patients enrolled in the ATRA trial were compared with those from 29 comparable cases treated with the same schedule without ATRA, according to patient risk class and an in vitro study. RESULTS ATRA was administered for a median of 52 days to the patients selected for study who achieved a remission. ATRA-related toxicity was mostly non-severe apart from high incidence of headache in conjunction with high-dose cytarabine. Complete remission (CR) rate after cycle 1 (54%), kinetics of hematological recovery, postremission treatment realization, disease-free survival (DFS 37.5% at three years) and overall survival (30% at three years) were not different between ATRA-treated and untreated patients. The only significant prognostic factor was the patient risk class, as defined by cytogenetics and other clinical criteria: DFS rate was 57% at three years in standard-risk cases compared to only 19% in the high-risk group, with no influx by ATRA in either category. The in vitro study, in patients with a definite clinical response, failed to document any inhibitory or pro-apoptotic effect of ATRA on AML blast cells. CONCLUSION As a consequence to these results, the pilot ATRA phase was closed. This study does not suggest a significant role for the present ATRA schedule as an adjunct to standard antileukemic therapy in adult AML.

Introduction Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia (CML), effectively prolonging overall survival. Because discontinuation of treatment is associated with molecular relapse, IM is required indefinitely to maintain operational cure. To evaluate the degree of response to therapy and to highlight the persistence of the disease after treatment, patients should be monitored routinely. The gold standard for diagnosing CML is the cytogenetic analysis, a direct not-sensitive method to detect Ph-positive cells. Quantitative real-time RT-PCR (qRT-PCR) provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells and cannot detect transcriptionally silent leukemic stem cells. Methods Here we will propose a new sensitive approach to directly detect the number of leukemic cells using a DNA-based biomarker specific for each patient. We applied targeted next-generation sequencing for the identification of genomic BCR-ABL1 fusion junctions, and we developed a sensitive new approach to detect the number of leukemic cells by a DNA Q-PCR assay based on the genomic break-point, with a formula to calculate the number of Ph+ cells. The percentage of the leukemic cells (LC) was calculated using the following formula: %LC= (2/(2Δct+1))*100, where ΔCt is the difference between the amplification cycles of the BCR-ABL1 and BCR reactions. The number of LC was calculated by multiplying the total number of cells analyzed in each sample by the percentage of LC calculated by the ΔCt formula. We then defined a limit of quantization and a limit of sensitivity in the evaluation of minimal residual disease (MRD), as described by guidelines for the detection of MRD by genomic Q-PCR in acute lymphoblastic leukemia (ALL). We defined a “quantitative range” of detection, the portion of the standard curve in which the MRD levels can be quantified reproducibly and accurately, and we defined the “limit of sensitivity”, the lowest MRD level that still can be detected, although not in all replicates. We thus calculated the exact number of leukemic cells only when the MRD fell within the range of quantization. The detection of MRD at the limit of sensitivity was indicated as positive but not quantified. Results We monitored eight CML patients treated with Imatinib for 8 years. We tested the same samples by patient specific Q-PCR, and in parallel by cytogenetic analysis and by standard qRT-PCR. In all samples positive for chimeric transcripts we measured corresponding chimeric genomic DNA (gDNA) by Q-PCR, confirming the sensitivity of the Q-PCR method. According to conventional criteria, undetectable levels of BCR-ABL1 mRNA assessed by qRT-PCR are indicative of complete molecular response (CMR), but in 33.3% (45/135) samples with undetectable levels of mRNA, we detected the persistence of transcriptionally-silent leukemic cells. However, we never found samples negative by gDNA Q-PCR and positive by RNA-based qRT-PCR (Figure 1). Thirty-six of 135 samples were also analyzed cytogenetically until the achievement of CCyR. As expected, Ph+cells were detected only in 25% (9/36) and 22,2% (8/36) of samples by CBA and I-FISH, respectively, whereas BCR-ABL1 mRNA was detected by qRT-PCR in 83.3% (30/36) of samples and Ph+ cells were detected by genomic Q-PCR in 91.7% (33/36) of samples (Figure 1). Finally, the separation of different cell populations from blood and bone marrow revealed the presence of a population of transcriptionally silent cancer stem cells. The gDNA based Q-PCR analysis performed on highly purified (immunomagnetically selected ) CD34+and CD3+ cells confirmed the presence of a population of transcriptionally silent cancer stem cells. Conclusions The demonstration of positive gDNA Q-PCR in 33.3% of samples negative for the RNA qRT-PCR could partially explain why some patients lose MMR and CMR and others do not, when IM is discontinued for brief periods. The gDNA based Q-PCR could be used to supplement conventional techniques, providing clinicians with additional information about disease status and response in determining whether to stop or alter therapy. Acknowledgments to AIRC and AIL. Disclosures: No relevant conflicts of interest to declare.

