Numerical Chromosomal Abnormalities in Patients with Acute Lymphoblastic and Myeloid Leukemia in Iran

The majority of cases of ALL demonstrate an abnormal karyotype, either in chromosome number or structural changes. Abnormal chromosome number in childhood acute lymphoblastic leukemia defines distinct biological subgroups with a different response to treatment. The tubes are cultured with three different protocols to save time if one protocol failed. Cultures are then harvested, and cells are fixed and chromosome spreads are prepared. Of 25 patient studied, one patient had psudodiploid karyotype, three patients had tetra-ploid karyotype, four patients had low hypo-diploid karyotype, four patients had high hyper-diploid karyotype, five patients had low hyper-d iploid karyotype and eight patients had normal karyotype. Some other factors like, Age, Sex, Consanguinity, Hemoglob in, W BC count, and Type of the leukemia cell also have been evaluated. We found excess number of patients having hypodiploid karyotype but still response to treatment protocols were satisfactory. By comparison between 4 different cultures methods, we find d irect method to be more efficient for ploidy analysis.


Introduction
Acute lymphoblastic leukemia (A LL) is characterized by clonal pro liferation, accumu lation, and tissue infiltrat ion of neoplastic cells. They are main ly regarded as childhood diseases, with an early incidence peak at two to five years of age, where they represent about 80% o f the childhood leukemia in the United States, and occur with an incidence of 30 cases per one million population per year [1].
A basic princip le of the WHO classification is that the diagnosis of myelo id and lymphoid malignancies should be based on the integration of the information derived fro m genetic, immunophenotypic, biological, and clinical features in order to better define specific disease entities. In fact, genetic findings may predict the prognosis and biologic properties of the leukemia more consistently than does morphology [2].
Chro mosomal abnormalities in childhood ALL had important significance related to prognosis, diagnosis and management. Chro mosomal classification in ALL is based on the number of chro mosomes in abnormal cells (p loidy pattern) as well as the structural changes.
Abnormal ch ro moso me n u mbers in ch ildhood acute ly mp h o b lastic leu kemia, d efin es d is tin ct b io lo g ical subgroups with a different response to treatment [3,4]. The largest subgroup within the non-T cells A LL (appro ximately 25%) is the hyper-diploid group, which is associated with favorable characteristics and outcome [3,4,5]. In contrast the hypo-diploid and near-haploid ALL is much rarer (1%) and has shown a very poor outcome with the few intensive protocols reported [6,7]. Near haplo id has been reported in around 41 children [5,8,9] and in six addit ional children within a clinical study [9].
The majority of cases of ALL demonstrate an abnormal karyotype either in chro mosome nu mber or as structural changes such as trans-locations, inversions, deletions, etc. These changes were detected in only half of A LL patients in the first banding studies [10]. The scantiness of information gained fro m chro mosomal findings in ALL has been in large part, due to technical difficult ies. The emerg ing theory for the role of constitutional trisomy 21 in leu kaemia predisposition is that genes on this chromosome contribute to the expansion of haematopoetic co mpart ments during early development that result in an increased pool of potential tumour precursor cells [11].
Chro mosome studies in ALL exh ibit poor morphology. Chro mosomes tend to spread poorly, and appear blurred and fuzzy with indistinct marg ins, making banding studies challenging or even impossible [12,13] Improvements in spreading and banding techniques have resulted in higher rates of detection, and most studies now report chromosomal changes in 60% to 85% of A LL cases. [14,15].
We have conducted this study to evaluate numerical abnormalities in these patients for better management of patients admitted to this hospital by using different cell culture and chemotherapy treatment protocols.

Material and Methods
This work was conducted on 25 cases (five cases with AML and 19 cases with A LL) referred to our laboratory for karyotype and ploidy determination of chro mosomes after getting a written consent from their parents. Only new diagnosed cases (from December 2007 to July 2008) were included in this survey and old cases which have been already started chemotherapy treatment were excluded. 0.5-1 ml of bone marrow aspirate were collected in centrifuges tube containing five ml RPMI co mplete with 1% preservative-free heparin . In few cases where the bone marrow samples were unavailable or failed to grow in culture, blood samp les were used. The optimu m cell density for a bone marrow cu lture is 10 6 cell/ ml. All samples were optimized for cell density, using hemocytometer (slide counter chamber).
In most cases, the preferred culture method was overnight colcemid (ONC), fo llo wed by flurodeo xy uridine (FudR) and 24 hour protocols. In AML samples the preferred technique was FUdR, followed by 24 hours cultures and then ONC. For each patient we set up at least three cu lture tubes including one d irect cu ltures method [12] and two out of the three fore-mentioned methods. Quality of spreads in these methods were compared. Good and analysable spreads could obtain with direct method for plo idy determination in most cases, but for structural chromosomal analysis, flurodeo xy uridine (FudR) method proved to be satisfactory.

Number of Patient, Age, Sex, Ethnic Groups, Consanguinity, Hemoglobi n Rate and WBC Count Factors
Bone marrow (19 patient) o r b lood (six patients) samples fro m 25 patients, suffering fro m leukemia referred fro m Shafa hospital have been analy zed. A mong 25 patients participated, 19 A LL (including two cases with T-ALL and seven cases with B-ALL), five AM L and one unidentified case was present.
Out of 25 patients, 16 males (64%) and nine females (36%) are recorded. The age of patients ranges fro m 1.5 to 12 with med ian age of 5.82 years.

