Follow-up examinations including sequential bone marrow biopsies in essential thrombocythemia (ET): A retrospective clinicopathological study of 120 patients

Abstract Background: the Registro Italiano Trombocitemia, that is a GIMEMA project, has been activated to registry Italian Essential Thrombocythemia (ET) patients, to improve the diagnosis appropriateness (WHO criteria), to verify the prognostic value of the clinical and biological parameters, to evaluate the compliance to the therapeutical Italian guidelines (1), and to create a network for activation of new studies. Objective: this analysis is mainly devoted to describe the ET patients registered in the RIT and to evaluate the therapeutic approach adopted in the 102 participating hematological centers. Material and methods: two thousand and fifteen ET patients have been registered after the written informed consent was obtained, and data validation by various expert panels is in progress. This preliminary report considers 1785 patients, diagnosed mainly (1078, 60.4%) since the publication in the year 2004 of the ET therapy Italian guidelines (1). Results: the patients, 678 (38%) males and 1107 (62%) females, showed at diagnosis: age 60.3 ± 16.8 years with higher values in males than in females (61.7 ± 15.3 vs. 59.4 ± 17.7, p<0.05), being the patients below 40 years 14% and those over 70 years 33% of cases; PLT count (109/L) 846 ± 309 with lower values in males than in females (813 ± 261 vs. 866 ± 334, p<0.002), and with values 1001–1500 and over 1500 in 16% and 4% of cases, respectively; WBC count (109/L) 9.1 ± 2.9, without difference by sex, and with values 12–15 in 10% and over 15 in 3% of cases; Hgb (g/dL) 14.2 ± 1.6 with higher values in males than in females (14.8 ± 1.5 vs. 13.8 ± 1.5, p<0.001), and with values over 16.5 in 8.5% of males and 2.7% of females, respectively; splenomegaly in 488 (27%), echo-documented in 324 cases (18%); history of hemorrhage and thrombosis in 90 (5%) and 325 (19%) of cases, respectively; disease-related symptoms in 41% and general thrombotic risk factors in 93% of cases, respectively. The WHO 2001 diagnostic criteria were reported for 33% of cases observed before the year 2004 and for 53 % of cases observed since the year 2004. Detailed data at diagnosis were reported as follows: bone marrow biopsy in 1087 cases (61%) with a frequency of 51% and 68% before and since the year 2004, respectively; bcr-abl study in 1045 cases (59%); cytogenetics in 828 cases (46%) with karyotype abnormalities in 27 patients (3%). The JAK2 V617F mutation, searched in 574 cases (32%), was observed in 320 of them (56%). The patient follow-up was 4.5 ± 4.5 years with a total of 5245 pt-yrs. During the follow-up the hemorrhagic events were 5.7% (1.3/100 pt-yrs), being the major events 1.9% (0.4/100 pt-yrs); the thrombotic complications were 14.9 % (3.3/100 pt-yrs), resulting the major arterial 9.4% (2.1/100 pt-yrs), the major venous 3.5% (0.8/100 pt-yrs) and the minor thrombosis 2% (0.4/100 pt-yrs). An antiplatelet treatment, almost always with low dose aspirin, was performed in 75% of the patients, without significant difference in the cases diagnosed before and since the 2004. A cytoreductive treatment was done with use of Hydroxyurea (HU, 64%), Interferon alpha (IFN, 16%), Anagrelide (ANA, 15%), Busulfan (BUS, 4%), and Pipobroman (PIPO, 2 %). In the ET patients diagnosed since the year 2004 respect those diagnosed before, it was observed a decrease in the use of all the cytoreductive drugs, particularly BUS (−62%), IFN ((−62%), and ANA ((−68%). The use of the cytoreductive drugs was related to the patient mean age (years): BUS (76), PIPO (72), HU (67), ANA (53), IFN (48). In the patients diagnosed since the 2004 as compared with those before 2004, the mean age of the treated patients increased for BUS (from 69 to 81 yrs, p<0.001) and for HU (from 64 to 69 yrs, p<0.001) while it decreased for IFN (from 49 to 46 yrs, p<0.05). Conclusion: in the analyzed patients of the ET Italian registry the diagnosis appropriateness resulted improved in the cases observed since the year 2004 respect those observed before, with an increase of bone marrow biopsies from 51% to 68% of patients. Moreover, in accord with the ET therapy Italian guidelines, the use of the cytoreductive drugs was less frequent in the patients diagnosed since the year 2004 than before (particularly for BUS, IFN, and ANA) and the more safe molecules IFN and ANA were preferentially deserved to the younger patients.

