Indian Journal of Pathology and Microbiology

HEMATOLOGY SECTION - ORIGINAL ARTICLE
Year
: 2008  |  Volume : 51  |  Issue : 1  |  Page : 97--101

Proliferative indices, cytogenetics, immunophenotye and other prognostic parameters in myelodysplastic syndromes


Neelam Varma1, Subhash Varma2,  
1 Department of Hematology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Correspondence Address:
Neelam Varma
Department of Hematology, PGIMER, Chandigarh - 160 012
India

Abstract

Thirty-five adult myelodysplastic syndrome (MDS) patients were included in this study: 11 refractory anemia (RA), 4 RA with ring sideroblasts (RARS), 9 RA with excess of blasts (RAEB), 10 RAEB in transformation (RAEB-T) and 1 chronic myelomonocytic leukemia (CMML). The ranges of survival were 4-51 months, 40-59 months, 7-38 months, 5-24 months and 5 days, respectively. Three patients died and 3 showed disease progression during the course of the study. A composite analysis of proliferative indices, cytogenetics, immunophenotype and other conventional/novel prognostic parameters in the context of Indian MDS patients was performed. The proliferative indices (AgNOR and Ki 67 positivity), immunophenotypic markers, serum LDH and ferritin levels revealed wide variations and great overlap among different FAB subtypes. The scoring systems (Bournemouth, Dusseldorf and Goasguen) did not correlate with the prognosis and survival (p > 0.05). Clonal cytogenetic abnormalities were detected in 24/35 (68.57%) patients, +8, −5 and −7 being observed commonly. Cytogenetic abnormalities were more frequent in RAEB (88.8%), RAEB-T (80.0%) and RA (63.6%) subtypes of MDS. By Using Mufti«SQ»s prognostic system and International prognostic scoring system (IPSS), a good positive correlation was found between low risk category and RARS with better survival as compared to other risk categories/FAB subtypes (p < 0.01). However, rest of the FAB subtypes were assigned into high, intermediate and low risk categories without any correlation with the survival and/or leukemic transformation. RARS subtype revealed itself as the better prognostic category according to the cytogenetic findings as well as Mufti«SQ»s grading system. This was possible due to the longer follow up available for these patients (40-59 months).



How to cite this article:
Varma N, Varma S. Proliferative indices, cytogenetics, immunophenotye and other prognostic parameters in myelodysplastic syndromes.Indian J Pathol Microbiol 2008;51:97-101


How to cite this URL:
Varma N, Varma S. Proliferative indices, cytogenetics, immunophenotye and other prognostic parameters in myelodysplastic syndromes. Indian J Pathol Microbiol [serial online] 2008 [cited 2023 Feb 3 ];51:97-101
Available from: https://www.ijpmonline.org/text.asp?2008/51/1/97/40416


Full Text

 Introduction



The entities included in the term "myelodysplastic syndromes" (MDS) cover a wide range of heterogenous clonal disorders of hematopoiesis, characterized by refractory cytopenias and morphological abnormalities of peripheral blood and bone marrow cells. The French-American-British (FAB) classification, [1] proposed in 1982, included five pathologic entities: refractory anemia (RA), RA with ring sideroblasts (RARS), RA with excess of blasts (RAEB), RAEB in transformation (RAEB-T) and chronic myelomonocytic leukemia (CMML). Despite the initial criticism that the classification lacks biological correlation, it is routinely employed all over the world. It has served as a cornerstone to facilitate communication among workers and acts as a baseline prognostic parameter. Generally, patients with RARS have better prognosis and those with RAEB-T entail worse prognosis. Rest of the subgroups possess intermediate prognosis. The blood cytopenias and leukemic transformation are the main factors that contribute to the morbidity and mortality of MDS patients. However, considering the marked variation in the prognosis (overall survival and leukemic transformation), many additional prognostic factors have been proposed and assessed. These include peripheral blood and bone marrow blast percentage, cytopenias, dysplastic features, scoring systems, bone marrow cellularity, abnormal localization of immature precursors (ALIP), chromosomal abnormalities, immunophenotypic features (myeloid and lymphoid), molecular genetic markers etc. Most widely applicable prognostic parameters include FAB classification, bone marrow blast percentage, scoring systems, BM trephine biopsy findings and cytogenetics. [2],[3],[4],[5],[6] These have been tried and reported in the MDS patients, mainly from the West. There is no uniformity in the opinion regarding the best prognostic parameter, although (IPSS) [6] is the most popular in the clinical setup all over the world. International prognostic scoring system incorporates cytogenetic findings in addition to cytopenia(s) and blast count. However, molecular genetic lesions hold a lot of promise in the near future.

