20.

idiopathic myelofibrosis

definition
The Idiopathic myelofibrosis is a chronic myeloproliferative disease which derives from
transformation of clonal hematopoietic stem cells and is characterized by marrow fibrosis
with disagreement leuco-eritroblastic and myeloid metaplasia with hepatosplenomegaly.
There is a form of idiopathic myelofibrosis or primitive (more frequently) and a secondary
shape in polycythemia vera (5-50% of cases) or essential thrombocythaemia (3-20% of
cases).

Epidemiology
The idiopathic myelofibrosis is more common in the elderly and affects more males. The
incidence in Europe is 0.7 x 100,000 people years in males, while it is 0.4 x 100,000 person-
years in sex women. The average age at diagnosis is 62 years and only 20% of patients
below the age of 55 years (age limit for eligibility procedures trapiantologiche- adj. of
transplant surgery, of implant surgery).

pathogenesis
The idiopathic myelofibrosis is considered a hematopoiesis clonal stem cell disease. There
are numerous experimental evidence to support this hypothesis:
First, the presence of recurrent genetic alterations (deletions of chromosome 20,
chromosome 13, chromosome 7, chromosome 12, and trisomies of chromosomes 1, 8 and
9); also Genetic studies on inactivation of the X chromosome in female patients demonstrate
the presence of only one type of allele at the level of cells affected by disease.

Pathogenesis of bone marrow fibrosis

The clonal proliferation of megakaryocytes and monocytes is accompanied by liberation of
inflammatory cytokines which determine a reaction of marrow stromal cells, bone polyclonal
nature with potential fibrogenetico, angiogenic and osteogenic.
The cytokines concerned are the TGF-β (Transforming Growth Factor β), the bFGB (basic
Fibroblast Growth Factor) and PDGF (Platelet Derived Growth Factor). These factors induce a
polyclonal proliferation of fibroblasts and osteoblasts associated with fibrous collagen and
osteosclerosis. Cytokines stimulate neo-angiogenic may contribute to these phenomena.

Pathogenesis of myeloid metaplasia

Progenitor cells emerge from the bone for a cause remains unknown and circulate
in the peripheral blood. The spleen and in the second instance, the liver act as a filter
favoring the maturation of circulating progenitors, becoming the outbreak of the
extramedullary hematopoiesis.

clinical
S debut

Approximately one third of patients are asymptomatic at the time of diagnosis, while the
remaining two thirds are having systemic symptoms such as fever, weight loss, fatigue,
dyspnea and joint pain.
On physical examination, splenomegaly is present in 85 to 100% of cases, and often reach
considerable size. Patients may report feeling of tension and abdominal pain in the left
hypochondrium, often secondary to splenic infarction. Splenomegaly which is evident in
patients with idiopathic myelofibrosis is attributable to the haemopoiesis and
extramedullary haematopoiesis; the hepatomegaly can be put in relation both to the
presence of outbreaks of extramedullary haemopoiesis and both hypertension secondary to
splenomegaly.
THE haemochromocytometric examination demonstrates anemia (50-70% of cases),
thrombocytopenia (35- 40 %) or piastrinosi (30 % ), and leukocytosis (50 % ). The smear of
peripheral blood are noticeable anisopoichilocitosi (variability in the size and shape of red
blood cells) , with the presence of dacriociti erythrocytes (teardrop) and disagreement
granule-eritroblastic (presence of circulating elements of immature erythroid series and
granuloblastic) .

Diagnosis
The criteria for the diagnosis of idiopathic myelofibrosis are reported in Table 1
The clinical and laboratory data which are given have great importance (defined criteria
necessary)
The presence of marrow fibrosis and the absence of the Ph chromosome 'in the cells of
bone marrow (specific marker of chronic myeloid leukemia). Considerable emphasis has
also the finding of splenomegaly. The diagnosis requires the presence of two necessary
criteria plus 2 optional criteria if there is splenomegaly, or of 2 needed plus 4 optional if
there is splenomegaly.
Table 1- page 99.

Differential Diagnosis
The differential diagnosis must be placed primarily with other diseases
myeloproliferative diseases (chronic myeloid leukemia and essential thrombocythemia),
with other causes of marrow fibrosis or alteration of bone structure (osteomyelitis, disease
Paget, osteopetrosis) and with different causes of splenomegaly.
Chronic myeloid leukaemia has, as the idiopathic myelofibrosis, splenomegaly and elements
of immature granuloblastic series in the peripheral blood: differential diagnosis is made by
checking the presence of Ph chromosome' (or rearrangement of bcr/abl) at the level of bone
marrow cells. The differential diagnosis of essential thrombocythemia can not such easy,
especially at the cellular stage of myelofibrosis: in fact both conditions have megakaryocytic
hyperplasia. The distinctive features of the two forms are the marrow fibrosis, the
disagreement leuco-eritroblastic and anisopoichilocitosi of red blood cells.
Finally, it should be noted that the framework of anisopoichilocitosi of red blood cells with
dacriociti and the presence of elements of the immature erythroid series and granuloblastica
in the peripheral blood is common in anemias mieloftisiche, determined by bone marrow
infiltration by neoplastic cells metastatic (lung, breast, prostate).

Clinical course and prognosis

The clinical course of patients with idiopathic myelofibrosis is highly variable. Observed
asymptomatic patients for a long period of time (years), and patients with course instead
clinical worsening that can quickly lead to the exitus.
With the progression of the disease is observed especially an increase of splenomegaly (and
hepatomegaly), worsening anemia, leukopenia (or of leukocytosis) and thrombocytopenia.
Sometimes the disease can be complicated by portal hypertension, thrombosis of the portal
vein and / or thrombosis spleen. An expansion of myeloid metaplasia outside of the branch,
and hepatic, spleen can induce the occurrence of cardiac tamponade, skin nodules,
spinal cord compression, pleural effusion, pulmonary hypertension.
The most frequent causes of death were identified in congestive heart failure, in bleeding
complications and evolution to acute leukemia. Some factors, individually or in combination,
adversely affect the prognosis and are represented by the presence of anemia with
hemoglobin <10 g / dL, leukopenic with values of white blood cells <4 x 109 / L and
Leukocytosis of white blood cells> 30 x 109/L.
Their combination [Lille score (Dupriez, 1996)] allows to identify three risk categories (low,
intermediate, high), which identify patients with survival median different.
Table p.100

The treatment of idiopathic myelofibrosis

In the absence of systemic symptoms, symptomatic splenomegaly, circulating blasts,
abnormal cytogenetics, peripheral cytopenias is indicated only clinical observation without
cytoreductive treatment.
By contrast, in the presence of leukocytosis, thrombocytosis, splenomegaly or progressive
clinical symptoms is useful to use cytoreductive therapy. The drug mainly used is
hydroxyurea, which allows to obtain a control of the disease in 40-80% cases, depending on
the stage of the disease. Other drugs used were busulfan or interferon.
Viable treatment option in young patients who have a donor is compatible allogeneic stem
cell transplantation. this procedure can lead to healing a number of patients, but with a high
risk of mortality peritrapiantologica.
Recently, thalidomide has demonstrated effectiveness obtaining control of anemia and
thrombocytopenia in 40% of patients with myelofibrosis.