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Novel Insights Regarding Pediatric Essential Thrombocythemia

Blood ◽

10.1182/blood-2019-123865 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4191-4191

Author(s):

Assaf Arie Barg ◽

Gili Kenet ◽

Tami Livnat ◽

Gal Goldstein ◽

Joanne Yacobovich ◽

...

Keyword(s):

Bone Marrow ◽

Clinical Data ◽

Essential Thrombocythemia ◽

Research Funding ◽

Complete Blood Count ◽

Myeloproliferative Neoplasm ◽

Bone Marrow Biopsies ◽

Acquired Von Willebrand Syndrome ◽

Von Willebrand

Background: Essential thrombocythemia (ET) is a chronic myeloproliferative neoplasm. As it is extremely rare in children, data regarding its clinical course are scarce and pediatric treatment guidelines are lacking. Aim: To evaluate diagnosis, treatment and clinical outcome in a group of pediatric ET patients. Methods: Medical files of all pediatric patients (age 0-18 years) diagnosed with ET between January 2010 and February 2019 in three tertiary hospitals were reviewed. Study was approved by all institutional ethics committees. Diagnosis was established according to the WHO criteria of ET. All patients had undergone bone marrow biopsy (BMB) and molecular evaluation for JAK2V617F. Patients with wild type JAK2V617F were also tested for JAK2 exon 12 mutation, calreticulin (CALR) mutations and thrombopoietin receptor (MPL) mutation. Complete blood count parameters at first evaluation and follow up were collected. Lag in diagnosis, defined as the period between the time at which thrombocytosis was first noticed until diagnosis of ET was documented. Patients were evaluated for acquired von Willebrand syndrome (AVWS) by testing for von Willebrand antigen level and activity. Clinical data included any adverse events particularly those related to thrombosis or bleeding. Initial treatment strategies and any need for therapy modifications were recorded. Results: Twelve children (5 males and 7 females) followed for a median time of 27.5 months (range 4-108 months) were included. Table 1 displays their demographic and clinical data. Family history of thrombocytosis was negative in all patients. Median age at which thrombocytosis was first noted was 8 years (range 1-14.5 years). In 5/12 patients thrombocytosis was detected as an incidental finding. In 7/12 patients CBC was performed due to symptoms including headache, visual disturbances, seizure and acroparesthesia (table 1). Patients who suffered from neurological symptoms had undergone cranial MRI; all were interpreted as normal. The mean lag period between the time in which thrombocytosis was first noted until diagnosis of ET was 36 months (range: 0.1-120 months). Molecular diagnosis yielded 5/12 patients who were positive for JAK2V617F, one patient with a JAK2 exon 12 mutation and 2/12 patients with mutations involving CALR (one with type 1 and one with type 2 mutation). No subjects with CMPL mutation were detected. Four children tested negative for all mutations. Bone marrow biopsies were compatible with ET and no chromosomal aberrations were identified in our cohort. Evaluation for AVWS was performed in nine of the panties. It was diagnosed in 67% of assessed patients. Median VWF:Rco/VWF:Ag 0.18 (range: 0.01-0.76). At diagnosis treatment with Aspirin was initiated in 4/12 patients. Cytoreductive therapy with Hydroxyurea was added at diagnosis in 2/4 patients, both symptomatic at presentation. One Patient underwent plateletpheresis at presentation due to severe headache and extreme thrombocytosis. In 3/8 untreated patients, therapy was added during follow up, with either Aspirin (n=1, due to increased severity of headaches and raising platelet count) or Hydroxyurea (n=2, following TIA). During follow up period neither leukemia nor myelofibrosis evolved in our cohort. One patient developed a provoked DVT, secondary to a femoral CVL. Three patients experienced TIA during study period. Two females experienced excessive bleeding (heavy menstrual bleedings and bleeding due to a raptured corpus luteum), both diagnosed with AVWS. Conclusions: Our study suggests that pediatric hematologists should increase awareness to ET as delayed diagnosis is common. Among children with ET, AVWS may be more prevalent as compared to adults and may increase the risk of bleeding. Further collaborative multicenter studies are required for robust data collection and may facilitate future ET treatment in children. Table 1 Disclosures Kenet: Alnylam: Consultancy, Honoraria, Research Funding; CSL: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Opko Biologics: Consultancy, Honoraria, Research Funding; BPL: Research Funding. Steinberg Shemer:Emendo bio: Consultancy. Revel-Vilk:Prevail therapeutics: Honoraria, Other: Travel, Research Funding; Sanofi: Honoraria, Other: Travel, Research Funding; Pfizer: Honoraria, Other: Travel, Research Funding; Takeda: Honoraria, Other: Travel, Research Funding.

