Massimiliano Postorino

Prognostic relevance of the expression of Tdt and CD7 in 335 cases of acute myeloid leukemia

We have analyzed the expression of Tdt and CD7 in 335 cases of unequivocal acute myeloid leukemia (AML). Tdt was expressed in 80 (25%) of 321 evaluable cases. Twenty-six of 77 (34%) Tdt+ patients assessable for response, entered complete remission (CR) vs 121 of 209 (58%) Tdt− cases (P < 0.001). cd7 was expressed in 102 of 332 (30%) evaluable cases; 37 of 93 assessable (40%) cd7+ patients attained a CR as compared to 114/204 (56%) CD7− (P = 0.013). Duration of survival was significantly shorter for patients with CD7+ or Tdt+ AML (P = 0.006 and 0.001, respectively). In a multivariate analysis, Tdt was found to significantly adverse achievement of CR (P = 0.018), while CD7 affected duration of CR (P = 0.037). Overall the expression of either Tdt or CD7 correlated with a relatively high expression of CD34 (P < 0.001), gp-170 (P = 0.003), lymphoid antigens (LyAg) (P < 0.001), t(9;22) or anomalies of chromosome 5/7 (P < 0.001). finally, we pooled the patients into four phenotypic classes, according to the presence of tdt, cd7 or both: [tdt−CD7−], [Tdt+CD7−], [Tdt−CD7+] and [Tdt+CD7+]. The category [Tdt+CD7+] was characterized by a more unfavorable outcome as suggested by a lower rate of CR (P < 0.001) and a shorter duration of survival as compared to cases [tdt−CD7−], [Tdt+CD7−] and [Tdt−CD7+] (P = 0.002). This figure is consistent with the frequent convergence in the subset [Tdt+CD7+] of GP-170 positivity (P = 0.003), translocation t(9;22), anomalies of chromosome 5 and/or 7 (P < 0.001) and signs of lineage infidelity (deviant expression of lymphoid antigens) (P < 0.001). we conclude that the expression of tdt or cd7 is associated with an unfavorable outcome and that the combination of both defines a clinical subset with a poorer prognosis due to the significantly higher association with mdr phenotype, and ‘poor prognostic’ chromosomal abnormalities.

An Allele-Specific RT-PCR Assay to Detect Type A Mutation of the Nucleophosmin-1 Gene in Acute Myeloid Leukemia

Nucleophosmin-1 (NPM1) mutations represent the most frequent gene alteration in acute myeloid leukemia (AML). The most common NPM1 mutation type, accounting for 75 to 80% of cases, is referred to as mutation A (NPM1-mutA). These NPM1 alterations have been shown to possess prognostic significance because they appear to identify patients who will benefit from chemotherapy. Given the high prevalence and stability of these mutations over the course of disease, NPM1 mutations may serve as ideal targets for minimal residual disease (MRD) assessment in AML. Current detection methods are costly, require sophisticated equipment, and are often not sufficiently sensitive. We report here an allele-specific (ASO)-RT-PCR assay that enables rapid and sensitive detection of NPM1-mutA. A semi-nested ASO-PCR method was also designed to increase the sensitivity of our assay for the monitoring of MRD. We analyzed bone marrow cells collected from 52 patients with AML at presentation. NPM1-mutA was detected in leukemic cells from 21 patients. Assay specificity was confirmed by capillary electrophoresis and DNA sequencing. ASO-RT-PCR and semi-nested ASO-PCR assays could detect NPM1-mutA with sensitivities of 10(-2) and 10(-5), respectively. Results obtained here verify that our ASO-RT-PCR assay is a specific and sensitive method for the routine screening of NPM1-mutA, as well as for MRD monitoring of AML patients with this alteration. This method is convenient and easily applicable in countries with limited resources and no access to real-time quantitative PCR-based technologies.

P-glycoprotein and BCL-2 levels predict outcome in adult acute lymphoblastic leukaemia