The awareness regarding MDS has been increasing over the years and lot more cases are being suspected and diagnosed in our country. Three of these reports have detailed the FAB subtypes and natural history of MDS, [7],[8],[9] and one dealt with the dysplastic and other morphological aspects of MDS. [10] As compared to the West, we encounter MDS in younger age group and the aggressive subtypes of MDS (RAEB and RAEB-T) predominate. With this background information, the present study was undertaken to analyze conventional and newer prognostic parameters in our MDS patients

 Materials and Methods



The prognostic markers in MDS patients were studied at different phases of the disease, e.g., at the time of diagnosis, during stable phase of disease and at the time of evolution into a worse prognostic subgroup of MDS or acute leukemia.

The complete blood counts, hemogram findings and LDH levels were used to derive scores according to three different scoring systems [3],[4],[5] [Table 1]. The bone marrow aspirates were used for cytogenetic analysis and making smears for morphological and immunocytochemical studies. Trephine biopsies were used for the assessment of ALIP, cellularity and dysplastic features.

Cytogenetic analysis

Metaphases were obtained by direct processing or short-term cultures of bone marrow or peripheral blood samples. [11],[12] From each patient, 0.2-0.5 ml of bone marrow aspirate or 1 x 10 6 cells/ml from peripheral blood, were used. The cells were cultured in the RPMI 1640 medium, containing L-glutamine, Penicillin, Streptomycin and supplemented with 15% fetal calf serum or 15-20% human AB serum. Two hours before harvesting, ethidium bromide was added to the samples (to prevent DNA condensation). The cells were treated with colcemid (0.05 µg/ml) for the last 15 min. After centrifugation and removal of the supernatant, the cells were treated with hypotonic KCl (0.075 M) for 20 min. Subsequently, the cells were fixed with methanol:glacial acetic acid (3:1 v/v). Cell suspension was dropped on to chilled clean slides and flame dried. GTG banding was performed by prior trypsinization (0.25% trypsin) and subsequent staining with Giemsa stain. The chromosome identification and karyotype designation was performed according to the recommendations of International system for human cytogenetic nomenclature (ISCN). [13]

The patients were assigned to low, intermediate and high risk categories according to Mufti's prognostic grading system and IPSS [Table 2],[Table 3] that incorporate cytopenias, blast count, ALIP and cytogenetic abnormalities.

Immunocytochemical studies

Immunocytochemical studies were undertaken by alkaline phosphatase anti alkaline phosphatase (APAAP) technique [14] by using monoclonal antibodies (MoAb) against CD34, CD33, CD 19, CD 7 and Ki 67 (Dako).

The percentage positivity of blast cells was noted.

AgNOR counts and serum LDH estimation were performed according to the standard methods. [15],[16] Serum ferritin was estimated using the ELISA kits.

Chi square test and Student's t-test were used for statistical analysis.

 Results



Thirty-five patients with primary MDS were included in this study from August 1995. Twenty six patients were studied twice, 5 patients thrice, 3 patients 4 times and one patient 5 times. The age ranged between 15 and 80 years, 13 of them (37.14%) being younger than 50 years. Male: female ratio was 2.18:1.

The patients were assigned into various FAB sub-types of MDS, depending upon the bone marrow blast percentage, ring sideroblasts, monocyte count and Auer rods. [1] Out of 35 patients included in this study, 11 patients were diagnosed as RA, 4 as RARS, 9 as RAEB, 10 as RAEB-T and 1 as CMML. The distribution of MDS patients in different FAB subtypes was found to be similar to a study reported earlier. [7] The inclusion of granulated blasts into total blast count changed the FAB subtype of #5 and #12 from RAEB into RAEB-T; however, it did not make any material difference in rest of the patients with RAEB or RAEB-T. Dysplastic features were very variable and quantitatively did not correlate with FAB subtype or prognosis. Similar results were observed in MDS patients, studied before the commencement of this study. [10] The abnormal localization of immature myeloid precursors (ALIP) was positive only in 5 patients: 2 RA, 2 RAEB and 1 CMML. Most of the RA or RARS patients did not reveal ALIP; therefore, these patients did not fall into a worse prognostic group on account of this parameter.