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Acute panmyelosis with myelofibrosis: a clinicopathological study on 46 patients including histochemistry of bone marrow biopsies and follow-up

Annals of Hematology ◽

10.1007/s00277-004-0881-8 ◽

2004 ◽

Vol 83 (8) ◽

Cited By ~ 18

Author(s):

J. Thiele ◽

H.M. Kvasnicka ◽

G. Zerhusen ◽

J. Vardiman ◽

V. Diehl ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Biopsies ◽

Clinicopathological Study

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BCR-ABL Transcripts in Bone Marrow Aspirates of Philadelphia-Negative Essential Thrombocythemia Patients: Clinical Presentation

Blood ◽

10.1182/blood.v90.7.2768 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2768-2771 ◽

Cited By ~ 32

Author(s):

Dorit Blickstein ◽

Adina Aviram ◽

Jacob Luboshitz ◽

Miron Prokocimer ◽

Pinhas Stark ◽

...

Keyword(s):

Bone Marrow ◽

Essential Thrombocythemia ◽

Clinical Presentation ◽

Molecular Level ◽

Philadelphia Chromosome ◽

Nested Polymerase Chain Reaction ◽

History Of ◽

Polymerase Chain ◽

Positive Groups

Abstract One of the diagnostic criteria of essential thrombocythemia (ET) is the absence of the Philadelphia chromosome (Ph-neg). On the molecular level, Ph-neg ET patients may carry BCR-ABL transcript. The natural history of BCR-ABL positive Ph-neg ET patients is undetermined. We examined the BCR-ABL status by reverse transcriptase two-step nested polymerase chain reaction in bone marrow aspirates of 25 Ph-neg ET patients. We found 12 BCR-ABL positive and 13 BCR-ABL negative patients in the study group. The comparison showed that the two groups had similar clinical and laboratory characteristics, except for a significant increased patients' age and decreased polymorphonuclear cell count in the BCR-ABL positive group. During a median follow-up of 20 and 22.5 months for the BCR-ABL negative and positive groups, respectively, there was neither blastic transformation nor unrelated death in both groups. We conclude that it is important to look for BCR-ABL transcript in Ph-neg ET patients and to follow them closely to investigate the nature of this translocation in this group of patients.

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Treatment of hairy cell leukemia with alternating cycles of pentostatin and recombinant leukocyte A interferon: results of a phase II study.

Journal of Clinical Oncology ◽

10.1200/jco.1990.8.4.721 ◽

1990 ◽

Vol 8 (4) ◽

pp. 721-730 ◽

Cited By ~ 22

Author(s):

A Martin ◽

S Nerenstone ◽

W J Urba ◽

D L Longo ◽

J B Lawrence ◽

...

Keyword(s):

