Neurological Complications Involving the Central Nervous System After Allogeneic Hematopoietic Stem Cell Transplantation During a Period of Evolution in Transplant Modalities: A Cohort Analysis.

BACKGROUND: Neurological complications (NC) after hematopoietic stem cell transplantation (HSCT) are rare events. The evolution of transplant procedures has resulted in improved survival and has allowed elderly patients or those with comorbidity to receive an HSCT. The risk of NC in these patients has still not been well defined. Therefore, we carried out an observational study to estimate the occurrence and identify the risks associated with NC. METHODS: The study cohort included 452 adult-allogeneic HSCT recipients, transplanted from 1997 to 2012. The median follow up was 1.3 year (0-15.7). A myeloablative regimen was used in 307 patients. Two hundred patients were grafted from matched unrelated donor (MUD), of these, 129 (64.5%) received an in vivo T-cell depletion. RESULTS: Out of 452 patients, 30 (6.6%) developed NC. Infections were the most frequent causes of NC (30%). Overall survival decreased in patients developing NC (P < 0.001). Univariate survival regression on the cumulative incidence of NC identified period of transplant, linear trend between 4-year periods (1997-2012) (P < 0.001), MUD (P < 0.001), and recipient's age (P = 0.034) as significant risk factors. In multivariate analysis, period of transplant (P < 0.001) and MUD (P = 0.004) remained significant independent risk factors. Matched unrelated donor recipients showed a 3.8-fold elevated risk of developing NC. CONCLUSIONS: Analysis highlights a temporal trend of incidence of NC that progressively increased over time and confirms a strong association between donor type and risk of NC. Our observations suggest that, although relatively uncommon, NC after allo-HSCT, may become more frequent due to the improved overall survival in recent years.

Independent prognostic impact of tumour-infiltrating macrophages in early-stage Hodgkin's lymphoma.

Although patients with early-stage Hodgkin's lymphoma have a high rate of cure, a portion of these are resistant to or relapse after standard treatment. Current prognostic criteria based on clinical and laboratory parameters at diagnosis do not allow to accurately identify the subset of patients with less favourable clinical outcome. An increased number of tumour-infiltrating macrophages was found to be associated with shortened survival in patients with classic Hodgkin's Lymphoma. The aim of this study was to assess the clinical significance of the proportion of CD68-positive infiltrating macrophages in patients with early-stage classic Hodgkin's lymphoma. By using immunohistochemistry technique, we evaluated for CD68 expression diagnostic biopsies of 106 patients affected by supradiaphragmatic early-stage classic Hodgkin's lymphoma treated at our institution since 2000 to 2010. All patients were treated with adriamycin, bleomycin, vinblastine, and dacarbazine chemotherapy followed by radiotherapy in the majority. The 2-year overall survival and progression-free survival (PFS) in the entire cohort were 97% and 83% respectively. The 2-year PFS was statistically different between patients with favourable and those with unfavourable prognosis according to the European Organisation for Research and Treatment of Cancer (EORTC) risk criteria (96% vs 79%, p = 0.039) and between patients having less than 25% of CD68-positive infiltrating macrophages and those with more than 25% (85% vs 67%, p = 0.012). All patients with favourable EORTC criteria had CD68 expression lower than 25%. Within those with unfavourable EORTC criteria, patients with a CD68+ count greater than 25% had a worse 2-year PFS than patients having values lower than 25% (64% vs 82%, p = 0.03). Moreover, in multivariate analysis, after adjusting for CD68+ macrophages count and EORTC score, only CD68+ macrophages count higher than 25% retained a prognostic effect on PFS (hazard ratio = 2.8, 95%CI: 1.1-7.6, p = 0.038). Our data show that a proportion of tumour-infiltrating macrophages greater than 25% is associated with unfavourable clinical outcome in patients with early-stage Hodgkin's lymphoma Copyright © 2016 John Wiley & Sons, Ltd.

Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis.

We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials.

Driver somatic mutations identify distinct disease entities within myeloid neoplasms with myelodysplasia.