21. Chronic myeloid leukemia

Definition
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic
stem characterized from the molecular point of view by the presence of gene rearrangement
cell bcr / abl and from the clinical point of view by progressive leukocytosis (with
accumulation in the peripheral blood of mature granulocytes and myeloid precursors), by
bone marrow hypercellularity and splenomegaly.

Epidemiology
The CML has an incidence of 2 cases per 100,000 inhabitants per year and constitutes about
15% of adult leukemias. The median age of onset is included between 45 and 55 years, with
a predominance in males. Although it is not the mechanism responsible for the neoplastic
transformation of hematopoietic stem cells, numerous studies show that exposure to
radiation- ionizing radiation induces an increase in the incidence of CML compared to the
expected frequency in the general population.

Pathogenesis
CML is characterized from the cytogenetic point of view by the presence of chromosome
Philadelphia or Ph '(Nowell P, Hungerford D. A minute chromosome in human chronic
granulocytic leukemia. Science 1960; 132:1497), or a chromosome 22 small size, which arises
from the balanced translocation between chromosome 9 and chromosome 22
t (9; 22) (q34; q11)] (Rowley JD. A new consistent chromosomal abnormality in chronic[.
myelogenous leukemia Identified by quinacrine fluorescence and Giemsa staining.
)Nature. 1973, 243:290)
This translocation leads in the molecular level to the rearrangement between the gene bcr
(Breakpoint cluster region) located at the level of band q11 of cromocsoma 22 and the
protooncogene abl (Ableson) at the level of band q34 of chromosome 9 (Figure 1).
At the level of the ABL breaking point located at the 5' in a region of about 300kb and
may be upstream or downstream of exon Ia or Ib exon or, more often, between both.
Even if one or both of these exons are translocated on chromosome 22 are rearranged in
accordance with they are not transcripts being removed from final transcript for action of
the phenomenon of "splicing" operating at the level of the mRNA.
Figure 1 - translocation t (9; 22) and bcr-abl gene rearrangement in CML p. 103

Therefore, in patients with CML on chromosome 22 rearranged creating a hybrid gene

BCR / ABL, format for the 5 'portion from BCR sequences and for the 3' portion from
sequences ABL. The hybrid gene BCR / ABL results in constitutive activation of the tyrosine
kinase of the ABL gene.
The tyrosine kinases belong to the family of protein kinases, enzymes that transfer
phosphate groups by adenosine triphosphate (ATP) to specific amino acids (in
this case the tyrosine) at the level of the substrate. The phosphorylation of these proteins
leads activation of signal transduction pathways that control a number of important
biological processes such as growth and cell differentiation, and apoptosis.
The protein p210bcr-abl through these mechanisms (in particular through the inhibition of
apoptosis) is able to prolong the survival of the cell and to determine the expansion of the
leukemic clone (Figure 1).

clinical
Approximately 50% of cases of CML is diagnosed through examination performed for other
reasons (incidental finding). The blood count shows leukocytosis with neutrophilis, presence
of myeloid precursors in peripheral blood, absolute basophilia and thrombocytosis.

Below is illustrated, by way of example, the blood count of an asymptomatic young woman
(highlighted in bold are the most significant parameters): (table p. 105).

Patients with more advanced disease may show symptoms that depend on the
hypermetabolism, which include anorexia, asthenia, weight loss and night sweats.
Splenomegaly is usually present, sometimes important symptomatic (sense of weight to the
left hypochondrium, feeling of repletion post-prandial glycemia, pain in the case of splenic
infarction)

Natural history of chronic myeloid leukemia

The natural history of CML is characterized by a chronic phase of variable duration (in
typically 3-4 years) asymptomatic or poorly symptomatic responsive to treatment
)control leukocytosis(.
At this stage follows a more aggressive, defined accelerated, characterized from
haematological point of view by the appearance of blasts (immature who have suffered
arrest ripening) in the bone marrow and in peripheral blood (10-20% of the cells nucleated)
poorly responsive to treatment with leukocytosis, anemia and thrombocytopenia. from a
clinical perspective is characterized by fever and bone pain. The analysis cytogenetics can
demonstrate the appearance of additional clonal chromosomal abnormalities.
This phase usually lasts a few months and evolves in blast crisis (myeloid in 2/3, lymphoid in
1/3 of cases), characterized by the increase of the share blast (≥ 20% of nucleated cells in the
bone marrow or peripheral blood) with severe anemia and thrombocytopenia (bone marrow
failure), while from the clinical point of view there has been a rapid deterioration of the
general conditions (marked fatigue, weight loss), associated with fever and bone pain. Blast
crisis is the terminal phase of the disease and poorly responsive to chemotherapeutic
treatment, hesitating in almost all nell'exitus cases of the patient.

Diagnosis
The investigations need be carried out for a correct diagnosis of the patient with CML:
the CBC (which shows in the chronic phase neutrophilic leukocytosis with
presence of myeloid precursors in peripheral blood, absolute basophilia and
thrombocytosis), the aspirate (showing hyperplasia of the granulocytic line), chromosome
analysis of marrow blood (which shows the presence of the Philadelphia chromosome) and
RT-PCR Reverse Transcriptase Polymerase Chain Reaction, which shows the presence of the
rearrangement ( bcr / abl).

Diagnostic criteria for accelerated phase (Vardiman JW, Harris NL, Brunning RD. The
World Health Organization (WHO) classification of the myeloid neoplasms).
To formulate the diagnosis of accelerated phase at least one of following criteria must be
present:
*percentage equal to 10-19% of blasts in peripheral blood or bone marrow
*percentage of basophils in peripheral blood ≥ 20%
*persistent thrombocytopenia (<100x109 / L) unrelated to therapy, or persistent
thrombocytosis (> 1.000x109 / L) unresponsive to therapy
*increased splenomegaly and leukocytosis unresponsive to therapy
*cytogenetic evidence of clonal evolution (appearance of a genetic abnormality
Additional that was not present at the time of diagnosis of CML in chronic phase)
*proliferation of megakaryocytes in clusters of large size, associated with marked
reticolin or collagen fibrosis and / or severe granulocytic dysplasia (these findings,
however, have not yet been analyzed in large clinical studies; therefore it is not clear
whether they are independent criteria of accelerated phase. They are often associated with
one or more of the other criteria listed Diagnostic criteria for blast crisis (Vardiman JW,
Harris NL, Brunning RD. The World Health Organization (WHO) classification of the blood
myeloid neoplasms)
for the diagnosis of blast crisis must have at least one of the following criteria:
*percentage of blasts in the peripheral blood or bone marrow ≥ 20
*extramedullary proliferation of blasts
*large foci or clusters of blasts detectable bone marrow biopsy

Differential Diagnosis
The differential diagnosis of chronic myeloid leukemia should be considered: reactive
leukocytosis (which are not found in immature myeloid series peripheral blood), and other
myeloproliferative disorders (polycythemia vera, thrombocythemia essential, idiopathic
myelofibrosis), in which there is no rearrangement of the bcr-abl.