Bone Marrow ◽

Hairy Cell Leukemia ◽

Pathologic Complete Response ◽

Hairy Cell ◽

Cell Leukemia ◽

Cell Infiltrate ◽

Recombinant Interferon ◽

Bone Marrow Biopsies ◽

Hairy Cells

Fifteen patients with hairy cell leukemia (HCL) were treated with deoxycoformycin (pentostatin; dCF) (4 mg/m2 intravenous [IV] every week x 3) and recombinant interferon-alpha 2a (rIFN-alpha 2a) (3 x 10(6) units subcutaneously [SC] daily x 4 weeks) in alternating months for a total of 14 months. Eleven patients had undergone splenectomy; four had received prior systemic therapy with chlorambucil and/or steroids. All 15 are evaluable for toxicity and peripheral blood response, while 14 are assessable for bone marrow response. Toxicity was tolerable with grade 3 or 4 nausea and vomiting in three patients, neutropenic fevers in five, transient but significant depression in eight, and localized cutaneous herpes zoster in four. Circulating hairy cells were undetectable by the end of the first month in 10 of 13 patients, and by the end of the second month in the other three. Fourteen patients had bilateral bone marrow biopsies performed at baseline after 6 months of treatment, at the end of treatment (14 months), and at 6-month intervals during follow-up. Before treatment, all patients had hypercellular marrows with hairy cels replacing normal marrow elements; all showed at least a 95% clearing of their hairy cell infiltrate by 6 months of therapy. However, small collections of residual hairy cells could be detected intermittently on at least one side of bilateral samples in all patients. All patients have completed treatment with a median duration of follow-up off therapy of 27 months (range, 15 to 31 months). To date, all peripheral counts and serum soluble interleukin-2 receptor (sIL2R) levels remain stable, and no patient has had progression of the hairy cell infiltrate in the bone marrow. Although no patient achieved a pathologic complete response, alternating monthly cycles of dCF and rIFN-alpha 2a produced durable partial remissions (PRs) in all patients. Continued follow-up is required to determine the length of such remissions.

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Clinical Relevance of Bone Marrow Biopsy in Staging and Follow-Up of Breast Cancer

Tumori Journal ◽

10.1177/030089168306900210 ◽

1983 ◽

Vol 69 (2) ◽

pp. 143-150 ◽

Cited By ~ 2

Author(s):

Giorgio Cruciani ◽

Gian Maria Fiorentini ◽

Giovanni Rosti ◽

Amelia Tienghi ◽

Daniele Bardella ◽

...

Keyword(s):

Breast Cancer ◽

Bone Marrow ◽

Bone Metastases ◽

Bone Marrow Biopsy ◽

Clinical Relevance ◽

Bone Biopsy ◽

Bone Marrow Biopsies ◽

Female Patients

Bone marrow biopsies by Jamshidi needle were performed in 106 breast cancer female patients. Sixty-four of them were in follow-up after mastectomy, and neoplastic involvement of marrow was found in 21 patients (32.8%). Among the 42 women undergoing staging before mastectomy, the incidence of marrow involvement was 11.9% (5 women, all with radiographic positivity). Of the 37 women, either in follow-up or in the staging phase, with bone metastases detected by roentgenographic and isotopic examination, the bone biopsy was positive in 23 (62.1%), and 7 histologically had micrometastases. Three women, without any radiographic or isotopic sign of metastases, had positive biopsies. A good correlation was found between the hydroxyproline:creatinine ratio and neoplastic involvement of bone marrow.

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Isolated Chromosome 20q Deletions in Patients with Non-Myeloid Disorders

Blood ◽

10.1182/blood.v120.21.1442.1442 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1442-1442

Author(s):

Dean Andrea ◽

Melissa Hayden ◽

Kathleen W. Rao ◽

Yuri D. Fedoriw ◽

Bahjat Qaquish ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Lymphocytic Leukemia ◽