Summary Routine Q-banding chromosome analysis detected the jumping behaviour of bright fluorescent chromosome 22 satellites (22s+) in two unrelated males (case 1 ascertained for recurrent abortions and case 2 for infertility), and in the mother of one of them, all with a normal karyotype. The 22s + was present in more than 90% of the cells. In a minority of the cells the polymorphism was present alternatively on another acrocentric, on one chromosome 22 and on another acrocentric, on both chromosomes 22 or it was absent. We take these findings as evidence of mitotic exchanges between the short arms of the acrocentric chromosomes. The presence of a stable 22s + in the fibroblasts of case 1 and in the lymphocytes of his son indicates that acrocentric short arm exchanges depend both on the type of tissue and on the genetic content of all the other acrocentrics.

The growth hormone-releasing factor (GHRF) gene has been mapped by different authors alternatively at 20p12 and 20q11.2. In situ hybridization of the relevant probe to metaphases of two patients with different translocations involving 20q has allowed us to map it definitively at 20q11.2.

We planned a prospective analysis involving 3 multicentric national studies of the GIMEMA (Gruppo Italiano Malattie Ematologiche Adulto) CML Working Party (WP) to evaluate the correlation between conventional cytogenetics (CC) and FISH response in CML pts in chronic phase (CP) treated with Imatinib. Karyotype and FISH analyses were performed on bone marrow (BM) cells in 26 local laboratories and in 12 WP reference labs. Peripheral blood samples for quantitative RT-PCR were centralized in Bologna. The data are reported in Figure 1 and in Table 1. Fig 1 shows the relationship between CC and FISH (r=0.91;p=0.008). Tab 1 shows the demonstration of FISH data according to CC data and the number of metaphases available for CC. Of 217 pts in CCgR by CC and > 20 metaphases studied, 81.6% were FISH negative, 15.2% showed a low rate of FISH positive cells (1–5%) and 3.2% an higher rate. Of 94 pts in CCgR by CC but with < 20 metaphases studied, 71.3% were FISH negative, 21.3% showed a low rate of FISH positive cells (1–5%) and 7.4% an higher rate. Of 43 pts in PCgR by CC and FISH positive, 48.8% showed 1– 5% positive cells. Moreover, 358 samples were performed simultaneously by CC, FISH and quantitative RT-PCR: 179 (50%) samples in CCgR showed major molecular response (MMolR, defined as a BCR-ABL × 100 ratio < 0.1%): 164 (91.6%) were FISH negative and 15 (8.4%) were FISH positive (1.3–10% positive cells). We conclude that interphase FISH is a very releable method of monitoring the CCgR once it has been achieved. The relationship of FISH on BM cells with molecular response is at least as good as the relationship of CC with molecular response. It remains to be shown if the same