Bournemouth (B) score and Goasguen score are derived from peripheral blood cytopenias and bone marrow blast count. B score and Goasguen score did not predict prognosis better than FAB subtyping. For example, #12 (RAEB-T) with a B score of 2 (a better prognostic group) evolved into acute myeloblastic leukemia (AML) over a period of 6 months and #13 (RAEB) with a B score of 4 evolved over a period of 8 months. Dusseldorf score in addition takes into account serum LDH levels. Serum LDH values were more than 200 IU/L in 12 patients; however, this did not correlate with survival or leukemic transformation. [Table 4] shows the details of proliferative indices (AgNOR and Ki67), immunophenotypic markers, serum LDH and ferritin in MDS patients.

Proliferative indices were higher in RAEB and RAEB-T patients as compared to RA and RARS subgroups. However, the difference of median values of Ki 67 and AgNOR did not reach statistical significance ( p > 0.05). AgNOR counts increased only in #12 (from 1.9 to 2.43) and #13 (from 2.2 to 2.57) while evolving into AML.

Out of the markers studied immunocytochemically, CD34 and CD33 showed wide ranges with overlapping values in FAB subtypes. Blast cells showed positivity only for myeloid markers, B (CD 19) and T (CD 7) cell lineage markers were not expressed in any of the patients, neither in the MDS (with excess of blasts) phase nor in the leukemic phase. The number of CD 34 and CD 33 positive cells showed an increasing trend from the spectrum of RA to RAEB-T, which reflected the increase in blast number. CD 34 positivity increased in #12 (from 12 to 16%), #13 (from 14 to 22%) and #14 (from 16 to 18%). #12 and #14 evolved into AML from RAEB-T whereas #13 progressed from RAEB to RAEB-T subgroup during the follow up. In rest of the patients, the number of CD34 and CD 33 positive cells remained stable during the follow up.

Median LDH values were higher in RAEB, RAEB-T and CMML patients as compared to RA and RARS ( p p > 0.05). An overlap in the range of serum LDH values was again found among different FAB subgroups; this was true for serum ferritin values as well.

Individually, the proliferative indices, immunophenotype, serum LDH and ferritin values did not discriminate between different FAB subtypes vis-à-vis their prognosis (survival and leukemic transformation).

Clonal cytogenetic abnormalities were detected in 24 out of 35 (68.57%) patients. These were present in 7/11 (63.63%) of RA, 1/4 (25.0%) of RARS, 8/9 (88.8%) of RAEB, 8/10 (80.0%) of RAEB-T and 0/1 (0%) CMML patients.

Trisomy 8 was the most frequent cytogenetic abnormality - detected in 7/24 (29.16%) patients, followed by monosomy 5 - detected in 6/24 (25.0%) and monosomy 7 - detected in 4/24 (16.6%) patients. Rest of the abnormalities were detected in one case each and these included; del 3q24, 5q−,7q−, +11, del 12p, −15, −19, del (20)(q11q13) and −X.

Cytogenetic abnormalities, in addition to peripheral blood cytopenias, marrow blast count and ALIP were taken into account in order to assign patients into low, intermediate and high risk MDS grades [Table 2],[Table 3]. These results are shown in [Table 5],[Table 6].

This grading system predicted better prognosis for RARS patients as all of them were in the low risk category. However, other patients showed a scatter into 2 or 3 categories and the prognosis did not correlate with the risk category assigned to them.

 Discussion



Leukemogenesis has long been considered to be a multistep process and MDS constituted the earliest recognizable stage, which was characterized by distinct hematologic, morphologic and cytogenetic abnormalities. [17] The accumulation of multiple genomic lesions, irrespective of their precise order in relation to one another, may be the critical determinant for manifestation of MDS and its progression into AML. [17] MDS offers the investigators an ideal opportunity to explore various diseases/cellular characteristics in the indolent (stable) phase and subsequently in the leukemic phase. This scenario is not reproduced in any other hematological disorder except chronic myelocytic leukemia.