Clinical Evidence ◽

B Cell Lymphoma ◽

Lymphocytic Leukemia ◽

Red Blood Cell Transfusion ◽

Bone Marrow Biopsies ◽

Chromosome 20 ◽

Dose Modifications

Abstract Abstract 1442 Background: Deletions of the long arm of chromosome 20 have conventionally been associated with myeloid neoplasms. The frequency of chromosome 20q deletion (del(20q)) is 1–10% in myeloid diseases and associated with good prognosis in patients with myelodysplastic syndromes (MDS) and poor response to treatment in patients with acute myeloid leukemia (Greenberg 1997; Campel 1994). Previously chromosome 20q deletions were thought to be pathognomonic for myelodysplastic syndrome. Due to the observation that del(20q) may be present in non-malignant clones in patients with non-myeloid cancers, the 2008 WHO Classification (Swerdlow ed.) stated that MDS could not be diagnosed solely on the basis of isolated del(20q). The significance of isolated del(20q) in non-myeloid disorders have yet to be defined. When this chromosomal abnormality is found incidentally in marrows of patients for non-myeloid cancers, some oncologist may consider altering plans for cytotoxic chemotherapy out of concern for developing MDS. The aim of this study was to determine if isolated del(20q) in non-myeloid disorders implies an impending myeloid disease and if treatment should be altered for such patients. Methods: We conducted a retrospective, single institution cohort study among patients who between January 2005 and July 2012 were found to have 20q deletions without other chromosomal alterations per conventional cytogenetics and non-myeloid disorders per clinical history or bone marrow biopsies. Patients with isolated 20q deletion were identified from the clinical cytogenetic laboratory database and results were reviewed per cytogeneticist for accuracy. Pathology reviewed initial and subsequent bone marrow biopsies for histopathologic or immunophenotypic evidence of a myeloid disease. If a subsequent bone marrow exam was not available, the patient's complete blood counts were reviewed to determine if they developed clinical evidence of a myeloid disorder. We defined clinical suspicion for MDS as the development of transfusion dependence (≥1 unit red blood cell transfusion every 8 weeks over 4 months), any grade = 2 anemia with either grade = 2 thrombocytopenia or grade = 2 neutropenia not related to chemotherapy or immunotherapy, or any grade = 3 cytopenia without known etiology. For patients undergoing chemotherapy, MDS was suspected if there was evidence of poor bone marrow reserve as manifest by dose modifications or delays for hematologic toxicity. Results: Thirty nine patients with isolated del(20q) were identified in the cytogenetic database from January 2005 to July 2012. Twelve out of thirty nine (31%) patients were found to have non-myeloid disorders. Three patients with multiple myeloma (25%), two patients with chronic lymphocytic leukemia (17%), two patients with autoimmune disorders (17%) and five others with breast cancer, diffuse large B cell lymphoma, monoclonal gammopathy of undetermined significance, Crohn's disease and melanoma. There were an equal number of men and woman with median age of 60 years (range 30–83 years) at the time of isolated del(20q) detection. Six patients were found to have del(20q) at the initial presentation of their disease and six developed del(20q) after undergoing treatment. Nine patients were treated with standard first line systemic therapies. Six of the nine patients were treated with chemotherapy and four of them did not have to undergo any dose modifications due to myelosuppression. In the patients with chronic lymphocytic leukemia, FCR (Fludarabine,Cyclophosphamide and Rituximab) was dose modified and later discontinued due to persistent neutropenia and thrombocytopenia. After a median follow up of twenty two months (range 2 – 64 months) no patients developed evidence of a myeloid disorder by bone marrow pathology or clinical evidence. Conclusion: Isolated deletion of the long arm of chromosome 20 in patients with non-myeloid disorders does not result in bone marrow failure or myeloid disease, therefore physicians should not alter their treatment plans. Further patient follow up is necessary to provide more insight on the prognosis and treatment of non-myeloid disorders with isolated chromosome 20q deletion. Disclosures: No relevant conflicts of interest to declare.

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Diagnostic Problems in Follow-Up Bone Marrow Biopsies of Patients Treated for Acute and Chronic Leukaemias and MDS

Pathobiology ◽

10.1159/000101710 ◽

2007 ◽

Vol 74 (2) ◽

pp. 115-120 ◽

Cited By ~ 5

Author(s):

Stephan Dirnhofer ◽

Philip Went ◽

André Tichelli

Keyword(s):

Bone Marrow ◽

Bone Marrow Biopsies ◽

Diagnostic Problems

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A Large Proportion of Patients With a Diagnosis of Essential Thrombocythemia Do Not Have a Clonal Disorder and May Be at Lower Risk of Thrombotic Complications

Blood ◽

10.1182/blood.v93.2.417 ◽

1999 ◽

Vol 93 (2) ◽

pp. 417-424 ◽

Cited By ~ 150

Author(s):

Claire N. Harrison ◽

Rosemary E. Gale ◽

Samuel J. Machin ◽

David C. Linch

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Essential Thrombocythemia ◽

Lower Risk ◽

Study Group ◽

Reactive Thrombocytosis ◽

Age Related ◽

Hemorrhagic Complications ◽

Thrombotic Complications

Abstract Essential thrombocythemia (ET) is traditionally considered to be a clonal disorder. No specific karyotypic abnormalities have been described, but the demonstration of clonality using X-chromosome inactivation patterns (XCIPs) has been used to differentiate ET from a non-clonal reactive thrombocytosis. However, these assays may be difficult to interpret, and contradictory results have been reported. We have studied 46 females with a diagnosis of ET according to the Polycythemia Vera Study Group (PVSG) criteria. XCIP results in 23 patients (50%) were uninterpretable due to either constitutive or possible acquired age-related skewing. Monoclonal myelopoiesis could be definitively shown in only 10 patients. Thirteen patients had polyclonal myelopoiesis, and in 8, it was possible to exclude clonal restriction to the megakaryocytic lineage. Furthermore, there was no evidence of clonal progenitors in purified CD34+CD33− and CD34+CD33+ subpopulations from bone marrow of 2 of these 13 patients. There was no difference between patients with monoclonal and polyclonal myelopoiesis with respect to age or platelet count at diagnosis, duration of follow-up, incidence of hepatosplenomegaly, or hemorrhagic complications. However, polyclonal patients were less likely to have experienced thrombotic events (P = .039). These results suggest that ET is a heterogeneous disorder, and the clinical significance of clonality status warrants investigation in a larger study.