results can be obtained on peripheral blood cells, that are already widely used for molecular monitoring. | | No | FISH negative | FISH 1–5% | FISH > 5% | |:-------------- | --- | ------------- | ---------- | ---------- | | CCgR ≥ 20 met | 217 | 177 (81.6%) | 33 (15.2%) | 7 (3.2%) | | CCgR < 20 met | 94 | 67 (71.3%) | 20 (21.3%) | 7 (7.4%) | | PCgR ≥ 20 met | 43 | | 21 (48.8%) | 22 (51.2%) | | CCgR and MMolR | 179 | 164 (91.6%) | 12 (6.7%) | 3 (1.7%) | ![Figure][1] Figure [1]: pending:yes

Extensive submicroscopic deletions adjacent to the breakpoint on derivative chromosome 9 [der(9)] have been reported in a subset of Chronic Myeloyd Leukemia (CML) patients and have been associated with an adverse outcome with conventional drugs and α-interferon (α-IFN). Huntly et al (Blood.2003; 102.2205–12) reported 275 CML pts who were treated with imatinib in CP, suggesting that der(9) deletions were associated with lower response rates and a shorter time to progression. Different data were reported by Quintas-Cardama et al (Blood.2005; 105:2281–6), who did not find any difference related with der(9) deletions in other 320 patients treated with imatinib. In these 2 studies, some patients began imatinib in early CP (51 and 152, respectively) while many patients (224 and 168, respectively) were treated in late CP. To establish the relationship of der(9) deletions with the response to imatinib in early CP patients, we planned a prospective study involving 3 consecutive multicentric national studies of the GIMEMA (Gruppo Italiano Malattie Ematologiche dell’Adulto) CML Working Party. 421 CML patients in early chronic phase were enrolled between January, 2004 and January, 2006; Fluorescence in situ hybridization (FISH) analysis of bone marrow cells was performed using BCR/ABL extra-signal, D-FISH or dual-color dual-fusion probes. At diagnosis, 52 (12%) of them had der(9) deletion and 369 (88%) had not. The 2 groups, with/without deletions, were comparable (no difference in age, Sokal risk, imatinib dose). Median observation time is 12 months. At 3 months, the CHR rates in with/without deletions patients were 87%/92%. At 6 months, the complete cytogenetic response (0% Ph-pos; CCgR) rates were 80%/80%, with major molecular response (MMolR, defined as a Bcr-Abl/Abl x 100 ratio < 0.1%) rates of 52%/51%, respectively. At 12 months, CCgR rates were 89%/86% and MMolR 52%/61%. No difference is significant. We conclude that the presence of der(9) deletions at diagnosis do not constitute a negative factor for response to imatinib: the haematological, cytogenetic and molecular response rates resulted equal between the 2 groups of patients with and without der(9) deletions. This finding is relevant to the long term effect of imatinib treatment, since both the CCgR and the MMolR are important and established indicator of long term survival.