Prognostic factors at diagnosis

Sokall et al. (Sokal JE, Cox EB, Baccarani M, et al. Prognostic discrimination in "goodrisk"
chronic granulocytic leukemia. Blood 1984, 63:789) proposed a score based on the
evaluation of four parameters (age, spleen size in cm from the arc costal, platelet count x109
/ L and percentile blasts in the peripheral blood) for calculate the relative risk (RR) of each
patient with CML.
The relative risk (RR) is calculated with the following formula:
]RR = ESP * {0.0116 x (age-43.4) + 0.0345 x (spleen-7.51) 0188 x} + [(piastrine/700) 2-0563
)x (blasts-2.10 0.0887 +
*ESP = exponential.
Patients with relative risk <0.8 (ie low) have a median survival and about half times more
'life span' than those with a relative risk of 1.2 (high risk).

Therapy
Meaning of clinical response, cytogenetic and molecular in the LMC.
In patients with CML, complete clinical remission is a condition defined by normalization of
the peripheral blood and bone marrow.

Cytogenetic response is evaluated based on the percentage of positive metaphases for the
Search the Ph chromosome in the bone marrow (on the minimum number of metaphases
analyzed equal to 20). The cytogenetic response: you define full remission (cytogenetic) if
the chromosome Ph' is absent in all metaphases analyzed, greater if is between 1 and 35 %,
lower if it is between 36 and 65 %, minimum if between 66 and 95 %, absent if greater than
95 %. The quality of the cytogenetic response plays a prognostic significance and is
correlated with the life expectancy of these patients.
Sensitivity of the method: conventional cytogenetics, which has the limit analyze the only
dividing cells, identifies a cell leukemic cells of 10-100 examined (sensitivity 10-1-10-2).

The molecular response is defined in patients with complete cytogenetic response as

a reduction> 3 logarithms in the amount of bcr abl transcript evaluated with RT-PCR. The
molecular remission is defined instead as a condition characterized by disappearance of the
bcr abl transcript analysis RT-PCR. Sensitivity of the method: the PCR technique has high
sensitivity compared to other laboratory techniques used for the evaluation of the amount
of residual disease after treatment, equal to 10-4-10-6.

Minimal residual disease (MRD): definition and clinical utility

Although considerable progress has occurred in the conventional treatment and transplant
of hematologic malignancies, a significant percentage of patients in complete remission
clinical relapse after a variable time interval. Therefore in a consistent number of patients
survive of a limited share of neoplastic cells, possibly in able to determine a location of the
disease. This small population of neoplastic survivor, quantitatively consists a number of
cells always inferior to 1010 MMR is defined and is demonstrated by several laboratory
methods to different sensitivity (flow cytometry, cytogenetics, FISH, PCR).
The examination of the BCR-ABL by RT-PCR has provided a model for the study of a possible
MMR and laid the foundation for the analysis of the latter in other onco-hematological
disorders. It has been observed that the majority of patients with CML underwent allogeneic
has a persistence of the transcript in the first six months, but thereafter at least two thirds of
patients become PCR negative for progressive elimination of leukemic cells to effect the
reaction which goes under the term "graft versus leukemia". Patients after one year or more
after Allogeneic transplantation have two consecutive: PCR positive samples are qualitative
at high risk of cytogenetic and hematological relapse. Therefore, this method was
able to predict recurrence at the individual level and, consequently, to establish an early
appropriate therapeutic interventions.

Treatment of CML
The treatment of chronic phase CML was previously conventionally based on the use of
hydroxyurea and subsequently of interferon alpha. Interferon allows obtain a greater
percentage of major and complete cytogenetic responses compared hydroxyurea (median
life expectancy by 3-4 to 5-6 years), guaranteeing patients who respond to an average life of
more than 8 years.

The treatment of blast crisis was based on the use of cycles polychemotherapeutic, with
The proportion of response which however does not exceed 20% in traformation myeloid in
lymphoid transformation, also the few patients who get treatment fall quickly or die from
disease progression. Allogeneic hematopoietic stem cell is considered the only approach
able to ensure healing of patients with CML, however, is encumbered with a mortality
peritrapiantological of 20-40%. Allogeneic hematopoietic stem cells is been applied with
absolute availability in young patients with a HLA-identical donor.

Since 2001, this therapeutic approach has been radically changed by the introduction
of imatinib mesylate (STI 571, Gleevec, Glivec), a specific inhibitor of tirosinakinase
mutated (molecular therapy). This drug was designed to compete with the ATP at the level
of the specific binding site in the kinase domain of the protein of P210 bcr-abl fusion (Figure
3). The link with the ATP allows the tyrosine kinase to phosphorylate the tyrosine residues at
the level of the substrate; in the presence of the drug (which mimics the ATP placing itself in
the binding pocket specifies) the enzyme is no longer able to transfer phosphates groups,
interrupting the activation of signal transduction pathways to the nucleus. As final event, the
cell goes into apoptosis. The aim of treatment with Imatonib is the suppression / elimination
of Ph positive clone and the restoration of hematopoiesis normal Ph negative.
Figure 3 p. 109.

The results of controlled clinical trials carried out so far show that treatment with
imatinib mesylate is much more effective compared to therapy with interferon and the drug
is very well tolerated. With the use of such a drug, the percentage of cytogenetic complete
responses in patients in chronic phase is over 80% and were also observed complete
molecular remission (absence of bcr-abl rearrangement detectable by RT-PCR)
In addition, at higher doses it has proved efficient also in steps and accelerated in crisis
blastiche.
For insofar as they are not yet available data on long-term effect, therapy with imatinib
mesylate should be considered today as a therapy of choice for the treatment of CML and
the allogeneic transplantation peripheral stem cell is reserved for patients who fail to obtain
a response of good quality to therapy with imatinib. there ongoing studies for evaluation of
MMR during therapy with Imatinib, in order to identify the patients with risk of recurrence
or progression of disease.
In the different published series, a variable percentage of patients (between the 5
and 15%) has or develops a resistance to imatinib therapy (defined as absence of
hematological or cytigenetic rispsonde at therapeutic doses of the drug). are
been identified some of the mechanisms underlying the resistance to imatinib: the presence
of mutations at the level of bcr-abl, the amplification of the gene bcr-abl and the increased
expression of the multidrug resistance gene.
We are studying novel inhibitors of bcr-abl (BMS-354825 or dasatinib and AMN107) who
shown efficacy against most forms of mutant bcr-abl tested in vitro.

Therapeutic decision algorithm for chronic myeloid leukemia.

Image page 110.