Our knowledge of the genetic basis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) has considerably improved. To define genotype/phenotype relationships of clinical relevance, we studied 308 patients with MDS, MDS/MPN, or acute myeloid leukemia evolving from MDS. Unsupervised statistical analysis, including the World Health Organization classification criteria and somatic mutations, showed that MDS associated with SF3B1-mutation (51 of 245 patients, 20.8%) is a distinct nosologic entity irrespective of current morphologic classification criteria. Conversely, MDS with ring sideroblasts with nonmutated SF3B1 segregated in different clusters with other MDS subtypes. Mutations of genes involved in DNA methylation, splicing factors other than SF3B1, and genes of the RAS pathway and cohesin complex were independently associated with multilineage dysplasia and identified a distinct subset (51 of 245 patients, 20.8%). No recurrent mutation pattern correlated with unilineage dysplasia without ring sideroblasts. Irrespective of driver somatic mutations, a threshold of 5% bone marrow blasts retained a significant discriminant value for identifying cases with clonal evolution. Comutation of TET2 and SRSF2 was highly predictive of a myeloid neoplasm characterized by myelodysplasia and monocytosis, including but not limited to, chronic myelomonocytic leukemia. These results serve as a proof of concept that a molecular classification of myeloid neoplasms is feasible.

Predictive factors for the outcome of allogeneic transplantation in patients with MDS stratified according to the revised IPSS-R.

Approximately one-third of patients with myelodysplastic syndrome (MDS) receiving allogeneic hematopoietic stem cell transplantation (HSCT) are cured by this treatment. Treatment failure may be due to transplant complications or relapse. To identify predictive factors for transplantation outcome, we studied 519 patients with MDS or oligoblastic acute myeloid leukemia (AML, <30% marrow blasts) who received an allogeneic HSCT and were reported to the Gruppo Italiano Trapianto di Midollo Osseo registry between 2000 and 2011. Univariate and multivariate survival analyses were performed using Cox proportional hazards regression. High-risk category, as defined by the revised International Prognostic Scoring System (IPSS-R), and monosomal karyotype were independently associated with relapse and lower overall survival after transplantation. On the other hand, older recipient age and high hematopoietic cell transplantation-comorbidity index (HCT-CI) were independent predictors of nonrelapse mortality. Accounting for various combinations of patient's age, IPSS-R category, monosomal karyotype, and HCT-CI, the 5-year probability of survival after allogeneic HSCT ranged from 0% to 94%. This study indicates that IPSS-R risk category and monosomal karyotype are important factors predicting transplantation failure both in MDS and oligoblastic AML. In addition, it reinforces the concept that allogeneic HSCT offers optimal eradication of myelodysplastic hematopoiesis when the procedure is performed before MDS patients progress to advanced disease stages.

CALR exon 9 mutations are somatically acquired events in familial cases of essential thrombocythemia or primary myelofibrosis.

Somatic mutations in the calreticulin (CALR) gene were recently discovered in patients with sporadic essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking JAK2 and MPL mutations. We studied CALR mutation status in familial cases of myeloproliferative neoplasm. In a cohort of 127 patients, CALR indels were identified in 6 of 55 (11%) subjects with ET and in 6 of 20 (30%) with PMF, whereas 52 cases of polycythemia vera had nonmutated CALR. All CALR mutations were somatic, found in granulocytes but not in T lymphocytes. Patients with CALR-mutated ET showed a higher platelet count (P = .017) and a lower cumulative incidence of thrombosis (P = .036) and of disease progression (P = .047) compared with those with JAK2 (V617F). In conclusion, a significant proportion of familial ET and PMF nonmutated for JAK2 carry a somatic mutation of CALR.

Impact of ruxolitinib on the natural history of primary myelofibrosis: a comparison of the DIPSS and the COMFORT-2 cohorts.

