|Year : 2018 | Volume
| Issue : 2 | Page : 63-67
Myelodysplastic syndrome among elderly patients with anemia: A Single institutional experience
Karuna Rameshkumar1, Meenakshi Arumugam2, LN Samaga3, Prashanth Shetty2, Jayaprakash Shetty1
1 Department of Pathology, KS Hegde Medical Academy, NITTE University, Mangalore, Karnataka, India
2 Department of Cytogenetics, KS Hegde Medical Academy, NITTE University, Mangalore, Karnataka, India
3 Department of Medicine, KS Hegde Medical Academy, NITTE University, Mangalore, Karnataka, India
|Date of Web Publication||18-Jun-2018|
Dr. Karuna Rameshkumar
Rainbow Children's Hospital, Marathalli, Bengaluru - 560 076, Karnataka
Source of Support: None, Conflict of Interest: None
BACKGROUND AND OBJECTIVES: Many geriatric patients with different cytopenias are symptomatically treated and miss the diagnosis of myelodysplastic syndrome (MDS). The study was done to estimate the prevalence of elderly presenting with cytopenias in hospital population and to assess karyotyping profile and gene expression in mTOR pathway and correlate with clinical course.
PATIENTS AND METHODS: Blood samples of patients (> 60 years) who visited outpatient department and who were in - patients being evaluated for anemias, were included. 15 patients were diagnosed with myelodysplastic syndrome. Karyotype profile and gene expression studies for S6K1 and 4E-BP1 and IPSS –R scoring were done.
RESULTS: Among 521 patients, normocytic normochromic anemias was most common (48.3%).15 MDS patients were classified (WHO classification) as - Single lineage dysplasia -6, multilineage dysplasia-5 multilineage dysplasia with excess blasts 1- and three with hypo plastic marrow. There was no significant correlation between the karyotype and subgroups of MDS and age. IPSS scoring showed patients, with very low score – n-1 (ARR-1.3) low score n-6 (ARR- 1.6-2.8) intermediate score n-4 (ARR 3.1-3.7). During follow up for two years one death was reported in the intermediate category. Quantification of mRNA expression of S6K1 and 4EBP1 showed absence or reduction in all cases.
CONCLUSIONS: An incidence of 2.8% MDS was observed. The median age reported is consistent with Asian literature. IPSS-R scoring with inclusion of cytogenetic analysis helped in effective risk stratification of patients for management. Though no definite conclusions can be drawn on expression of S6K1 and 4EBP1 of mTOR pathway due to limited sample size, their role in the cytopenia cannot be ruled out.
Keywords: Cytopenia, International Prognostic Scoring system scoring, karyotypic profile, mammalian target of rapamycin pathway, myelodysplastic syndromeCytopenia, International Prognostic Scoring system scoring, karyotypic profile, mammalian target of rapamycin pathway, myelodysplastic syndrome
|How to cite this article:|
Rameshkumar K, Arumugam M, Samaga L N, Shetty P, Shetty J. Myelodysplastic syndrome among elderly patients with anemia: A Single institutional experience. J Appl Hematol 2018;9:63-7
|How to cite this URL:|
Rameshkumar K, Arumugam M, Samaga L N, Shetty P, Shetty J. Myelodysplastic syndrome among elderly patients with anemia: A Single institutional experience. J Appl Hematol [serial online] 2018 [cited 2019 Jun 18];9:63-7. Available from: http://www.jahjournal.org/text.asp?2018/9/2/63/234557
| Introduction|| |
Many of the geriatric patients who present with morbidities such as anemia, leukopenia, and thrombocytopenia are symptomatically treated and miss the diagnosis of myelodysplastic syndrome (MDS). MDSs are characterized by cytopenias due to defects in hemopoietic stem cell differentiation. The pathophysiology of MDS is a multistep process involving cytogenetic changes and/or gene mutations and widespread gene hypermethylation at advanced stages., Diagnosis of MDS is based on the blood and marrow examination, showing blood cytopenias, hypercellular marrow with dysplasia, with or without an excess of blasts.
