The Hemato Oncology Clinic is
directed by Prof. Alessandra Biffi and pertains to the Department of
Woman and Child Health, University of Padua, Padua, Italy.
The Laboratory of Pediatric
Hemato Oncology is part of the Clinic and was started by Prof. Giuseppe Basso in 1989. The greatest effort was and continues to be placed on introducing the
most sophisticated technology available today, in order to provide the highest
level of diagnosis and care possibile to every child afflicted with a malignant
hemopathy regardless of birth place or center where daily care is being
administered.
The main strategic research areas
are periodically evaluated to determine their corrispondance to the needs of
the patients and excellence they offer as well as technological advances.
The areas presently active are
grouped as follows (click a group to open the activity info, click 'hide content' to close it):
Cytomorphology plays a pivotal role for diagnosis of acute leukemia as well as for all other hematological diseases. After morphological evaluation of bone marrow and/or peripheral blood smears, the biological samples are divided and sent to all necessary diagnostic and research areas within the Laboratory.
Morphological analysis is usually performed on all samples entering the Laboratory and always carried out whenever there is suspicion of acute leukemia in order to discriminate disease origin (lymphoid or myeloid) based on the different characteristics of the cells.
This classification is redone/updated at all treatment phases in order to evaluate patient response to therapy. Additionally, at stop therapy and during the first period after that, the analysis is necessary in order to verify the maintenance of the state of complete remission.
For other hematological diseases such as Myelodysplastic Syndrome, Chronic Myeloid Leukemia, Shwachman-Diamond Syndrome, Aplastic Anemia, Piastrinopenia, etc, the morphological analysis is very important for formulation of the diagnosis and for evaluation of possible disease evolution into Secondary Acute Myeloid Leukemia during or after treatment.
Our group performs morphological analysis to all pediatric patients at our Clinic and, since we are the national reference center for diagnostics, for a large number of patients from other Italian pediatric hospitals.
As the diagnostic reference lab, based on our morphological evaluation, we advise the laboratories which analyses need to be performed such as cell immunophenotyping, molecular/genetic characterization, evaluation of cytogenetic aberrations, etc in order to complete the diagnosis. All results obtained are correlated and evaluated during all therapy phases.
Morphology is helpful in understanding the pathological mechanisms of leukemia and other emathological diseases both for clinical and research purposes.
Clinical Diagnostics
The immunophenotyping of acute leukemias today is the preferred method to perform the identification, enumeration and characterization of blast cells at diagnosis. The commercial availability of large panels of high quality reagents facilitates the applications of this analytic method in diagnosis, classification, prognostic evaluation and minimal residual disease detection. FlowCytometry allows the analysis of a large number of cells (usually between fifteen thousand cells per sample, and three hundred thousand/one million in minimal residual disease studies) with high statistical accuracy. The simultaneous analysis of several different parameters (two related to physical properties of the cells and 5 to 8 to the immunophenotype) contributes to increase both the specificity and sensitivity of the test. Furthermore, the results can be stored in list-mode files and analyzed by other investigators to facilitate objective interpretation.
Diagnosis and MRD assessment
MRD assessment in ALL
MRD assessment in AML
DNA index
General Research Areas
MRD detection in Acute Lymphoblastic Leukemia
Standardization of FC MRD in ALL
Diagnosis in ALL
MRD detection in Acute Myeloid Leukemia
Immunophenotypic analysis of Myelodysplastic Syndromes
Analysis of cell cycle associated alterations in paired diagnosis-diagnosis-relapsed acute lymphoblastic leukemia
Clinical / diagnostic aspects
Definition of the acquired chromosomal order of paediatric patients with:
Acute Lymphoblastic Leukaemia (ALL)
Acute Myeloid Leukaemia (AML)
Chronic Myeloid Leukaemia (CML)
Lymphomas
Myelodisplastic Syndromes (MDS)
Solid Tumors
Other haematological disorders (SDS, piastrinopenias and other)
General Research Lines
The cytogenetic analysis of oncohematologic neoplasie is one of useful analysis for the diagnostic classification of these pathologies and for the characterization of new alterations in relation to defined types or subtype of tumor. This “macrocospic” surveying allows, moreover, to define regions or geniuses implied in the development of these tumors.