22. hypereosinophilic syndrome

Definition
It is a clonal disorder of hematopoiesis characterized by the presence in the peripheral blood
of an absolute number of eosinophils greater than 1.5x109 / L and an increase of
eosinophils in bone marrow hematopoietic tissue for a period of time exceeding six months
and in the absence of clinical conditions capable of determining eosinophilia.
The latter may in fact be secondary to allergic disease, autoimmune, parasitic,
dermatological and cancer. Eosinophils secondary to the release of cytokines are been
reported not only in patients with chronic myeloid leukemia Ph1 positive, with acute
lymphoblastic leukemia and non-Hodgkin lymphomas, but also in patients who apparently
did not have a lymphoproliferative disease. In the latter group of patients with frequent
episodes of pruritic dermatitis and elevated IgE levels, was observed a clonal population of T
lymphocytes that produced various cytokines but especially interleukin 5, necessary for the
differentiation of eosinophilic in cell myeloid lineage.
Pathogenesis
The hypereosinophilic syndrome is always caused by a acquired somatic mutation
arising in hematopoietic stem cells. In some cases the cellular differentiation is mainly
eosinophilic oriented (this is called eosinophilic leukemia), in other is instead to all myeloid
cell lines. In the latter group of patients eosinophilia is part of a larger neoplastic disorder of
hematopoiesis, However in both cases the increased production of eosinophils is induced by
an increased production of interleukin-5, interleukin-3 and growth factor
granulocitomacrofage (GM-CSF).

Clinic
At the time of onset the patient presents with symptoms caused by the fact that
the eosinophils infiltrate the various tissues and release cytokines contained in their
granules.
This explains the intense itching often associated with the presence of cutaneous nodules,
the profound fatigue with frequent retrosternal pain of anginal episodes and the rarest of
profuse diarrhea.
On physical examination appreciate important infiltrated skin lesions type exfoliative
pustules and localized angioedema, there is often a significant hepatosplenomegaly,
rarer are the enlarged lymph nodes. A visit cardiology often shows congestive heart failure,
arrhythmias, angina, and echocardiography is observed a marked deficit of the contractile
myocardium and alterations at the level of heart valves. At the level of the central nervous
system is observed suffering of type popular with frequent transient ischemic attacks, and
peripheral neuropathy; level a marked alteration of pulmonary lung function due to the
important pulmonary fibrosis, at the level of the gastrointestinal tract mucosal infiltration,
are the cause of the diarrhea complained of by the patient.
In 16% of patients after a chronic phase lasting 6-9 months develops a phase acute clinical
picture similar to that of acute myeloid leukemia.

Diagnosis
The emocitometric examination usually shows leukocytosis with normal hemoglobin
and platelets. Microscopic examination of the peripheral blood smear is detected
dacriocitosi and an increase in eosinophils, which are normal size and cytoplasmic vacuoles.
The aspirate shows a hematopoietic tissue with normal -or hypercellulitic eosinophilic
hyperplasia of the jamb.
The cytogenetic and molecular analysis is now an absolutly not only essential for a correct
diagnosis of the syndrome, but also for a correct treatment.
It has been shown that the hypereosinophilic syndrome is not a homogeneous entity but
includes various sub-entities associated with specific cytogenetic and molecular alterations.
The chromosomal translocation that first was characteristically associated with a
myeloproliferative disease with a marked increase in eosinophils was the t (5; 12) (q31-33,
-p13-p12).
The anomaly, which has an incidence of about 1%, determines the rearrangement between
gene coding for the receptor of Beta "Platelet Derived Growth Factor" (PDGFRB), mapped to
band 5q33, and the ETV6 gene, mapped to 12p13. The gene encoding PDGFRB for a receptor
protein tyrosine kinase with activity, which develops only happened with the ligand binding,
represented by PDGF. The fusion gene ETV6-PDGF, produced by the translocation
determines the constitutive activation of the kinase in the absence of ligand.
Another translocation associated with a framework of eosinophilic syndrome is one that
involves the FGFR1 gene, which encodes for the protein "Fibroblast Growth Factor Receptor
to tyrosine kinase. In the translocation t (8; 13) the FGFR1 gene, mapped to" 1
chromosome 8 to the p11 band, rearranges with the ZNF198 gene, mapped to band
q12. The chimeric gene ZNF198-FGFR1, produced by translocation Case13
constitutive activation of the kinase in the absence of ligand.
The last translocation most recently demonstrated by Molecular cytogenetic techniques
is the one that determines the rearrangement between the gene for the receptor of the Alfa
PDGF and the gene FIP1L1, both mapped to band q12 of chromosome 4. Even in this case
the translocation generates a fusion gene that causes constitutive activation PDGFRA.

Therapy
Until the recent past, the drugs most often used in patients with hypereosinophilic
syndrome were corticosteroids and cytotoxic.
The recent demonstration that in the majority of patients with hypereosinophillic syndrome
occurs constitutive activation of a particular tyrosine kinase in following a specific
chromosomal translocation has radically altered the treatment of these patients by directing
them towards a molecular therapy.
The latter consists of the administration of imatinib mesylate (STI571, Gleevec,
Glivec), a molecule already proved effective in the treatment of chronic myeloid leukemia
Ph1 positive. Recent studies indicate that imatinib is able to induce durable remissions
also in patients with t (5; 12) or with rearrangement FIP1L1-PDGFRA.

23. myelodysplastic Syndromes.

definition

Myelodysplastic syndromes are a heterogeneous group of disorders of the primitive spinal

bone marrow typically affecting the elderly and is characterized by anemia, usually
refractory to treatment, persistent neutropenia and thrombocytopenia (or various
combinations of the preceding cytopenias), and from a risk (of varying degree) of evolution
in acute myeloid leukemia.

Epidemiology
The median age at diagnosis is between 65 and 70 years. The overall incidence of these
conditions is about 8 cases per 100,000 people per year. In patients under the age 30
is 1 case per 100,000 people per year, while over 70 years old is 35 cases per 100,000 people
per year. there are more affected males Exposure to toxic factors such as organic solvents,
pesticides, ionizing radiation or intake cytostatic drugs-based therapies are risk factors for
the development of a myelodysplastic syndrome.

pathogenesis
Myelodysplastic syndromes are clonal disorders of hematopoietic stem cells that
retain the ability to differentiate and mature, but they do so in a disorderly manner
)hematopoietic dysplasia) and inefficient (ineffective hematopoiesis) (Figure 1(.
Figure 1 page 113. 'Pathophysiology of myelodysplastic syndromes'.

A blood disease (such as myelodysplastic syndromes) is defined as clonal when cell

proliferation that characterizes it takes its origin from a single sick progenitor. The
demonstration of clonal origin of myelodysplastic syndromes is based on the identification
of acquired cytogenetic abnormalities, and, limited to female population, by demonstrating
random inactivation of one chromosome X using different expression of DNA methylation in
patients heterozygous for gene polymorphisms PGK (phosphoglycerate kinase) and
HUMARA (hormone receptor human androgen).
The genetic and molecular mechanisms responsible for neoplastic transformation of
hematopoietic stem cells in myelodysplastic syndromes remain largely not clarified. Some
chromosomal abnormalities occur more frequently in myelodysplastic syndromes. The most
frequent abnormalities are of chromosome 5, the chromosome 20, chromosome Y
(associated with favorable prognosis) of chromosome 7 (associated with prognosis Sevara)
and trisomy 8 (intermediate prognosis), which represents the most frequent numerical
abnormality in myeloid disorders (myelodysplastic syndromes acute myeloid leukemia,
myeloproliferative disorders). Approximately 40-60% of patients represent a normal
cytogenetic karyotype in conventional analysis technique. (G banding). L' uses of more
sensitive approaches (molecular cytogenetics fluorescent in situ hybridization, FISH) allows
to detect the presence of cryptic cytogenetic lesions in a certain percentage (10-20%) of
patients with karyotype normal.