The international prognostic scoring system (IPSS) provides reliable risk assessment in patients with primary myelofibrosis (PMF). Recent clinical trials in PMF patients with intermediate-2 or high IPSS risk have shown a survival advantage of ruxolitinib over placebo (COMFORT-1) or best available therapy (COMFORT-2). Because crossover was allowed in these studies, we analyzed the cohort of ruxolitinib-naive patients used for developing the dynamic IPSS (DIPSS). By adopting ad hoc statistical analyses, we compared survival from diagnosis of 100 PMF patients receiving ruxolitinib within COMFORT-2 with that of 350 patients of the DIPSS study. Subjects were properly matched, and both left-truncation and right-censoring were accounted in order to compare higher IPSS risks exclusively. Patients receiving ruxolitinib had longer survival (5 years, 95% confidence interval [CI]: 2.9-7.8 vs 3.5 years, 95% CI: 3.0-3.9) with a hazard ratio of 0.61 (95% CI: 0.41-0.91; P = .0148). This observation suggests that ruxolitinib may modify the natural history of PMF.

JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes.

Patients with essential thrombocythemia may carry JAK2 (V617F), an MPL substitution, or a calreticulin gene (CALR) mutation. We studied biologic and clinical features of essential thrombocythemia according to JAK2 or CALR mutation status and in relation to those of polycythemia vera. The mutant allele burden was lower in JAK2-mutated than in CALR-mutated essential thrombocythemia. Patients with JAK2 (V617F) were older, had a higher hemoglobin level and white blood cell count, and lower platelet count and serum erythropoietin than those with CALR mutation. Hematologic parameters of patients with JAK2-mutated essential thrombocythemia or polycythemia vera were related to the mutant allele burden. While no polycythemic transformation was observed in CALR-mutated patients, the cumulative risk was 29% at 15 years in those with JAK2-mutated essential thrombocythemia. There was no significant difference in myelofibrotic transformation between the 2 subtypes of essential thrombocythemia. Patients with JAK2-mutated essential thrombocythemia and those with polycythemia vera had a similar risk of thrombosis, which was twice that of patients with the CALR mutation. These observations are consistent with the notion that JAK2-mutated essential thrombocythemia and polycythemia vera represent different phenotypes of a single myeloproliferative neoplasm, whereas CALR-mutated essential thrombocythemia is a distinct disease entity.

Somatic mutations of calreticulin in myeloproliferative neoplasms.

BACKGROUND: Approximately 50 to 60% of patients with essential thrombocythemia or primary myelofibrosis carry a mutation in the Janus kinase 2 gene (JAK2), and an additional 5 to 10% have activating mutations in the thrombopoietin receptor gene (MPL). So far, no specific molecular marker has been identified in the remaining 30 to 45% of patients. METHODS: We performed whole-exome sequencing to identify somatically acquired mutations in six patients who had primary myelofibrosis without mutations in JAK2 or MPL. Resequencing of CALR, encoding calreticulin, was then performed in cohorts of patients with myeloid neoplasms. RESULTS: Somatic insertions or deletions in exon 9 of CALR were detected in all patients who underwent whole-exome sequencing. Resequencing in 1107 samples from patients with myeloproliferative neoplasms showed that CALR mutations were absent in polycythemia vera. In essential thrombocythemia and primary myelofibrosis, CALR mutations and JAK2 and MPL mutations were mutually exclusive. Among patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 or MPL, CALR mutations were detected in 67% of those with essential thrombocythemia and 88% of those with primary myelofibrosis. A total of 36 types of insertions or deletions were identified that all cause a frameshift to the same alternative reading frame and generate a novel C-terminal peptide in the mutant calreticulin. Overexpression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with mutated CALR had a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. CONCLUSIONS: Most patients with essential thrombocythemia or primary myelofibrosis that was not associated with a JAK2 or MPL alteration carried a somatic mutation in CALR. The clinical course in these patients was more indolent than that in patients with the JAK2 V617F mutation. (Funded by the MPN Research Foundation and Associazione Italiana per la Ricerca sul Cancro.).

Long term evaluation of the impact of autologous peripheral blood stem cell transplantation in multiple myeloma: a cost-effectiveness analysis.

