Vincenzo Rossi

Molecular evaluation of residual disease as a predictor of relapse in acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) is characterised by a unique fusion transcript, PML/RAR alpha. We tested for this transcript in 35 APL patients who were in apparent remission after various treatments. 11 of 13 patients who tested positive 4 months after achieving remission were in relapse 1-4 months later. All 22 patients who tested negative at 4 months were disease-free after a further 3 months to five years. The test may therefore prove useful in determining the need for additional treatment during clinical remission.

Extramedullary involvement in patients with acute promyelocytic leukemia: A report of seven cases

Extramedullary involvement is only occasionally observed in patients with acute promyelocytic leukemia (APL) but has been said to occur more frequently after treatment with all‐trans retinoic acid (ATRA) than after treatment with cytotoxic drugs. In the literature, 37 well‐documented cases have been reported.

FLT3 internal tandem duplication in childhood acute myeloid leukaemia: association with hyperleucocytosis in acute promyelocytic leukaemia

Summary. We evaluated the incidence of FLT3/internal tandem duplication (ITD) mutation in childhood acute myeloid leukaemia (AML) diagnosed over 15u2003years. FLT3/ITD was found in 10 of 45 (22·2%) non‐acute promyelocytic leukaemia (non‐APL) patients. The 5‐year event‐free survival of non‐APL patients was higher in FLT3/ITD‐negative versus ‐positive patients (48·9%, SEu20038·9, vs 20·0%, SEu200316·1, Pu2003=u20030·03). In childhood APL, FLT3/ITD incidence was higher than in non‐APL, although not statistically significant (10 out of 29 patients, 34·5%, Pu2003=u20030·29). In APL patients, FLT3/ITD was strongly correlated to a higher white blood cell count at diagnosis and the M3 French–American–British subtype.

Clinical relevance of residual disease monitoring by polymerase chain reaction in patients with ALL-1/AF-4 positive-acute lymphoblastic leukaemia

In this study we used reverse transcriptase‐polymerase chain reaction (RT‐PCR) for the longitudinal monitoring of minimal residual disease in 12 patients with ALL‐1/AF‐4 positive ALL. Of these, seven also showed at presentation a typical t(4;11) cytogenetic translocation. Seven patients were infants <18 months of age and five were adults.

Prospective molecular monitoring of BCR/ABL transcript in children with Ph+ acute lymphoblastic leukaemia unravels differences in treatment response

Summary. Children with Philadelphia‐chromosome‐positive (Ph+) acute lymphoblastic leukaemia (ALL) represent a subgroup at very high risk for treatment failure, despite intensive chemotherapy. However, recent retrospective studies showed that Ph+ childhood ALL is a heterogeneous disease with regard to treatment response. We have prospectively monitored, by reverse transcription polymerase chain reaction (RT‐PCR) during follow‐up, the presence of the BCR/ABL fusion transcript in Ph+ ALL children diagnosed in the Italian multicentre Associazione Italiana Ematologia Oncologia Pediatrica ALL‐AIEOP‐95 therapy protocol. To our knowledge, this is the first report on the evaluation of minimal residual disease (MRD) in childhood Ph+ ALL prospectively enrolled in an intensive, Berlin–Frankfurt–Munster (BFM)‐type treatment protocol. Twenty‐seven of 36 (75·0%) Ph+ patients consecutively enrolled into the high‐risk group of the AIEOP‐ALL protocol between May 1995 and October 1999 were successfully analysed. Twenty were good responders to the pre‐phase of prednisone/intrathecal methotrexate treatment (PGR) and seven were poor responders (PPR). Within the PPR group, the RT‐PCR monitoring constantly showed positivity for the BCR/ABL fusion transcript and all the patients died of disease progression. In contrast, highly sensitive qualitative RT‐PCR monitoring revealed heterogeneity within the PGR group of Ph+ childhood ALL patients. Three different subgroups could be defined, according to the clearance of Ph+ cells within the first 5u2003months of treatment. This provides useful information on the capability of chemotherapy to reduce the leukaemic clone, with prognostic implications.

Transcriptional and post‐transcriptional regulation of IL‐1β, IL‐6 and TNF‐α genes in chronic lymphocytic leukaemia

The present study was designed to define the mechanisms of interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and tumour necrosis factor (TNF‐α) gene regulation in chronic lymphocytic leukaemia of B cell origin (B‐CLL). By nuclear run‐on analysis, all B‐CLL cases displayed high levels of nuclear transcription of the IL‐6 and TNF‐α genes, whereas IL‐1β gene transcription was only barely detectable. Upon in vitro culture for 1 h, B‐CLL cells from different patients were substantially heterogeneous in terms of expression of steady state mRNA levels of IL‐1β, IL‐6 and TNF‐α even though the pattern of nuclear transcription of these cytokines was only marginally affected by in vitro culture. mRNA stability was then examined and cytokine gene transcripts showed a half life of more than 2 h in cultured B‐CLL cells and treatment with cycloheximide (CHX) did not affect cytokine transcript levels in B‐CLL cells. These results indicate that: steady state levels of each mRNA do not reflect the rate of nuclear transcription of these cytokines in fresh or cultured B‐CLL cells, that purification and in vitro culture of leukaemic cells may amplify cytokine gene expression in B‐CLL, and that cytokine gene transcripts are relatively stable in B‐CLL.