Clinical
At diagnosis, patients report symptoms most commonly correlated to anemia, such as
fatigue (asthenia) and varying degrees of respiratory distress (dyspnea), especially in
conjunction with physical exertion. Less frequent symptoms are infectious episodes and
hemorrhagic manifestations. Infections (resulting from the neutropenia) are for the most
part of bacterial type, in relapsing character and slow resolution. In the event of a fall in
platelet count (thrombocytopenia), the most important Clinical event is the appearance of
purpura, ecchymosis or hematoma at trauma, more rarely, epistaxis, gengivorragia or
bleeding of the gastrointestinal tract. Finally in a certain percentage of patients, the
diagnosis is occasional, ie suspected on the basis of changes emerged from a blood count
performed in the course of an inspection routine. On physical examination, in a small
percentage of cases (about 15%) are having hepatomegaly, splenomegaly or enlarged lymph
nodes, The blood count shows mono-cytopenia trilinear: anemia normal-or more often with
macrocytic reticulocytes rather than increased reticulocytes, neutropenia (<1.8 x 109 / L)
thrombocytopenia (<100 x 109 / L). The peripheral blood smear reveals the presence of
dependent morphological abnormalities of neutrophils, which may have hyposegmentation
nucleus (Pelger-Huet anomaly type) and the hypogranulation cytoplasm, and / or platelets,
from giants cell.

The bone marrow aspirate showed generally hypercellularity, with morphological anomalies
dependent on one or more lines of maturation:
*dyserythropoiesis: mitosis, nuclear abnormalities (internuclear bridges, binuclearità),
abnormal cytoplasmic (intense basophilia, Howell-Jolly), ringed sideroblasts. (Figure
detectable by Perls staining ,2
*disgranulopoiesi: nucleo-cytoplasmic asynchronized maturation, hypogranulation, blasts
)elements which have undergone maturation arrest(
*dismegacariopoiesi: micro-megakaryocytes, mononuclear megakaryocytes, hypogranultion

Figure 2: erythroblasts ferritinici and ringed sideroblasts page 115.

The ringed sideroblasts are defined based on the presence of a number of granules of iron
(Perls staining). available perinuclear, that indicates a location in mitochondrial level.
Erythroblasts ferritinici have a smaller number of granules that localizied is in the cytoplasm.

Classification
Myelodysplastic syndromes have been classified to date according to the criteria
formulated by the French-American-British (FAB) Cooperative Group in 1982 (Table 1).
This classification that relies exclusively on morphological criteria (cytopenia, bone marrow
dysplasia, percentage of immature cells or blasts in peripheral blood and
bone, percentage of bone marrow ringed sideroblasts). It distinguishes five forms:
refractory anemia (RA), refractory anemia with ringed sideroblasts (ASIA), anemia refractory
anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation
(RAEB-t) and chronic myelomonocytic leukemia (CMM).

Table 1 - FAB classification of myelodysplastic syndromes (p.116)

In October 2002, was made the new WHO classification, which incorporates many of the
criteria and definitions of the FAB system, but clarifies some subtypes (Table 2). It
distinguishes six main forms: refractory anemia (RA) refractory anemia with ringed
sideroblasts (ASIA), refractory cytopenia (with or without ringed sideroblasts) with
multilineage dysplasia (RCMD, RCMD-RS), refractory anemia with excess blasts (AREB1 and
AREB2), 5q-syndrome (5q).
The main differences compared to the previous classification concerning the exclusion
subtype RAEB-t, which is equivalent to the category of acute myeloid leukemia.
The elimination of the subtype CMML placed in a group of disorders with myeloid
features of both myelodysplastic syndromes and myeloproliferative diseases
(MDS / MPD), the establishment of a new clinical entity, the 5q-syndrome associated with
favorable prognosis.

Syndrome 5q
This syndrome is defined as a de novo MDS with an isolated cytogenetic anomaly
which consists in the deletion of the bands q21 and q32 of chromosome 5.
It affects more frequently women. From the hematologic point of view is presents as anemia
(macrocytic) refractory, with a normal or increased number of platelets and an increased
number of megakaryocytes, many of which with nuclei ipolobati (polylobate nuclei-?). The
number of blasts in the marrow and peripheral blood is less than 5%, this is associated with
favorable prognosis.
Table 2 - WHO Classification of MDS P.117

Mielodisplastici Disorders / myeloproliferative (MDS / MPD)

Chronic myelomonocytic leukemia has been removed from the category of syndromes
Myelodysplastic and placed in a group of disorders with features of both myeloid
myelodysplastic syndromes and myeloproliferative diseases (MDS / MPD). this
group includes the following diseases:
*chronic myelomonocytic leukemia
*mielide atypical chronic leukemia
*juvenile myelomonocytic leukemia
*disease myelodysplastic / myeloproliferative, not classifiable

The criteria for the diagnosis of chronic myelomonocytic leukemia (LMMC) include: the
presence of persistent monocitosi > 1x109/l, the absence of Philadelphia chromosome or
rearrangement BCR/ABL, the presence of blasts in the peripheral blood or marrow in less
than 20 %, and the presence dysplasia in one or more lines myeloid.

prognosis
The WHO classification showed a significant prognostic value, layering is
survival is the risk of evolution of leukemic patients with syndrome
MDS (Table 3).

Table 3 - Overall survival and risk of leukemic evolution of the subtypes of

myelodysplasia defined by the WHO classification P.118

The currently prognostic most widely used is the International Prognostic Scoring
System (IPSS), defined in 1997 (therefore before the introduction of the new
WHO classification) by an international cooperative group.
This system takes into account the cytopenias, percentage of BM blasts (the which
stratification criterion was adopted with minor changes from the WHO classification),
and the karyotype (Table 4). The IPSS idenfica 4 Different risk groups (low, Intermediate-1,
Intermediate-2, high risk) that differ significantly in risk of leukemic evolution and survival.
Patients with low-risk IPSS have a median survival of 5.7 years, those with intermediate risk-
1 to 3.5 years, with intermediate risk-2 to 12 months, and with high risk of 4.5 months. 25%
of progression to acute myeloid leukemia is reached in 9.4 years for the low risk, in 3.5 years
for the intermediate risk-1, in 12 months for the risk Intermediate-2 and 4.5 months for the
high risk.

Table 4 - International Prognostic Scoring System (IPSS) variables P.119

therapy
The treatment of MDS uses several approaches to assess depending on the characteristics of
the patient (age, performance status) and the disease (IPSS).
Among the therapeutic tools that can potentially give healing there are the allogeneic
peripheral blood stem cells and aggressive chemotherapy, as reserve in young patients
(younger than 55 years for the transplant donor family HLA-identical and less than 65 years
for chemotherapy) with forms of in intermediate or high risk myelodysplasia.
The low-risk patients or in elderly or poor performance status are candidates for supportive
care: red blood cell transfusions, prevention and treatment of infection, prevention and
treatment of bleeding, or experimental treatments (EPO + GCSF), inducers of differentiation,
drug anticitochinici.