The blast count is of vital importance for the classification of MDS into FAB subgroups and also in order to differentiate MDS from acute leukemia. [1] An increasing blast count indicates progression into worse prognostic category of MDS or frank acute leukemia, over a period of time. Various factors affecting/reflecting the growth potential of blasts in an individual MDS patient are likely to have a bearing on the ultimate prognosis. There exists a marked variation of the natural course and response to treatment modalities; therefore, the necessity to study more prognostic parameters in MDS patients in order to understand the biology of preleukemia and leukemia better. Since the patients diagnosed in our country have been found to be different from those in the West, in certain aspects, it is more pertinent to study the disease characteristics of MDS in our setup.

The break up of FAB subtypes of our 35 adult MDS patients revealed a preponderance of patients belonging to RA, RAEB, RAEB-T subgroups. Thirteen out of 35 (37.14%) patients were younger than 50 years of age. These results are similar to those reported earlier from India [7],[8],[9] and somewhat dissimilar from the experience in the West. [2] Morphological features such as the inclusion of granulated blasts into the total blast count, dysplastic features and ALIP did not correlate with the FAB subtype or prognosis. Similar results were obtained earlier on a different set of patients. [10]

Bournemouth score was not found to be a good prognostic parameter in the earlier study. [7] In the present analysis, Bournemouth, [3] Dusseldorf [4] and Goasguen [5] scores did not correlate with the prognosis. Since the conventional parameters were found wanting in the previous experience, we decided to explore the usefulness of other, hitherto infrequently tried prognostic markers. These included cytogenetics, proliferative indices (AgNOR and Ki67), immunophenotypes and serum markers (LDH and ferritin).

Clonal cytogenetic abnormalities were found in 68.5% MDS patients, mostly belonging to the commonly encountered FAB subtypes. The overall incidence of cytogenetic abnormalities (22-79%) is concordant with that reported in the literature. [2] The incidence in the RAEB and RAEB-T (16/19, 84.4%) is similar to the reported experience of 60-90%. However, a larger number of our RA patients (63.6%) revealed clonal cytogenetic abnormalities, the reported incidence being 30-50%. Trisomy 8, monosomy 5 and monosomy 7 were the commonly detected cytogenetic abnormalities.

Proliferative indices (AgNOR and Ki67) showed higher values in RAEB and RAEB-T patients as compared to the RA and RARS subgroups; however, the differences in median values did not reach a statistical significance. Ki67 and AgNOR counts have been extensively reported in solid tumors, lymphomas and acute leukemias. However, there are no published reports regarding these indices in relation to MDS.

MDS and acute leukemia are two closely related disorders. While the immunophenotypic profile of AML blasts has been the focus of attention, surprisingly only few studies have addressed this aspect in the blasts of MDS patients. Clark et al. [18] studied the cells from 21 MDS patients and found that granulocyte and macrophage progenitors did not develop along two divergent lines but differentiate with emergence of dual characteristics. The present study could not address this aspect because a monocyte specific marker was not included in the antibody panel. Hokland et al. [19] studied 32 patients with RA and 10 patients with RARS. An increased number of CD 33 positive cells were found to correlate with higher probability of progression to RAEB, AML or CMML. Guyotat et al. [20] observed the marrow expression of CD 34 almost exclusively in the RAEB and RAEB-T groups. Thirty-five MDS patients were studied, of whom 23 were diagnosed as RAEB and RAEB-T and only 6 as RA. These authors found CD 34 positivity to be associated with poor prognosis. Oertel et al. [21] performed immunocytochemical studies on the marrow blast cell population of 16 newly diagnosed RAEB and RAEB-T patients and 12 patients of AML evolving from RAEB and RAEB-T. CD 34 positivity was found to increase with the progression to RAEB-T and AML. In the present study, the number of CD 34 and CD 33 positive cells showed an increasing trend from the spectrum of RA to RAEB-T, which reflected the increase in the number of blasts. However, CD 34 positivity did not correlate with the disease characteristics. This was true for serum LDH and ferritin values also.