BACKGROUND: High-dose therapy with autologous peripheral stem cell transplantation represents today the standard approach for younger multiple myeloma patients. This study aimed to evaluate the long term economic impact of autologous transplantation with respect to conventional therapy. METHODS: We retrospectively reviewed the charts of multiple myeloma patients diagnosed at our department between 1986 and 2003 and treated according to the therapy considered standard at the time of diagnosis. Analysis of costs was done by assessing resource utilization and direct costs were measured and monetized before proceeding with the analysis, based on public health service tariffs. RESULTS: Group A including 78 patients treated with Melphalan and Prednisone was compared with Group B including 74 patients who received an autologous transplant. The median overall survival was 3.2 and 5.4 years respectively (p = 0.0002). Mean cost per patient was significantly higher in group B with respect to group A (102373€ vs 23825€; p<0.001). The final quality-adjusted-life-year gain in group B patients as compared to group A was 1.73 QALY, with an incremental cost-effectiveness ratio of 45460€. With a threshold of 75000€ per QALY gained, the cost effectiveness acceptability curve indicated that the probability that autologous transplantation in multiple myeloma is a cost-effective intervention is 90%. CONCLUSIONS: The cost of autologous transplantation remains high. The calculated incremental cost-effectiveness ratio, however, given the significant prolongation of overall survival obtained with autologous transplantation, is within an acceptable threshold. Notwithstanding, its high cost should be taken into account when considering the whole cost of multiple myeloma.

Associated cancers in Waldenström macroglobulinemia: clues for common genetic predisposition.

BACKGROUND: Several population-based and cohort studies have reported an increased risk of second cancers in lymphoproliferative disorders (LPDs). The cause of second cancers in LPDs is probably multifactorial, and the relative contribution of treatments, genetic predisposition, and immune dysfunction typical of LPDs is still unclear. PATIENTS AND METHODS: We retrospectively studied 230 patients with Waldenström macroglobulinemia (WM) to assess the frequency, characteristics, and predictive factors of second cancers and to evaluate whether patients with WM are at higher risk of second cancers compared with an age- and sex-matched control population. RESULTS: In a competing-risk model, the cumulative incidence of solid cancers was 6% at 5 years, 11% at 10 years, and 17% at 15 years, whereas the incidence of hematologic malignancies was 4% at 5 years, 7% at 10 years, and 8% at 15 years. Compared with an age- and sex-matched population, the overall risk of second cancers was 1.7-fold higher than expected (95% confidence interval [CI], 1.22-2.38; P = .002). Patients with WM were at increased risk for diffuse large B-cell lymphoma (DLBCL) (standardized incidence ratio [SIR], 8.64; 95% CI, 3.88-19.22; P < .0001), myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) (SIR 9.5; 95% CI, 3.6-25.3; P < .0001), and brain cancer (SIR, 7.59; 95% CI, 1.9-30.4; P < .0001). The risk of a second hematologic malignancy was 5-fold higher in treated than in untreated patients (P = .08). CONCLUSION: Patients with WM are at increased risk of DLBCL, MDS/AML, and brain cancers compared with the general population. Further studies are needed to clarify whether the increased incidence of second cancers is related to treatments, to the immunologic impairment associated with the disease, or to genetic predisposition.

Monoclonal gammopathy of undetermined significance: a new proposal of workup.

OBJECTIVE: Diagnostic criteria for monoclonal gammopathy of undetermined significance (MGUS) require quantification of bone marrow plasma cells (BMPCs) and skeletal survey to discriminate between MGUS and multiple myeloma (MM). By contrast, recent published guidelines suggest that these procedures could be avoided in the presence of serum monoclonal spike (M-spike) of small amount (≤1.5 g/dL). Aim of this study is to better quantify the risk of missing a diagnosis of MM, not performing bone marrow aspirate and skeletal survey in patients with M-spike ≤ 1.5 g/dL asymptomatic for bone pain. METHODS: We reviewed data of 2282 patients consecutively observed from January 1974 to December 2010 in our single hematology department. We considered eligible for this study 1271 patients with grade <2 NCI bone pain, confirmed to have an MGUS or an MM after extensive standardized diagnostic workup including bone marrow biopsy, skeletal bone survey and laboratory tests. RESULTS: The risk of finding a BMPC infiltration ≥10% in patients with an M-spike ≤ 1.5 g/dL was very low (7.3%), although significantly different according to IgH isotype (4.7% for IgG vs. 20.5% for IgA). The risk of finding bone lesions with M-spike ≤ 1.5 g/dL was negligible (2.5%), regardless of IgH isotype. CONCLUSION: In asymptomatic patients with M-spike of small amount (≤1.5 g/dL): (i) BMPC evaluation may be reasonably avoided in patients with IgG M-spike, while should always be part of diagnostic workup in the presence of IgA M-spike and (ii) skeletal survey, less predictive for MM, should not be routinely indicated irrespective of IgH isotype.