A (15;17) translocation not associated with acute promyelocytic leukaemia

We report a woman with acute myeloid leukaemia (AML) type M2 according to FAB classification, showing a t(15;17) apparently identical to that of acute promyelocytic leukaemia (APL) on conventional cytogenetic analysis. Fluorescence in situ hybridization (FISH) using cosmidic probes specific for RARα and PML regions did not show a fusion signal as in APL. The breakpoints were assigned to 15q24.3 and 17q21.1. Detailed molecular analyses did not reveal any involvement of RARα and PML genes. The patient was resistant to several front‐line AML treatments and to all‐trans retinoic acid (ATRA). These findings reinforce FISH and RT‐PCR as useful tools for the characterization of a t(15;17) as the translocation specifically associated with APL.

Monitoring of minimal residual disease in leukemia, advantages and pitfalls

The term ‘minimal residual disease’ (MRD) defines the level of disease detectable in patients in clinical remission during therapy, below the detection limit of conventional methods. Very sensitive methods can be used, able to identify one leukemic cell out of 10,000 normal lymphocytes. In vivo measurements of leukemia cytoreduction reflect the combined effect of clinical and biological variables, thus providing direct information on the effectiveness of treatment in each patient. Thus, these methods can potentially be used for tailoring treatment and personalize the cure. Although MRD studies are becoming an integral part of the modern management of patients with leukemia, several parameters are critical for the application and interpretation of MRD studies, including therapeutic context, timing of sampling, target genes and sensitivity of the polymerase chain reaction (PCR) assay, inter‐laboratory standardization (particularly relevant in multicenter studies), selection of patients, retrospective or prospective nature of the study. Methodologies and pitfalls as well as results of clinical uses of MRD will be reviewed in this article by selecting significant examples of its clinical impact in the management of patients with leukemia.

Monoclonal antibody-defined surface markers of effector cells involved in human monocyte cytotoxicity

Human adherent peripheral blood mononuclear cells were cytotoxic in vitro against the murine TU5 line in a 48-hr [3H]thymidine-release assay. Monocyte-enriched adherent cell preparations contain a small and variable (usually less than 5%) contamination with large granular lymphocytes as assessed by morphology and staining with monoclonal antibody markers B73.1 and HNK1. To assess whether killing was in fact mediated by monocytes, mononuclear cells or monocyte-enriched preparations were separated using monoclonal antibodies directed against mononuclear phagocytes (Mo2, UCHM1, B44.1) or natural killer (NK) cells (B73.1 and HNK1), and a fluorescence-activated cell sorter. Cells positive for monocyte markers were highly cytotoxic against TU5, whereas negative cells were not. B73.1+ or HNK1+ cells had little or no activity. Cytotoxicity of cells positive for monocyte markers (Mo2, UCHM1, B44.1) was augmented by in vitro exposure to lymphokines or less frequently to interferon (IFN). However, cells negative for these monocytes markers were also stimulated to kill TU5 by lymphokine or IFN to an extent similar or greater than that of positive ones. IFN or lymphokines induced killing of TU5 by monocyte-depleted, B73.1-positive, lymphoid cells. These observations demonstrate that human monocytes do kill tumor cells, either in the absence of deliberate stimulation or after exposure to agents such as lymphokines. However, the possible contribution to monocyte cytotoxicity of minor NK cell contaminants must be taken into account particularly when agents such as IFN and lymphokines are applied, even when a relatively NK-cell-resistant target such as TU5 is used.

Spinal cord retrograde perfusion: review of the literature and experimental observations.

Abstractu2003 Background: Spinal cord damage represents a devastating complication of thoracic and thoracoabdominal aortic surgery. Retrograde perfusion as an alternative route to protect the spinal cord has recently been investigated with controversial results. We reviewed the literature and analyzed additional experimental observations. Methods: Ten juvenile pigs were divided into control and study groups (A and B, respectively). Through a lateral thoracotomy the distal aortic arch was cannulated and connected to a cardiotomy reservoir. All animals underwent 40‐minute single cross‐clamping of the proximal descending aorta while keeping proximal systolic arterial pressure above 100 mmHg. In group B, normothermic arterial blood was delivered retrogradely through the azygos vein, maintaining perfusion pressure within 25–30 mmHg. Animals were allowed to recover to perform a primary neurologic evaluation. Results: Flaccid paraplegia was uniformly observed in group A. In group B, all animals showed mild‐to‐moderate voluntary hind limb movements on awakening (p = 0.007). Controls also showed urine incontinence short after cross‐clamping, and this was not observed in group B (p = 0.008). A different veno‐arterial oxygen step‐down was observed in blood collected from the excluded aorta in the two groups (p < 0.001). Conclusions: Preliminary results indicate that controlled retrograde normothermic perfusion alone through the azygos system provides some degree of protection from spinal cord ischemia. Bladder dysfunction may represent a simple test to detect massive cord damage intraoperatively. Retrograde spinal cord perfusion warrants further investigation.