HES (hypereosinophilic syndrome with organ damage), CEL (chronic eosinophilic leukemia with chromosome marker and/or myeloproliferation/dysplasia) and CIH (chronic idiopathic hypereosinophilia) are diseases that pose a therapeutic challenge and are potentially Glivec-sensitive. This drug inhibits BCR-ABL type tyrosine kinase (TK) as well as Platelet-Derived-Growth-Factor Alpha and Beta (FIP1L1-PDGFRA and ETV6-PDGFRB, detectable in HES and allied conditions) and c-kit related TKs. A study was planned to assess 1) the response to escalating doses of Glivec (starting dose 100 mg/d, maximum 400 mg/d, weekly increment 100 mg/d as necessary) and 2) the diagnostic profile of Glivec-responsive cases. Eligible patients had BCR-ABL- HES, CEL, or CIH with eosinophilia >5x10e9/l. Diagnostic work-up included marrow morphology and cytogenetics, PDGFRA/B rearrangement study, and immunophenotype for the detection of expanded (Vbeta) T-cell clones (TCC). Glivec was kindly provided by Novartis Oncology, Italy, to be administered for 12 consecutive weeks. A complete remission (CR) was defined by an eosinophil count Starting 11/’04, 18 patients were identified (14 M), aged 26–80 years (median 54), with a disease history of 0.2–12 years (median 3.4). Blood eosinophilia was 0.67–17.5x10e9/l (median 2.1), with diagnosis of HES (n= 8), CEL (n= 6) or CIH (n= 4). Cytogenetics was abnormal in 5/14 (36%): 1 t(4;8), 1 der(5) t(1;5)(q25;q34) del(5)(q?), 1 del(5)(q32) i(17), and 2 -Y as possible constitutional anomaly; a TCC was detected in 3/9 (33%); FIP1L1-PDFGRA and ETV6-PDGFRA rearrangements were found in 4/17 (24%) and 0/9 (0%), respectively. As of 07/’05, 16 cases are evaluable (2/4 PDGFRA+), 8 having completed the study. Four patients were withdrawn very early because nonresponders at increased dosage (n=3) or noncompliant (n=1), and 4 are still on study. A CR was documented in 6/8 who completed the trial, 3 with CEL and t(4;8) or an involved chromosome 5, 2 with PDGFRA+ HES, and 1 with PDGFRA-/B- HES. No patient with TCC responded. As concerns dose-/time-response relationships, 4 cases were fully responsive to 100 mg/d through the 12 weeks of the study, and 2 required 200 mg/d from the 6th and 11th week, respectively (cases with longer HES history: 12 and 11 years; one with prior blastic transformation), whereas eosinophil blood counts (x10e9/l) varied as follows from baseline to 1st and 4th weeks: 1.95 (0.88–17.5) to 0.20 (0.02–2.09) to 0.15 (0.00–0.4). However, both PDGFRA+ cases remained molecularly positive, while the single evaluable patient achieved only a transient cytogenetic remission. Time to progression (>0.35) was short following drug withdrawal (5 patients: 1, 1, 3, 3, 3 months). Glivec is highly active in hypereosinophilic syndromes with PDGFR rearrangements and other gene lesions that need to be properly identified. These cases respond very early even at the lower dosage. However, an early stopping of therapy can be followed by rapid progression as indicated also by the persistence of cytogenetic/molecular lesions in some cases. This dictates a continuing molecular/cytogenetic monitoring for dose titration like in chronic myelogenous leukemia.

In adult AML, effective postremission consolidation is obtained with HD-ARA-C and either autologous or allogeneic stem cell transplantation (SCT). HD-ARA-C is highly myelotoxic, causing remissional deaths and nonlethal toxicity that often precludes the administration of repetitive courses especially in older patients. We are evaluating a risk-oriented strategy that specifically considers the use of multiple, stem cell-supported HD-ARA-C courses. After double induction (ICE/IC or ICE/sequential HD-ARA-C-I when primary resistance is documented), ARA-C 1 g/m2/bd is given on dd 1–4, followed by G-CSF to collect autologous CD34+ blood stem cells. Then, patients with high risk cytogenetics, i.e. -5/del(5q), -7, t(11q23), t(9;22), abn 3q, 9q, 11q, 20q, 21q, 17p, iso(17q), t(3;5), t(6;9), or >3 unrelated clonal anomalies, and those with intermediate risk cytogenetics (normal or not known, non-high, non-favourable) plus any additional risk feature (WBC >50, MDS/secondary, late CR, FLT-3 mutation, hepatosplenomegaly, FAB M0,6,7) are grouped together in the high-risk (HR) category and are