The most commonly used prognostic tool in the evaluation of patients is the international prognostic scoring system (IPSS), which stratifies the disease as low risk, intermediate-1 and intermediate-2 risk, and high risk on the basis of the percentage of myeloblasts in bone marrow, cytogenetic findings, and the number of hematopoietic lines affected. With one additional risk category, IPSS-R provides a better prognostic value helpful in the management.
Gene expression studies of progenitor cells from patients with MDS have reinforced the heterogeneity of the disease at a molecular level, differences in gene expression between low-and high-risk disease and differences among specific cytogenetic subcategories of MDS., Mammalian target of rapamycin (mTOR), a protein kinase (serine/threonine group), incorporates responses from a wide variety of signals to regulate cell growth, metabolism, and survival. This important kinase forms two distinct protein complexes, mTOR complex 1 (mTORC1), and mTORC2. The rapamycin-sensitive mTORC1 complex regulates multiple biosynthetic cellular processes (protein synthesis, cell cycle progression, cell growth, and proliferation ). It was observed that mTOR and its downstream intermediates, S6K1 and 4E-BP1, were activated in high-risk MDS patients and were not activated in low-risk patients.
In this background, the study was undertaken to estimate the prevalence of the elderly presenting with anemia and/or cytopenias in the population screened in KS Hegde Hospital, Nitte University, Mangalore and to study the karyotyping profile and gene expression in the mTOR pathway to understand the pathogenesis of cytopenias which occur in MDS causing morbidities.
| Patients and Methods|| |
Blood samples of patients (>60 years) including both male and female who visited the outpatient department and/or who were in-patients being evaluated for anemia in KS Hegde hospital were included in the study. The details regarding age, sex, clinical presentation, and therapeutic modalities were collected. Consent was taken as per the protocol approved.
RBC morphology by peripheral smear was used to classify anemia. This was supported by cytometric evaluation-mean corpuscular volume (MCV) mean corpuscular hemoglobin (MCHC) through the automated cell counter.
The results were tabulated and analyzed for the type of anemia. Hemoglobin levels, RBC morphology, and RBC indices were compared and analyzed with each other. Pearson's correlation coefficient was used to correlate between hemoglobin and age.
Among the patients who were screened for anemia, 20 patients were clinically suspected with MDS. Based on morphology, after ruling out vitamin B12 and folic acid deficiency, 15 patients were confirmed with a diagnosis of MDS.
A volume of 2 ml of bone marrow sample from each patient was collected in sodium heparin vacutainer. Karyotyping was carried out with direct and 24 h unstimulated cultures by using 5 ml of the Roswell Park Memorial Institute - 1640 media with 20% of fetal bovine serum. The optimum sample volume was calculated based on the number of WBC's in the received marrow sample. 24 h culture tube was incubated in 37°C CO2 incubator.
To the direct culture tube, 100 μl of KaryoMAX COLCEMID (10 μg/ml) (Gibco by life technologies™) was added and incubated at 37°C. After 30 min of incubation, the culture was spun at 2000 rpm for 10 min. Then, the cells were treated with the hypotonic solution (0.075M KCl) for 20 min at 37°C and fixed with Carnoy's fixative (3:1 ratio of methanol and glacial acetic acid).