Summary. Concurrent resistance mechanisms, such as P‐glycoprotein (PGP) and bcl‐2, may contribute to a worse outcome in adult acute lymphoblastic leukaemia (ALL). Between 1990 and 2000, we analysed PGP and bcl‐2 by flow cytometry, using two anti‐PGP (C219 and JSB‐1) monoclonal antibodies (mAbs) and an anti‐bcl‐2 mAb in 115 de novo adult ALL patients. Both a longer overall survival (OS) and longer disease‐free survival (DFS) were observed in PGP‐negative patients (23%vs 0% at 3 years, P = 0·011 and 29%vs 0% at 2 years, P = 0·006 for C219 respectively; 42%vs 0% at 1·5 years, P = 0·004 and 53%vs 0% at 8·5 months, P = 0·00006 for JSB‐1 respectively). Bcl‐2 positivity was associated with a significantly higher complete remission rate (90%vs 66%, P = 0·01). Moreover, in 69 patients not presenting with either t(9;22) or B‐mature immunophenotype, PGP negativity (JSB‐1) maintained its significant favourable prognostic impact with regard to OS (41%vs 0% at 1·5 years, P = 0·009) and DFS (83%vs 0% at 6 months, P = 0·0005). Importantly, within a subset of 62 patients with normal (n = 31) or unknown (n = 31) karyotype, PGP (JSB‐1)‐negative patients showed both a significantly longer OS and DFS (63%vs 0% at 1·4 years, P = 0·018 and 84%vs 0% at 6 months, P = 0·001 respectively). In multivariate analysis, JSB‐1 (P = 0·008) and cytogenetics (P = 0·02) were found to be independent prognostic factors with regard to DFS. Therefore, in adult ALL, PGP and bcl‐2 represent sensitive indicators of clinical outcome, and potential targets of novel molecules aimed at overcoming chemoresistance and recurrent relapses.

CD90/Thy-1 is preferentially expressed on blast cells of high risk acute myeloid leukaemias*

Different transformation mechanisms have been proposed for elderly acute myeloid leukaemia (AML) and secondary AML (sAML) when compared with de novo AML or AML of younger patients. However, little is known regarding differences in the immunophenotypic profile of blast cells in these diseases. We systematically analysed, by flow cytometry, 148 patients affected by de novo (100 cases) or sAML (48 cases). By defining a cut‐off level of 20% of CD34+ cells co‐expressing CD90, the frequency of CD90+ cases was higher in sAML (40%) versusde novo AML (6%, P < 0·001), elderly AML (>60 years) (24%) versus AML of younger patients (10%, P = 0·010) and poor‐ versus good‐risk karyotypes (according to the Medical Research Council classification, P < 0·001). The correlation between CD90 expression, sAML and unfavourable karyotypes was confirmed by analysing the subset of CD34+ AML cases alone (91/148). Consistently, univariate analysis showed that expression of CD90 was statistically relevant in predicting a shorter survival in CD90+ AML patients (P = 0·042). Our results, demonstrating CD90 expression in AML with unfavourable clinical and biological features, suggest an origin of these diseases from a CD90‐expressing haemopoietic progenitor and indicate the use of CD90 as an additional marker of prognostic value in AML.

Infections increase the risk of central venous catheter-related thrombosis in adult acute myeloid leukemia

INTRODUCTION Central venous catheters (CVC) related thrombosis (CRT) represents a well known complication in patients with acute myeloid leukemia (AML) receiving intensive chemotherapy but the efficacy of antithrombotic prophylaxis still remains controversial. PATIENTS AND METHODS We analyzed 71 consecutive AML patients whose CVC was inserted before each chemotherapy cycle for an overall number of 106 CVC placements. In 47/106 insertions, a prophylaxis with 100 IU/kg/day low molecular weight heparin (LMWH) was administered for 7 days after CVC insertion and additional 7 after CVC removal. This unconventional dose of LMWH, although higher than usual, appeared adequate for a short-course approach. LMWH was delivered regardless of the platelet (PLT) count once provided that it should have been maintained above 20 x 10(9)/L by transfusions. RESULTS Of 106 insertions, we observed 19 (18%) episodes of CRT, 58 (54%) of sepsis and 50 (47%) infections of CVC-exit site with no difference between LMWH and no-LMWH group. Occurrence of CRT was significantly associated with CVC-exit site infections (14/19, p=0.01) and sepsis (16/19, p=0.005) with no difference between LMWH and no-LMWH group. In multivariate analysis, both CVC-exit site infections and sepsis were confirmed to be independent risk factors for CRT development. CONCLUSION Our retrospective study, although based on a small sample size, suggests that the occurrence of CVC-exit site infections and neutropenic sepsis following chemotherapy significantly increases the risk of CRT in AML, independently from the use of LMWH prophylaxis.