The introduction of molecular cytogenetic techniques like FISH, M-FISH (multi FISH) and arrayCGH allow a better definition of the kariotype (FISH on metaphase, M-FISH, arrayCGH) or in some cases offered the possibility to obtain partial information on its definition (FISH interfasica, arrayCGH). During the last few years the development of the array CGH open new perspective in this field with the definition of anomalies inferior at 1 Mb not always visible with the techniques in use.
Cytogenetic studies in paediatric age are limited from the low number of cases so are of fundamental importance multicentric or cetralized studies. From 1990 this it is made also in Padova for AIEOP centres with results for acute lymphoblastic and myeloid leukaemias comparable, in terms of resolution and in terms of incidence of chromosomic anomalies, to those of important international groups like the POG, BFM and MRC.
The main activity of our group is the cytogenetic diagnosis of the pediatric acute leukaemias. Cytogenetic analyse was performed for the cases from the Clinical of Pediatric Oncoematology of Padova and the other centers of AIEOP’s group.
General Research Lines
The development of (acute myeloid leukaemia) AML is associated with accumulation of acquired genetic alterations and epigenetic changes in hematopoietic progenitor cells which alter normal mechanism of cell growth, proliferation, death and differentiation. Currently, acute myeoid leukemia (AML) is one of the cancer for which only about half of children and young adults are cured of this disease. Most patients with AML achieve remission after therapy, but nearly half of these patients experience relapse. In addition, despite improvements in supportive care, treatment-related morbidity and mortality remain significant problems. Therefore, the overall goal of current AML programs for children is to explicate the mechanism of leukemogenesis discovering other oncogenes and molecular targets and to develop novel therapies that overcome drug resistance, decrease relapse rates, and reduce the short- and long-term adverse effects of treatment. The probability of survival for children with cancer has improved greatly during the last half century, for AML the 5-year event free survival range between the 49% to 63% in some of the more successful clinical trials. Nowadays, resistance to anthracycline and ara-C-based chemotherapy is a major cause of treatment failure in this disease. These aims may be answered by reaching a better understanding of the mechanism of leukemogenesis and the biology of the disease. Malignant transformation may be characterized by the failure of cells to differentiate, increased invasiveness and metastatic capacity, and decreased drug sensitivity. Tumorigenesis reflects the accumulation of excess cells that results from increased cell proliferation and decreased apoptosis. Apoptotic process in fact play a key role in the control of tissue homeostasis and drug resistance, and studies carried out in the last twenty years have improved the crucial role of apoptotic mechanism in the control of cancer cell survival. The majority of cases of AML are associated with non-random chromosomal translocations, point mutations, viral insertions, gene amplifications, deletions or rearrangements. The final results of these aberrancies often contribute to activate and promote malignant transformation of proto-oncogenes. Conversely, tumor suppressor genes and genes that regulate cell death are inactivated. A wide variety of different studies have provided evidence for the central role of gene rearrangements in the initiation and progression of leukemia, as well as treatment failure. Potential targeting of these alterations has become a major focus for development of novel therapeutics. Moreover, recent genome-wide studies have begun to enlighten novel target genes that might further improve the possibility to advance the biology and treatment of leukemia. The study of the alteration of gene activation/inactivation and expression typical of cancer, led to the generation of prognostic information that has been incorporated into current risk stratification protocols. If the hallmark of cancer is dysregulated gene expression, together with genetic lesions, the epigenetic modifications and factors must be considered. These mutations are not sufficient to cause the development of AML and additional mutations, occurring at the level of signal transduction, might cooperate. DNA methylation, histone/chromatine modifications and microRNAs are in fact key mechanisms that are becoming increasingly recognized in human cancer both for their frequency and importance. Many progresses are reached in recent years, agents that are showing promise in the treatment of pediatric AML include targeted immunooconjugates, inhibitors of signalling molecules (e.g. FMS-FLT3, farnesyltransferase, m-TOR), agents that target epigenetic regulation (DNA methyl transferase inhibitors and histone deacetylase inhibitors), and proteasome inhibitors (Bortezomib). In many instances, one of the partners in gene rearrangement or in a mutated gene functions as aberrant transcriptional or epigenetic regulators. The resulting aberrant expression profile interferes with the process of myeloid differentiation, suggesting that ultimately their unique molecular properties converge into a common molecular mechanism to transform cells. A plausible explanation of how this can be achieved would be that similar pathways are affected concurring together to trigger leukemia or resistance to therapy. In most cases, even if the main important pathways have been identified (PI3K, AKT, ERK, TNF, BCL-2) the central players leading to tumorigenesis are not identified, and how they collaborate to induce AML or overcome drugs have never been discovered.