Optimal timing of allogeneic hematopoietic stem cell transplantation in patients with myelodysplastic syndrome.

Allogeneic hematopoietic stem cell transplantation (HSCT) represents the only curative treatment for patients with myelodysplastic syndrome (MDS), but involves non-negligible morbidity and mortality. Registry studies have shown that advanced disease stage at transplantation is associated with inferior overall survival. To define the optimal timing of allogeneic HSCT, we carried out a decision analysis by studying 660 patients who received best supportive care and 449 subjects who underwent transplantation. Risk assessment was based on both the International Prognostic Scoring System (IPSS) and the World Health Organization classification-based Prognostic Scoring System (WPSS). We used a continuous-time multistate Markov model to describe the natural history of disease and evaluate the effect of allogeneic HSCT on survival. This model estimated life expectancy from diagnosis according to treatment policy at different risk stages. Relative to supportive care, estimated life expectancy increased when transplantation was delayed from the initial stages until progression to intermediate-1 IPSS-risk or to intermediate WPSS-risk stage, and then decreased for higher risks. Modeling decision analysis on WPSS versus IPSS allowed better estimation of the optimal timing of transplantation. These observations indicate that allogeneic HSCT offers optimal survival benefits when the procedure is performed before MDS patients progress to advanced disease stages.

Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms.

We studied mutations of MPL exon 10 in patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF), first investigating a cohort of 892 consecutive patients. MPL mutation scanning was performed on granulocyte genomic DNA by using a high-resolution melt assay, and the mutant allele burden was evaluated by using deep sequencing. Somatic mutations of MPL, all but one involving codon W515, were detected in 26/661 (4%) patients with ET, 10/187 (5%) with PMF, and 7/44 (16%) patients with post-ET myelofibrosis. Comparison of JAK2 (V617F)-mutated and MPL-mutated patients showed only minor phenotypic differences. In an extended group of 62 MPL-mutated patients, the granulocyte mutant allele burden ranged from 1% to 95% and was significantly higher in patients with PMF or post-ET myelofibrosis compared with those with ET. Patients with higher mutation burdens had evidence of acquired copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p in granulocytes, consistent with a transition from heterozygosity to homozygosity for the MPL mutation in clonal cells. A significant association was found between MPL-mutant allele burden greater than 50% and marrow fibrosis. These observations suggest that acquired CN-LOH of chromosome 1p involving the MPL location may represent a molecular mechanism of fibrotic transformation in MPL-mutated myeloproliferative neoplasms.

Clues to pathogenesis of Waldenström macroglobulinemia and immunoglobulin M monoclonal gammopathy of undetermined significance provided by analysis of immunoglobulin heavy chain gene rearrangement and clustering of B-cell receptors.

We characterized immunoglobulin heavy chain (IGH) gene rearrangements and searched for clusters of stereotyped B-cell receptors in 123 patients with Waldenström macroglobulinemia (WM; n = 59) or immunoglobulin M monoclonal gammopathy of undetermined significance (IgM-MGUS) (n = 64). A productive monoclonal IGHV-D-J rearrangement was obtained in 99/123 patients (80%). Immunoglobulin heavy chain variable (IGHV) genes were mutated in 94/99 patients (95%) with a median somatic hypermutation rate of 6.7% (2.1-14.5). Compared with the normal B-cell repertoire, patients with WM/IgM-MGUS showed an over-representation of the IGHV3 subgroup (83% vs. 55%, p < 0.0001) and an under-representation of IGHV1 (7% vs. 14%, p = 0.04) and IGHV4 (7% vs. 23%, p = 0.0001) subgroups. At the gene level, in WM/IgM-MGUS there was an over-representation of IGHV3-23 (24% vs. 12%, p = 0.0003), IGHV3-64 (3% vs. < 1%, p = 0.003), IGHV3-7 (12% vs. 4%, p = 0.0001) and IGHV3-74 (9% vs. 2%, p < 0.0001), while IGHV4-39 was never used (0 vs. 5%, p = 0.03). Intra-WM/IgM-MGUS search for HCDR3 similarity showed no association fulfilling criteria for stereotyped receptors. WM/IgM-MGUS sequences were unrelated to known chronic lymphocytic leukemia (CLL), splenic marginal zone lymphoma (SMZL) or mantle cell lymphoma (MCL) subsets. In conclusion, the IGHV gene usage in WM and IgM-MGUS is remarkably biased as compared to the normal B-cell repertoire. WM and IgM-MGUS-specific HCDR3 clusters do not occur with a frequency detectable with currently available databases, not supporting a B-cell receptor-driven pathogenesis in WM and IgM-MGUS.

Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstrom's macroglobulinemia and related lymphoid neoplasms.

A study has shown that MYD88 (L265P) is a recurring somatic mutation in Waldenström's macroglobulinemia (WM). We developed an allele-specific polymerase chain reaction (PCR) for this mutation, and analyzed bone marrow or peripheral blood samples from 58 patients with WM, 77 with IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), 84 with splenic marginal zone lymphoma (SMZL), and 52 with B-cell chronic lymphoproliferative disorders (B-CLPD). MYD88 (L265P) was detected in 58/58 (100%) patients with WM, 36/77 (47%) with IgM-MGUS, 5/84 (6%) with SMZL, and 3/52 (4%) with B-CLPD. Compared to IgM-MGUS patients with wild-type MYD88, those carrying MYD88 (L265P) showed significantly higher levels of IgM (P < .0001) and presented Bence-Jones proteinuria more frequently at diagnosis (P = .002). During follow-up, 9 patients with IgM-MGUS progressed to WM or to marginal zone lymphoma. Using a case-control approach, the risk of evolution of patients carrying MYD88 (L265P) was significantly higher than that of patients with wild-type MYD88 (odds ratio 4.7, 95% confidence interval 0.8 to 48.7, P = .047). These findings indicate that the allele-specific PCR we developed is a useful diagnostic tool for patients with WM or IgM-MGUS. In this latter condition, MYD88 (L265P) is associated with greater disease burden and higher risk of disease progression, and the mutation may therefore also represent a useful prognostic marker.

Chronic lymphocytic leukemia with del13q14 as the sole abnormality: dynamic prognostic estimate by interphase-FISH.

This study analyzed 140 patients with isolated del13q14 on interphase FISH (I-FISH), to identify subsets with a different progression risk and to assess the acquisition of additional chromosomal abnormalities (clonal evolution) in treatment-naïve del13q14 patients. A monoallelic deletion (del13qx1) was detected in 123 cases (88%), a biallelic deletion (del13qx2) in eight and a mosaic of monoallelic and biallelic deletions (del13qx1/del13qx2) in nine. In 33% of cases, deletion encompassed the Rb1 locus The median percentage of abnormal nuclei was 50% (15%-96%), and it was higher in patients with a biallelic/mosaic pattern in comparison with patients with monoallelic deletion. Sixty two patients (44%) have been treated; 5-year treatment free survival rate was 56% and the median treatment free survival was 65 months. The baseline percentage of deleted nuclei, as a continuous variable, was related to progression (HR: 1.02; p = 0.001). According to deletion burden, three groups were identified: 64 cases (46%) had <50% deleted nuclei, 47 (33%) had 50-69% deleted nuclei, and 29 (21%) had ≥70% deleted nuclei. The 5-year untreated rate was 70.5% , 52.6% and 28.7% (p < 0.0001), respectively. In multivariate analysis using IGHV mutational status, presence of a nullisomic clone, CD38 expression and percentage of deleted nuclei as covariates, only IGHV mutational status and the percentage of deleted nuclei were independent risk factors for treatment. In 103 patients serially monitored by I-FISH before starting any treatment, we observed a significant increase in the proportion of del13q14 cells, and this increase affected the risk of subsequent treatment requirement (HR 2.54, p = 0.001). The appearance of a new clone was detected in 16 patients (15.5%) and chromosome 13 was involved in 14 of them. I-FISH monitoring proves worthwhile for a dynamic risk stratification and for planning clinical surveillance.