PERCUTANEOUS KYPHOPLASTY: INDICATIONS AND TECHNIQUE IN THE TREATMENT OF VERTEBRAL FRACTURES FROM MYELOMA

Aims and background The purpose of our research was to assess the effectiveness and safety of percutaneous kyphoplasty, the new method of treatment for pain deriving from vertebral compression fractures due to myeloma. Methods We treated 3 patients with pain refractory to conventional medical therapy (analgesics, bed-rest, bracing with orthopedic devices for more than 4 weeks), localized in the lumbar area, painful to the touch and in the absence of neurological signs. Results The method demonstrated a swift pain relief associated with an evident augmentation in the resistance and restoration of the vertebral bodys physiological shape. Polymethylmethacrylate leaks were not observed in the epidural or foraminal area, nor were complications such as pulmonary embolism for involvement of the venous plexus or related to phenomenon of infections due to the procedure, or toxicity due to the polymethylmethacrylate. Conclusions Kyphoplasty was found to be an effective alternative, simple and safe in the treatment of vertebral collapse consequent to multiple myeloma. The same injection of polymethylmethacrylate can be done before the radiotherapy treatment, thereby synergizing its delayed analgesic action to pain, or after the failure or in the case of local recurrence.

Fibrin glue for refractory hemorrhagic cystitis after unrelated marrow, cord blood, and haploidentical hematopoietic stem cell transplantation.

BACKGROUND: Patients undergoing hematopoietic stem cell transplant (HSCT) are particularly exposed to the risk of developing hemorrhagic cystitis (HC), which is characterized by symptoms ranging from macroscopic hematuria to renal failure. Although HC significantly affects the quality of life and in a few cases becomes intractable leading to patient death, its therapeutic management has not been established. Fibrin glue (FG), a hemostatic agent derived from human plasma, has been largely employed in different surgical settings including urologic procedures.

Sezary syndrome in relapse after reduced intensity allogeneic transplant successfully treated with donor lymphocyte infusion.

Sezary syndrome in relapse after reduced intensity allogeneic transplant successfully treated with donor lymphocyte infusion

A cluster of Geotrichum clavatum (Saprochaete clavata) infection in haematological patients: a first Italian report and review of literature

Invasive fungal infections, usually Aspergillus and Candida, represent a major cause of morbidity and mortality in patients with malignant haematological diseases, but in the last years rare fungal infections have more frequently been reported. Here, we report the clinical history of three patients affected with haematological malignancies who developed an infection caused by Geotrichum (G.) clavatum. Two out of three patients were affected by acute myeloid leukaemia (AML), and one by mantle cell lymphoma (MCL). All patients received cytarabine‐based chemotherapeutic regimens and developed G. clavatum infection within 3 weeks from therapy initiation. In all cases, G. clavatum was isolated from central venous catheter and peripheral blood cultures. In vitro susceptibility test confirmed an intrinsic resistance to echinocandins and, in all cases, visceral localisations (spleen, liver and lung) were documented by total body computed tomography (CT) scan. A prolonged antifungal therapy with high doses liposomal amphotericin‐B was necessary to obtain fever resolution. Only the patient with MCL died while the other two AML recovered, and one of them after received an allogeneic stem cell transplantation. We consecutively reviewed all published cases of infection caused by G. clavatum. Our experience and literature review indicate that G. clavatum can cause invasive infection in haematological patients, mainly in those with acute leukaemia.

Clonal evolution in therapy-related neoplasms

Therapy-related myeloid neoplasms (t-MN) may occur as a late effect of cytotoxic therapy for a primary malignancy or autoimmune diseases in susceptible individuals. We studied the development of somatic mutations in t-MN, using a collection of follow-up samples from 14 patients with a primary hematologic malignancy, who developed a secondary leukemia (13 t-MN and 1 t-acute lymphoblastic leukemia), at a median latency of 73 months (range 18-108) from primary cancer diagnosis. Using Sanger and next generation sequencing (NGS) approaches we identified 8 mutations (IDH1 R132H, ASXL1 Y591*, ASXL1 S689*, ASXL1 R693*, SRSF2 P95H, SF3B1 K700E, SETBP1 G870R and TP53 Y220C) in seven of thirteen t-MN patients (54%), whereas the t-ALL patient had a t(4,11) translocation, resulting in the KMT2A/AFF1 fusion gene. These mutations were then tracked backwards in marrow samples preceding secondary leukemia occurrence, using pyrosequencing and a NGS protocol that allows the detection of low variant allele frequencies (≥0.1%). Somatic mutations were detectable in the BM harvested at the primary diagnosis, prior to any cytotoxic treatment in three patients, while they were not detectable and apparently acquired by the t-MN clone in five patients. These data show that clonal evolution in t-MN is heterogeneous, with some somatic mutations preceding cytotoxic treatment and possibly favoring leukemic development.