The decision to characterize genetic abnormalities and signalling pathways in pediatric leukemia is an integral part of scientific research that we followed from many years, in order to better stratify patients for risk assignment and to discover new target genes. Only a portion of patients has a known disease marker which is currently being considered for both clinical and prognostic significance; for a few of them new genetic drugs are being under investigation. The availability of increasingly sophisticated techniques to study genes, protein and cellular biology has allowed the identification of specific mutation of the cell which allowed in some cases the transition from a specific antiblastic pharmacology to a directed targeted therapy. In particular we have identified the clinical and biological role of MLL rearrangements, and the mutational status of novel genes as IDH1, IDH2, DNMT3A, the role of ERG expression and novel rare translocations. We are currently investigating the functional role of the chimera MLL-AF6, whose expression has been shown to severely compromise the survival of a subgroup of AML patients by inactivating RAS pathway. These findings confirm the hypothesis that DNA/RNA lesions together with aberrancies in molecules of different pathways take to leukemogenesis process. Our interest in gene mutations and AML involvement make us confident that clarifying the genetic abnormalities of AML we’ll really contribute to overcome drug failure and patient death.
General Research Lines
The cure rate of childhood acute lymphoblastic leukemia is near to 90% merit of improved supportive care, more precise risk stratification, and personalized chemotherapy based on the characteristics of leukemic cells and hosts (pharmacokinetics).
Minimal residual disease (MRD) is the level of tumour residual cells not identifiable with the light microscopy. The prognostic significance of MRD during therapy has been already clearly demonstrated in paediatric ALL. The MRD is quantifiable by sensitive and specific technique as like as the real-time quantitative PCR of immunoglobulin (Ig) or T cell receptor (TCR). This method use the characteristic that during the normal process of lymphocyte differentiation, V, D and J segments recombine in a casual but stochastic way producing insertion and deletion of nucleotide in the junction region giving the so called N region. Each lymphocyte is characterize by a specific junction region at the level of Ig and TCR genes consequence of the process of gene recombination. This region can be used as a clone marker for each lymphocyte and it is patient specific.
Today the diagnostics carried out
in the Lab are reference points for all the European cooperative groups. In
fact there have been great advances in the immununophenotypic aspects due to
the introducuction of 8-10 colour multiparametric analyses. Additionally, we
have introduced routine use of molecular biology in diagnosis and in disease
monitoring of Leukemia. The cytogenetics group has developed molecular
diagnostic examinations through FISH and CGH, as well as CGH array. Gene Expression Profiling (GEP) and Next Generation Sequencing (NGS) are also perfomed for clinical and reaseach purposes.
Additionally the Lab carries out a lead role in applied methodologies for the
analyses of minimal residual disease (a methodology used in the new European
therapeutic protocols) and participates in international standiardized quality
controls in molecular and flow cytometry diagnostic applications. The Lab is
also the reference center for the morphological, immunophenotypic, cytogenetic and
molecular diagnoses for AML patients.
The Lab is of no doubt one of the most complete
diagnostic laboratories worldwide: it was chosen as one of
11 international laboratories (and the only European pediatric laboratory) to
carry out a collaborative study of 4000 patients afflicted with leukemia for
the determination of a new diagnostic standard for the most common
hematological pathologies (MILE study).
The Flow Lab participates in training programs of the AIEOP-BFM Community to promote a world-wide maturation program in MRD analysis for clinical application of Flow MRD in clinical Labs.