In the present study, RARS subtype was found to be the better prognostic category, based on the cytogenetic findings as well as grading systems. A longer follow up of patients of other FAB subtypes is being recorded. This might yield a better correlation between newer prognostic parameters and overall survival in the near future.

 Acknowledgement



The authors gratefully acknowledge the ICMR project grant (no. 56/2/94-BMS II), which made this study possible, and the technical assistance rendered by Ms. Kiran Malik and Mr. K. L. Kapur.

References

1Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al . Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 1982;51:189-99.
2Mufti GJ. Primary myelodysplastic syndrome: Prognostic value of FAB classification, scoring systems, bone marrow histology and karyotypic analysis. In : Mufti GJ, Galton DA, editors. The Myelodysplastic Syndromes . Churchill Livingstone: London; 1992. p. 207-23.
3Mufti GJ, Stevens JR, Oscier DG, Hamblin TJ, Machin D. Myelodysplastic syndromes: A scoring system with prognostic significance. Br J Haematol 1985;59:425-33.
4Aul C, Derigs G, Schneider W. Abstracts of the first international symposium on myelodysplastic syndromes: Innsbruck, June 20-23. Blut 1987;56:C1-23.
5Goasguen JE, Garand R, Bizet M, Bremond JL, Gardais J, Callat MP, et al . Prognostic factors of myelodysplastic syndromes: A simplified 3-D scoring system. Leuk Res 1990;14:255-62.
6Greenberg P, Cox C, Le Beau MM, Fenaux P, Morel P, Sanz G, et al . International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997;89:2079-88.
7Dash S, Varma N, Sarode R, Marwaha N. Dysmyelopoietic features and bone marrow histology in 30 cases of primary myelodysplastic syndromes. Leuk Lymphoma 1991;3:419-22.
8Garewal G, Marwaha RK, Ray R, Marwaha N. Clinicohematological profile and natural history of childhood myelodysplastic syndromes. Indian J Pediatr 1993;60:573-81.
9Nair R, Iyer RS, Nair CN. Myelodysplastic syndromes: A clinical and pathological analysis of 88 patients. Indian J Cancer 1993;30:169-75.
10Varma N, Varma S, Khajuria A, Marwaha N, Garewal G. Evaluation of auer rods, blast morphology and dysplastic features in myelodysplastic syndromes. Indian J Haematol Blood Trans 1997;15:20-2.
11Garson OM. Cytogenetics of leukemic cells. In : Henderson ES, Lister TA, editors. Leukemia . 5 th ed. WB Saunders Company: Philadelphia; 1990. p. 131-52.
12Sandberg AA, Ake S. Cytogenetic techniques in Hematology. Clin Hematol 1980;9:19-38.
13Mitelman F, editor. ISCN 1995. International system for human cytogenetic nomenclature. Basel: Karger; 1995. p. 5-74.
14Mason DY. Immunocytochemical labeling of monoclonal antibodies by APAAP immunoalkaline phosphatase technique. In : Bullock GR, Petrusz P, editors. Techniques in immunocytochemistry. Academic Press: New York; 1985. p. 25-42.
15Crocher J, Nor P. Nucleolar organiser regions in lymphomas. J Pathol 1987;151:111-8.
16Wooton ID. Microanalysis in Medical Biochemistry . 4 th ed. J and A Churchill: London; 1964. p. 114-8.
17Bartram CR. Molecular genetic aspects of myelodysplastic syndromes. Hematol Oncol Clin North Am 1992;6:557-70.
18Clark RE, Hoy TG, Jacobs A. Granulocytic and monocytic surface membrane markers in the myelodysplastic syndromes. J Clin Pathol 1985;38:301-4.
19Hokland P, Kerndrup G, Griffin JD, Ellegaard J. Analysis of leucocyte differentiation antigens in blood and bone marrow from preleukemia (RA) patients using monoclonal antibodies. Blood 1986;67:898-902.
20Guyotat D, Campos L, Thomas X, Vila L, Shi ZH, Charrin C, et al . Myelodysplastic syndromes: A study of surface markers and in vitro growth patterns. Am J Hematol 1990;34:26-31.
21Oertel J, Kleiner S, Huhn D. Immunotyping of blasts in refractory anaemia with excess of blasts. Br J Haematol 1993;84:305-9.