24. Acute myeloid leukemia

definition
The acute myeloid leukemia (AML) is a clonal stem cell disease hematopoietic characterized
by uncontrolled proliferation and maturation arrest with accumulation of immature myeloid
cells (blasts) in the bone marrow and suppression normal hematopoiesis.

Epidemiology
The incidence of acute myeloid leukemia increases significantly with increasing age
is a total of 2-3 cases per 100,000 people per year. the LAM account for about 15-20% of
acute leukemia of children and 80% of leukemias acute adult.

pathogenesis
some risk factors have identified for the development of acute myeloid leukemia, which
include environmental factors, acquired diseases, hereditary diseases (Table 1).

Table 1 - Risk factors for the development of acute myeloid leukemia P.120-121

Acute myeloid leukemia were classified until October 2002 in accordance to the
morphological and immunocytochemical criteria- FAB (French-American-British Classification
Group) In all cases, for the diagnosis of acute myeloid leukemia, Centre should that the
number of blastic cells present in the bone marrow is greater than or equal to 20% of the
total cellularity.

The FAB classification distinguishes between the following subtypes of acute myeloid
leukemia:
*Acute leukemia M0 (undifferentiated): is characterized by blasts without granules
cytoplasmic and Auer rods. The cytochemical conventional reactions (myeloperoxidase,
sudan black) were negative. For the diagnosis it must be detected the positivity for one or
more myeloid markers (monoclonal antibodies anti-CD13 and anti- CD33) in at least 20% of
leukemic blasts. Is not associated with specific Cytogenetic abnormalities.
*Acute leukemia M1 (without maturation) is characterized by myeloid blasts without
signs of aging: there are no cytoplasmic granules, the nuclear chromatin is order. To
diagnose it is necessary to highlight the positive to myeloperoxidase and the Sudan black in
at least 3% of the leukemic blasts. The component with granulocyte signs of maturation
must be equal to or less than 10%. Is not associated with specific cytogenetic abnormalities.
*Acute leukemia M2 (with maturzione): is characterized by myeloid blasts in which
cytoplasm is possible to observe azzurophilic granules or Auer rods; at the nuclear level
are clearly visible nucleoli. The component with mature granulocyte is more than
10%; monocyte component must be less than 20%. This subtype associated with the
translocation t (8; 21) with the involvement of genes AML / ETO (prognosis favorable).
*Acute leukemia M3 (promyelocytic): almost all of the leukemic cells is consists promilociti
atypical cytoplasm rich in azzurrophilic granules and bodies Auer (variant hypergranulated).
The reaction to myeloperoxidase is intensely positive. This subtype is associated with very
high frequency at t (15; 17) rearrangement PML / RARα (prognosis).
There is a variant hypogranulate on acute leukemia M3, in which the granules are not
visible in light microscopy but can be demonstrated by electron microscopy.
The cytogenetics is the same as the variant in hypergranulate
*Acute leukemia M4 (myelomonocytic): for the diagnosis of this form it must be present
of in addition to a proportion of blasts greater than 20%, there must be a granulocyte
medullary component in various stages of differentiation greater than 20% and a monocyte
medullary component not less than 20%. Positive for the myeloperoxidase and chloro-
acetate esterase (specific esterase) is found in granulocyte component, and a net positive
for non-specific esterases (alfanaftil-acetate-esterase) is present in monocytic cells. A inv
(16), with prognosis positive, it is frequently associated with a variant of AML-M4 with that
eosinophilic component. Eosinophils are abnormal and in addition to the granules in the
cytoplasm specific basophil granules are particularly prominent.
*Acute leukemia M5a (poorly differentiated monocytic): the monocytic cell
must constitute at least 80% of leukemic cells; the monoblasts must constitute
at least 80% of the monolithic component, the component granulocyte if present
must be less than 20% of leukemia cells. The monoblasts are negative for
myeloperoxidase and positive for alpha-naphthyl acetate esterase (nonspecific esterase).
This form is not associated with specific cytogenetic abnormalities.
*Acute leukemia M5b (with monocytic differentiation) is necessary for the diagnosis
monoblasts that are less than 80% of the monocyte component. The promonocytes
are predominant.
*Acute Leukemia M6 (erythroleukemia) is characterized by the coexistence of blasts
myeloid and abnormal erythroblasts in the bone marrow. The erythroid precursors are
at least 50% of the cells; at least 30% of the cells not-erythroid consists of myeloblasts. The
erythroid precursors are dysplastic and PAS positive. Alterations cytogenetics are extremely
variable.
*Acute leukemia M7 (megakaryocytic): The diagnosis of this form is exclusively
immunophenotypic: the blastic element must be positive for antigens CD41, CD42 and CD61
(platelet antigens GPIb, GPIIb / IIIa and GPIIIa). In addition, the nature megakaryocytic
leukemia can be detected with microscopy electronically through the demonstration of
platelet peroxidase cells.
In 2002 he was drafted to the WHO classification of myeloid neoplasms, which is based
on many criteria included in the previous FAB classification, however assigning relevance
some diagnostic molecular abnormalities (Table 2)
Table 2 - WHO classification of acute myeloid leukemia P.123.

clinical
The clinical picture of acute myeloid leukemia is characterized by symptoms and signs
bone marrow failure: anemia (pallor, weakness, fatigue, palpitation) thrombocytopenia
(petechiae, bruising, bleeding skin and mucous membranes), granulocytopenia
)infections(.
In particular subtypes myelomonocytic lineages and monocyte (LAM M4-M5 in the
classification FAB) you may experience signs and symptoms of infiltration
(hepatosplenomegaly), enlarged lymph nodes, infiltrated skin, gingival hypertrophy,
involvement central nervous system). The onset of the disease can also be characterized by
a disseminated intravascular coagulation (DIC), in particular in acute leukemia promyelocytic
(AML-M3) (see below), where this complication is present in more than 90% of cases.

diagnosis
The grading of the patient with acute myeloid leukemia involves primarily the blood count,
which in most cases presents leukocytosis associated with anemia and thrombocytopenia,
but can also be demonstrated anemia and thrombocytopenia leukocytes with normal or
pancytopenia.
Of fundamental importance is the assessment of coagulation (prothrombin activity,
aPTT, Quick time, fibrinogen, FDP), in order to quickly identify the possible presence of
disseminated intravascular coagulation that predominantly associated with acute
promyelocytic leukemia.
To establish the diagnosis process, a aspirate, with morphological evaluation blood smear
bone marrow, with quantification of blasts (which must be larger or equal to 20% of the
total cellularity and with characterization in immunocytochemistry of leukemia cells.
Modern diagnostics of acute myeloid leukemia in addition provides the performance
immunophenotypic analysis of bone marrow cells (the major antigens of diagnostic utility
are given in Table 3) and cytogenetic and molecular analysis for the definition of
disease risk and prognosis.