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Ph-Abnormal Clones Emerged during Imatinib Therapy: Clinical Report and Clonal Analyses on 23 Patients from GIMEMA Working Party (GWP) in CML Registry.

Blood ◽

10.1182/blood.v104.11.2936.2936 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2936-2936

Author(s):

Elisabetta Abruzzese ◽

Alessandro Gozzetti ◽

Alfonso Zaccaria ◽

Nicoletta Testoni ◽

Sara Galimberti ◽

...

Keyword(s):

Cell Culture ◽

Bone Marrow ◽

Biological Significance ◽

Specific Inhibitor ◽

Chronic Phase ◽

Working Party ◽

Clinical Report ◽

Bone Marrow Biopsies ◽

Cytogenetic Abnormalities

Abstract Imatinib mesylate (Glivec, Novartis) is a tyrosine kinase specific inhibitor that kills BCR-ABL cells in vitro and in vivo. Cytogenetic abnormalities in Ph-negative cells emerging after treatment-induced suppression of the neoplastic clone have been described. A registry through the GWP in CML has been set and data on 23 patients collected. To acquire insights into the origin of the Ph-negative clone as well as the evolution of the coexisting Ph− and Ph+ cell populations, we have analyzed bone marrow cell segregation, cell culture and morphologic features. Patients characteristics and 28 months follow up are presented. The emergence of a cytogenetic abnormal clone in Ph-negative cells was evidenced in 23 patients after a median of 14.5 months after starting Imatinib. Median age was 51 years, median time from diagnosis 36 months. All patients started Imatinib while in chronic phase and none of the patients had ever presented accelerated or blastic phase. Five patients were treated with Imatinb at onset. Cytogenetics at diagnosis was characterized by the presence of Ph chromosome, except for one patient which presented with normal karyotype, but BCR-ABL B3A2 transcript. No additional abnormalities were evidenced except for one patient which presented with the Ph and a dup(1q)(q11q21). All patients achieved a good response to Glivec with 16 complete, 4 major and 3 minor cytogenetic remissions when additional abnormalities were noticed in Ph-negative cells. The clonal cytogenetic abnormalities included +8 in 13 patients, -Y in 2 patients, one −7, del(5q), del(7q), del(13q), t(6;7)(p24;q21), t(2;6)(p25;q23), and one patient presenting with both +8 and +21. The patient with dup(1q) maintained the abnormality while clearing the marrow from Ph positive cells (constitutional karyotype was normal). Retrospective analyses of stored pellet using FISH in patients presenting +8, −7, or −Y, did not evidence abnormalities in previous samples. Patients that lost cytogenetic response showed that the percentage of the Ph+ cells inversely correlated to the abnormal clone. In 5 patients the abnormal clone was not evidenced in subsequent controls, suggesting the possibility that the abnormalities could be temporary. We performed cell culture on a subgroup of patients demonstrating normal growth in four patients and an abnormal growth in one patient with reduced CFU formation affecting BFU-Es, CFU-GM, and colony size microclusters. FISH analyses on separated CD34+ and CD34-negative cells evidenced that the abnormal clone segregated into the CD34+ compartment suggesting the stem cells involvement. FISH on cultured cells did not demonstrate a growth advantage for Ph+ cells or for the new clone. Bone marrow biopsies presented with reduced cellularity, normal differential and mild dysplastic signs as documented in patients responding to Imatinib. No increased angiogenesis was evidenced. While a longer follow up observation and laboratory analyses are required, we remark that after >2 years follow up the Ph-negative abnormal clone did not tend in our patients to evolve in MDS, nor it seems to be associated with CML clonal evolution and disease progression. Hypothesis regarding the biological significance of these abnormalities are formulated.

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