Res ponse to Treatment
Of these 25 leukemic cases, 23 (92%), ach ieved comp lete remission but with different resistant. Two patients (8%) d id not respond to treatment and survival, one of them with hypo-diploid karyotype had Philadelphia chro mosome, and other one had complex karyotype of hypo-diploid and near-haploid . Both of them were among AML patients with M3 and M4 subgroup, Table 3.
Regarding hematolog ical parameters, cases with lower Hb (< 8 g m/dl) and high WBC (>50000/ mm3) showed a better remission rate. Cases with age < 5 years, male sex and couples with positive consanguinity also showed a better remission rate, although not significant.
Although some of these patients are hypo-diploid (three cases), or pseudo-diploid (one case), most of them (92%) had good remission with treat ment protocols (U.K.C.C.S.G) for ALL, and (B.F.M) AML patients. Only two patients (8%) had no responses to treatment, both of which were among AML leukemic cases.
Of 17 patients with abnormal karyotype, eleven B-cells ALL and five AM L with M 1 to M 5 subgroups were recorded. Classification of one of the patient could not be determined. Eight patients had complex karyotype with more than two clonal chro mosomal abnormalities. Details are available on request.

Discussion
Most of the leukemia patients have cytogenetical or mo lecular abnormality and it is shown that there are so me relations between chromosomal abnormalities and morphological and immunological characteristics of the cancer cells in these patients. More important are karyotype changes which show important prognosis without any relations to other variables like age, sex and primary leukocyte count, and so permits to differentiate between high risk and lo w risk patients at the time of d iagnosis for proper treatment.
Cytogenetic analysis of 25 cases in our study shows 8 patients (32%) to have normal d iplo id karyotype, similar to many reports . Pseudo-diploid on the other hand were found in only one case (4%) which is much lo wer than the earlier reports which suggested that pseudo-diploid co mprised the largest cytogenetic group (41.5%) in childhood ALL [16]. This could be due to misclassification of pseudo-diploid karyotype by failure of detection of some submicroscopic genetic abnormalities at the conventional cytogenetic analysis.
Low hypo-diploid karyotype (30-44 chro mosomes) were present in 12% of the our cases, which are h igher than those reported by others who considered hypo-diploidy to be a relatively uncommon (>9%) finding in ALL [17,18]. Many studies reported low percent of patients in hypo-diploid group, but some studies [19][20], including our study (12%) found much higher percent of the patient in this group with good remission. In studies in which only cytogenetic investigations are carried out, some hypo-diploid cells may not diagnosed correctly due to presence of many hyper-diploid cells and only after relapse of the disease and repeat investigation of these patients with FISH, or other advanced molecular methods, the true karyotype can be detected.
Hyper-dip loid (lo w + h igh hyper-diploid ) represented a majority of ALL cases in our study and many other authors have also reported same higher frequency [21,22].
The remission rates in our study cases were almost same among hypo-diploid, pseudo-diploid and hyper-diplo id groups, but the normal dip loid cases had better remission rate.
Dip loid g roup was reported to have the best remission by some authors [19] although others considered it to be of an intermediate remission rate [23,24].
Response to treatment rates varied among chro mosome ploidy groups. The highest response rates were seen in patients with normal karyotypes and a modal number >50 chromosomes. In our study, after normal karyotype cases, we also found the best remission rate in this group. Hyper-dip loidy was found by some authors to have a good prognosis [25,26] unless associated with structural aberrations making it less favorable [19,27,28].
In our study, we had two resistant cases, one mixed numerical aberration (near haploid + low hypo-diploid) and the other one with low Hypo diploid, both of which were among AML subtype.
Near-haplo id (< 30 chro mosomes) A LL, were not present in our case, except the one with mixed nu merical aberration (near haploid + low hypo-diploid) between two resistant patients. Near-haploid is a rare and unique subgroup associated with a very poor outcome. It may be underestimated being masked by a coexisting hyper-diploid line.
In our study although any chromosome could be involved in tetrasomy, most of chromosomes especially 5,9,10, 12,18, 20,22 and X were duplicated mo re frequently.
Of four Majors Ethnic groups in Khozestan province including Persians, Iran ian Arabs, Bakhtiari Lu rs, and Behbahanis, 14 Arab (56%), 9 Lurs (36%) and two Farsis (8%) are recorded. Prevalence of Arabs were significant in these four major ethnic groups, although it may be unreliab le due to small samp le size, Table 1. We could not find any data showing prevalence of leukemia in these Ethnic groups in Iran.
The most common FA B type among our cases (48%) were L2 subtype with better remission compared to other immunophenotypes [29]. So me studies reported a poor prognosis associated with L2 and other studies reported that significance of L2 remained controversial [30].
Although full analy zing of karyotype spreads for determination of structural, nu merical and submicroscopic genetic abnormalities at the conventional cytogenetic analysis and its easier detection by the molecular and FISH technique is mandatory, but determination of only numerical chromosomal changes (ploidy pattern) for typing of patients into different plo idy groups can also be useful as a primary step. Therefore we decided to conduct this study because of increasing number of ALL patients and need for immediate report for plo idy determination.
We compare three standard methods including, overnight colcemid (ONC), flurodeo xy urid ine (FudR) and 24 hour protocols, with a modified direct culture method (12), and found the modified direct culture method to be easy and satisfactory for plo idy pattern determination in most (68%) cases.

Conclusions
We found excess number of patients having hypo-diploid karyotype but still response to treatment protocols were satisfactory. Variat ion in number of hypo-diploidy in different studies could be due to difference in ethnic group. Out of four different cultures methods used, we found the direct method to be satisfactory for ploidy analysis.