Table 3. CD antigens main utility in the diagnosis of acute myeloid leukemia P.124

prognosis
The factors that most significantly affect the clinical course of patients with LAM are age,
which is associated with poor prognosis if more than 55-60 years, and cytogenetics, enabling
you to locate some forms with a good prognosis (low risk), with t (8; 21), inv (16), t (16; 16), t
(15; 17) and variants, and forms a poor prognosis (high risk) if abnormalities of chromosome
5, chromosome 7 or a complex karyotype (≥ 3 abnormalities).

Algorithm for defining the risk of acute myeloid leukemia not M3 P.125

Therapy
The treatment of patients with acute myeloid leukemia involves processing of support,
which consists in hydration and alkalinization of the urine to prevent kidney damage due to
the products of cellular degradation, prophylaxis and treatment of infections, and
transfusion therapy.
The anti-leukemia therapy is divided into a phase of induction of remission, that provides a
chemotherapy with the aim of reducing the leukemic cells to a value less than 5% of marrow
cells morphological examination (complete remission hematologic).
At this stage follows the consolidation therapy of remission, that has the purpose of
prevent the re-expansion of minimal residual disease (not morphologically detectable) that
persists at the time the complete remission.
The chemotherapy regimen most used at the stage of remission induction is the so-called "3-
7", that provides for the association of daunoblastina (45 mg/m2) or idarubicin
)mg/m2) for 3 days and cytosine arabinoside (ara-C) (200 mg/m2 continuous infusion 12(
for 7 days.
The consolidation phase of remission can make use of chemotherapy or the autologous
hematopoietic stem cell transplantation or allogeneic.
The most commonly used chemotherapy regimen in this phase involves high-dose ara-C (2-3
g/m2/12 hx 4-8 doses), which allows to obtain a disease-free survival of 30-40%.
Autologous haematopoietic stem cell transplantation involves the administration of doses
higher chemotherapeutic agents and provides a lower incidence of recurrence. It
however, be applicable to patients under the age of 65 years and is burdened with a
mortality peritrapiantologica about 5% (due to drug toxicity and complications
infectious). The survival disease-free with this procedure is about 40-50%.
Allogeneic hematopoietic stem cells (donor family or not sibling) bases its effectiveness, as
well as the effect of the cytotoxic regimen preparation of radio-chemotherapy, anti-
leukemic effect of the immune system donor against residual leukemic cells (graft-versus-
leukemia, GvL). The procedure, however, apply to patients aged less than 55 years old and in
good condition general and is still burdened by a transplant-related mortality of 20-40%, due
principally to the toxicity of radio-chemotherapy, infectious complications and the
graft versus host disease (graft-versus-host disease GvHD), acute and chronic. The
disease-free survival in patients undergoing the transplant is about 50-60%.

Acute promyelocytic leukemia

For biological and clinical characteristics deserves a separate discussion for acute leukemia
promyelocytic (AML-M3 of the FAB classification), which represents approximately 10% of
acute myeloblastic leukemia in adults.

Pathogenesis
In 98% of patients with AML-M3 is observed balanced translocation, without loss of
material, formally defined as t (15; 17) (q22, q21), 1% of patients do not show the
rearrangement conventional cytogenetic examination (cryptic rearrangement). In
methods of molecular biology show that the translocation determines the 1991
juxtaposition of the PML gene (promyelocitic leukemia), mapped to band 15q22, and
RARα (retinoic acid receptor α subunit), mapped to band 17q21, with the creation of
chimeric protein PML-RARA.
The breaking point at the level of PML is variable, localizing in three different "breakpoint
cluster regions" (BCR) and being subject to the effects of "splicing " alternative. The points of
breaking most common are as follows:
-Bcr1 (Incidence: 70%): is located towards the 3 'end of the gene and contains the sequences
encoded by exons 5 and 6 of PML. Determines the creation of a chimeric protein
PML / RARα weight of 110-120kD, which is defined so L = long
-Bcr2 (incidence 10%) is located in or around exon 6 of PML. Protein fusion of variable
length, defined therefore V = variable
-Bcr3 (incidence 20%) is the most frequent and falls at the 5 'end of the gene
PML, PML fuses exons 1-3 of the exon 3 of RARα. Fusion protein small weight of 90-103kD,
defined S = small.

The breaking point in the RARα gene is always constant being localized at the level of
the first intron of the gene. The RARα gene is a transcription factor that plays a role
role in normal hematopoiesis leading to a normal differentiation cellular and blocking cell
proliferation .. LAP in the chimeric protein PML/ RARα is able to recruit a complex of
repression of transcription which acts through a histone deacetylation. In this way the
chromatin assumes a conformation less accessible to the factors necessary to promote
transcription. The acid in the form trans retinoic acid (ATRA) at physiological concentrations
is able at least in part of remove the differentiation block in an almost physiological, since
the gene RARα coding for the receptor of ATRA.
There are variant translocations: t (11; 17) (q13, q11) with rearrangement PLZF-RARα,
t (5; 17) (q31, q11) and NPM-RARα rearrangement.

Clinical
The clinical presentation is characterized by more than 90% of patients from a severe
syndrome bleeding due to disseminated intravascular coagulation triggered the activity
procoagulant of the granules of promyelocytes, and which, moreover, is frequently
aggravated from the initial cytolysis induced by chemotherapy.

Diagnosis
The grading of the patient with acute promyelocytic leukemia include:
-Physical examination with special reference to hemorrhagic manifestations in place or
suspicious outbreaks actual or suspected
-The CBC, which shows general anemia, thrombocytopenia, leukocytosis / leukopenia
-Coagulation (PT, PTT, fibrinogen, FDP, D-dimer), which shows an increase of
FDP (from secondary fibrinolysis), a reduction of fibrinogen, an elongation of PTT and
thrombin time (for consumption)
-The aspirate: morphology shows respectively in the classic form ipergranulare
ipergranulari promyelocytes with Auer rods and variant ipogranulare promyelocytes
microgranular core folded and lobulated
-The immunological phenotype of bone marrow: in the classical form hypergranulate the
pathological promyelocytes are HLA-DR-, CD34-, CD11b-, CD9-, CD33 +, CD13 +, in
variant hyprpogranulates: leukemic cells frequently express positivity antigen CD2
-Cytogenetics (conventional and / or FISH) on bone marrow for the detection of
translocation t (15; 17) (q22, q21)
-Molecular biology of bone marrow or peripheral to search the transcript PMLRARα.
Therapy
The treatment of patients with acute promyelocytic leukemia involves the treatment of
CID with platelet concentrates, fresh frozen plasma, fibrinogen. After documented diagnosis
therapy is based on acid retinoic acid (all-trans-retinoic acidATRA), which induces
differentiation of leukemic cells, in combination with chemotherapy with anthracyclines.
A pilot study of the Italian Group for the Study of Malignant Hematologic Diseases
the Adult (GIMEMA) used a protocol based on the association of ATRA and idarubicin
(Protocol "AIDA") that includes a phase of induction of remission Full (ATRA and idarubicin),
a consolidation phase with various chemotherapy
)Idarubicin and Ara-C, mitoxantrone and etoposide, idarubicin, Ara-C and thioguanine(.
The results of the study showed the achievement of complete remission in
of cases with resolution of coagulopathy in 7-10 days and overall survival 85-90%
The ATRA therapy may present some side effects. Among the most serious recall
hyperleukocytosis, a result of the maturation of promyelocytes and ATRA syndrome
(detectable in 15-23% of cases) is characterized by fever, pulmonary infiltrates payment
pleuropericardico, kidney failure and heart failure. The treatment of syndrome ATRA
provides for the suspension of ATRA and the use of dexamethasone to high doses.