preferentially submitted to an allogeneic SCT. In the remaining cases, i.e. the standard-risk (SR) group, three HD-ARA-C-based cycles (2 g/m2/bd on dd 1–5, plus idarubicin) are planned, each one followed by the reinfusion of 1–2x106/kg CD34+ cells on day 6, and by G-CSF; HR patients unable to undergo SCT are transferred to HD-ARA-C, and those with an insufficient CD34+ cell yield are given one-two courses with intermediate-dose ARA-C. Ninety-one unselected patients (males 46) aged 17–68 years (median 50 years, 38 >55 years) with either de novo or secondary/MDS-related (23%) non-M3 AML (cytogenetics: favourable 10, intermediate 13, normal 45, unfavourable 15, unknown 8) are thus far evaluable: 67 HR and 24 SR. CR rate was 76% (96% in SR, 69% in HR). Stem cell harvest was successful in 43/58 (74%). So far, SCT was performed in 16 HR cases, while 89 HD-ARA-C courses were administered to 34 other patients (16 SR and 18 HR), with a median intercycle time of 39 days (only 17% >45 days), a median duration of neutropenia In conclusion low-dose autologous CD34+ cell rescue (plus G-CSF) may sensibly limit HD-ARAC-related toxicity in unselected adults with AML, up to an age of 68 years in this study, allowing an effective increase in dose intensity. This translated into a DFS rate >50% at 4 years in the SR group (mainly cases with normal/intermediate risk cytogenetics without additional risk factors), whereas an allo-SCT was more beneficial to the HR group. These results document the role of blood stem cell-supported, repetitive HD-ARA-C courses, particularly in intermediate-risk patients and/or those aged >55 years or with contraindications to allogeneic SCT.

To asses cytogenetic pattern of early diagnosed chronic phase chronic myeloid leukemia (CML) patients and to evaluate the role of either conventional (CC) and molecular cytogenetics in three multicentric studies, karyotype and interphase fluorescence in situ hybridization (FISH) analyses were performed in 372 enrolled patients between April 2004 and July 2005 by the GIMEMA CML Working Party (WP). Local investigator laboratories (25 labs) or WP reference labs (12 labs) performed both analyses. Cytogenetic examinations was performed at baseline; after 6 and 12 months of imatinib treatment; thereafter every 6 months and in case of failure or disease progression. At the baseline, 257 patients have been studied and 237 (92%) are valuable for both analyses (CC and FISH). Additional abnormalities in Ph+ clone have been observed in 12 patients (5%). Moreover, 18 (8%) cases showed variant Ph translocations and in 23 (10%) patients the derivative of chromosome 9 was deleted. As yet, cytogenetic response (CR) was evaluated in 188 samples and 156 cases were valuable (83%): 20 at 3 months, 101 at 6 months and 35 at 1 year of treatment. One hundred and eighteen (76%) patients achieved complete CR (CCR) established with more than 20 metaphases in 84 cases, meanwhile in 34 CCR cases the number of examined metaphases was lower. In the first group, 70/84 (83%) samples showed absence of bcr/abl rearrangement in FISH, meanwhile 13/84 (16%) carried a low rate of positive cells (1–5%) and the last one showed the rearrangement in 12% of cells. In the latter group, 23/34 (68%) didn’t show any rearrangement in FISH, in 8/34 (24%) the amount of Ph+ cells was low (1–5%), in 2 was higher (7% and 10%) and the last one carried an high rate (72%) of rearranged cells. In this latter case the RCC was evaluated on 10 metaphases. Twenty-three patients in major CR (MCR), but not in CCR, showed retention of persisting Ph+ cells ranging from 2 to 21% in CC study and from 2 to 16% in FISH analysis. Moreover we found a patient with 2% of Ph+ metaphases and 53% of Ph+ cells in FISH: in this case the CC evaluation was established with 10 metaphases. We can suggest there was a good correlation between cytogenetic and FISH tests in terms of the kinetics of disappearance of the bcr/abl rearrangement. FISH is a reliable method to reveal submicroscopic deletions and to monitor the size of the Ph + clone in treated CML patients. However, a good CC analysis remains an excellent approach to the evaluation of response to Imatinib. Moreover it can detect the emergence of other abnormalities in Ph positive or negative clone.