Molecular targets in the treatment of acute leukemia

Chromosomal translocations are present in about 40% of patients with acute myeloid
leukemia (AML). There have been reports two types of translocations: those that activate a
particular tyrosine kinases and those that modify chromatin structure, activating a
complex that represses transcription. The remaining 30-50% of patients may instead
present a point mutation of a gene that encodes a receptor activities tyrosine kinase or a
gene coding for a protein involved in the processes of signal transduction. Molecular therapy
should correct then directed towards these targets.

Tyrosine kinase
Activation Can be determined by a chromosomal translocation or by a gene mutation. A
leukemia characteristically associated with a Chromosomal translocation that involves the
activation of a specific tyrosine kinase is leukemia Ph1 positive chronic myelogenous. Other
translocations that cause constitutive activation of a tyrosine kinase have been reported in
the hypereosinophilic syndromes.
In 50% of AML the activation of a kinase can be determined by a mutation gene. 37% of
patients have a duplication ("internal tandem duplication," ITD) Flt3 gene, which encodes a
transmembrane receptor with tyrosine kinase activity.
The receptor, encoded by a gene Flt3 normal, binds to its ligand, it dimerizes and frees its
tyrosine kinase activity. When instead the gene presents a Flt3 ITD, the receptor tends to
dimerize spontaneously in the absence of ligand and it has a constitutive activation of the
kinase. 7% of patients with LAM also features Another type of mutation in the gene Flt3. It is
a point mutation that affects the aspartic acid in position 835, contained in a region that
plays a control function of the receptor kinase activity. Therefore a mutation in this seat
eliminates this adjustment and induces activation of the kinase.
Approximately 10% of patients with LAM may have a point mutation in the load c-KIT gene,
which codes for another receptor kinase activity. Mutations that affect this gene are similar
to those reported for Flt3 and cause the activation constitutive kinase.

Molecular therapy. Imatinib mesylate (Gleevec) is a molecule with anti-tyrosine kinase. Such
action is carried out because the imatinib competes with ATP for binding to the region with
tyrosine kinase activity of the fusion gene BCR-ABL, produced by the t (9; 22) and specific
marker of Ph1 positive CML. Several studies have demonstrated the effectiveness of Glivec
in patients with this type of leukemia. The Glivec exerts its inhibitory also against other
kinases involved in hypereosinophilic syndromes and against gene c-KIT normal or affected
by a particular type of mutation. The Glivec, however, has no effect on Flt3 mutations.
Therefore, for patients with AML with mutation Flt3 have been researched and developed
new molecules with anti-tyrosine kinase (PKC412). Direct investigative studies to test the
therapeutic efficacy of such molecules are still in progress. It 'was, however, shown that
these inhibitors when administered alone are able to induce a clinical improvement of 20%
of patients with AML with Flt3 mutation resistant to conventional chemotherapy protocols.

Changes in chromatin structure

Chromosomal translocations. Chromosomal translocations closely related to a specific
citotipo FAB AML that activate a protein complex that represses transcription of specific
target genes. A translocation is acting through this mechanism in the t (15; 17),
characteristically associated with acute promyelocytic leukemia. The translocation results in
the rearrangement of the PML gene, mapped to 15q22, the gene RARA, encoding the
retinoic acid receptor alpha, mapped to 17q21. The gene chimeric PML-RARA, formed on
chromosome 17, which operates a complex that is formed by two repressors of transcription
and a histone deacetylase. This complex represses the transcription of target genes
important for the differentiation of myeloid cell. The translocation then determines and
block mitogenesis and differentiation. The latter is exceeded giving the patient all-trans
retinoic acid (ATRA), at doses higher than physiological ones. The ATRA restores the
transcription of target genes by eliminating the link between RARA-PML and repression
complex.
The RARA gene, as well as with PML, can rearrange with other genes, among these we must
remember PLZF, Which mapped on chromosome 11 at band q23, rearranges
with RARA in the translocation t (11; 17). Patients with this translocation have
a LAM resistant to ATRA. The resistance is determined by the fact that the fusion gene
PLZF-RARA has two binding regions to the aforementioned repression complex of
Transcription: one provided by PLZF and the other provided by RARA.

Molecular therapy. Experimental data obtained from mouse models and anecdotal data
obtained in a few patients with LAM-resistant to ATRA indicate that resistance to ATRA can
be overcome by administering ATRA and inhibitors of histone deacetylation. These is
the last act on the histone deacetylase complex that participates in the aforementioned
repression. Histones are proteins that together form the nucleosome DNA, subunits
fundamental chromatin. The transcription of the nucleosome depends on the degree of
histone acetylation. In case of deacetylation the nucleosome is contracted and has a
block transcription while in the case of acetilation of histones the nucleosome
released and is favored transcription. Inhibitors of histone deacetylases blocking
this enzyme should restore the transcription of target genes and the reinduce
cell differentiation. Based on this rational, whereas the repression complex above is
activated not only in patients with AML-M3 but also in those with other cytotypes, patients
with disease that is refractory to chemotherapy were initiated in the investigative protocols
therapy based on the use of inhibitors histone deacetylases.

Inhibitors of signal transduction

Mutations. The most frequent target is the RAS gene. This gene encodes for a protein linked
to the cytoplasmic membrane and equipped with GTPase activity. The RAS protein, which is
capable of forming a bond with GDP and GTP, cycling between a state active (GTP-bound)
and an inactive state (bound to GTP). Also to be activated protein must be bound to the cell
membrane. This binding is induced by a modification of the protein at the level of its lipid
portion, process that goes under the name of prenylation. The latter takes place by the
action of two enzymes farnesyl-and geranylgeranyl transferase. Point mutations of RAS,
which affect the 20%, affecting the codons 12, 13 and 61 and maintain RAS in its active form
linked to GTP.

Molecular therapy.The prenylation of RAS can be blocked by using inhibitors farnesylation

(Zarnestra). So it is believed to block the binding of RAS to the membrane cell and its
subsequent activation. Farnesylation inhibitors may have a wide use in AML, inducing a cell
growth arrest and apoptosis. Despite numerous experimental studies the mechanism of
action of these molecules is yet to be defined. Since the response to inhibitors of
farnesylation notcorrelates with the state of RAS, it is believed that they act by preventing
the farnesylation of other proteins which in turn play a crucial role in various cellular
processes.However, in vitro studies have shown that inhibitors of farnesylation may be
employed effectively in Ph1 positive CML resistant to therapy with TSI and Ph1 positive
acute lymphoblastic leukemia. Moreover, the few studies investigating hitherto conducted
in patients with LAM resistant to various protocols of chemotherapy showed a clinical
response, which consisted of a partial remission in 32% of cases.

25. Lymphocytic leukemia (or lymphoblastic) acute p.131