After 24 h, 100 μl of KaryoMAX COLCEMID (10 μg/ml) (Gibco by life technologies™) was added to the 24 h culture tube and incubated at 37°C for 40 min. After incubation, the culture tube was spun at 2000 rpm for 10 min. Then, the cells were treated with the hypotonic solution (0.075M KCl) for 20 min at 37°C and fixed with Carnoy's fixative (3:1 ratio of methanol and glacial acetic acid). The cell pellet suspension was dropped on pre-chilled slides and dried at 45°C. Then, the slides were aged at 60°C in a dry oven for overnight. Next day, slides were treated with standard trypsin (1:250) (Gibco by life technologies™) solution and stained in 1% Giemsa (Merck) solution. A minimum of 20 well-spread metaphases were analyzed using Olympus BX53 microscope. 5 good quality metaphase spreads with 350–450 band resolution were captured using the CCD camera attached with a microscope. Analysis and karyogram of each metaphase were performed using the GENASI S software. Karyotypes were interpreted according to the International system for human cytogenetic nomenclature (ISCN) (2013).,
RNA extraction and real-time polymerase chain reaction
The total RNA was isolated with the total RNA Purification Kit (Jena Bioscience)– Isolation of total RNA by silica-gel membrane adsorption, according to instructions provided by the manufacturers. Purified RNA was dissolved in nuclease-free water and stored at-70°C. RNA integrity and concentrations were assessed with Biospectrometer (Eppendorf).
Reverse transcription was performed using the Verso cDNA Synthesis Kit (Thermo Scientific) with 200 ng total RNA in reactions of 20 μl according to the manufacturer's instructions. mRNA expression of S6K1 and 4EBP1 was quantified with an AreaMx Real-Time PCR System (Agilent Technologies). TaqMan assays (Applied Biosystems) for S6K1 (Hs00177357_m1), 4EBP1 (Hs0060705_m1), and the endogenous controls β-actin (ACTB; part number 4453320 G) were handled according to the manufacturer's instructions. Quantitative PCR was performed in duplicate with 10 μl reaction volume in the 1XTaqMan fast universal master mix (Applied Biosystems) using the following thermal conditions: 95°C for 15 s; 40 cycles of 95°C for 1 s, and 60°C for 1 min. To confirm specificity, reactions without reverse transcriptase as well as template controls were included.
Revised International Prognostic Scoring system score
In patients confirmed with MDS, the IPSS-R score was calculated based on hemoglobin levels, absolute neutrophils count, platelet count, number of blasts in the bone marrow, and the cytogenetics category. Age-related risk (ARR) was calculated. All patients were followed up.
| Results|| |
A total of 521 patients were screened. NC/NC anemia was the most common (48.3%) followed by MCHC anemia (29.9%). Among 20 patients suspected of MDS, 15 were confirmed by morphology [Figure 1]. Four of them presented with pancytopenia. The age ranged from 90 years to 60 years and the mean age was 65.9 years [Figure 2]. The male:female ratio was 8:7.
|Figure 1: Bone marrow biopsy showing dysplastic uni lobated megakaryocyte Leishman's stain ×1000|
Click here to view
The anemia was graded as mild, moderate, and severe based on hemoglobin levels. When the severity of anemia was correlated with age, it showed a significant value of 0.0089, which indicated the inverse relationship between age and hemoglobin levels.
The patients were classified according to the WHO classification – single lineage dysplasia-6, multilineage dysplasia-5 multilineage dysplasia with excess blasts-1 and 3 showed hypoplastic marrow. All three who had hypoplastic marrow had presented with pancytopenia.
Among the 15 patients diagnosed with MDS, in only one patient the metaphase spreads were inconclusive. Single cell, numerical, and structural abnormalities were clubbed together as SA. Eight patients had normal karyotype. 1 patient among them had in addition SA, whereas one patient had SA and polyploidy which for IPSS scoring was considered as normal. Four patients had only SA. Two patients had complex karyotype. When loss of Y was seen in few metaphase spreads, it was only noted down. There was no significant correlation between the karyotype and the subgroups of MDS and the age.
International Prognostic Scoring system
During the follow-up for 2 years, one death was reported in the intermediate category.
Quantification of mRNA expression of S6K1 and 4EBP1 showed absence or reduction in all cases. As age-matched controls also showed similar results, no conclusions were drawn.
| Discussion|| |
Given the high prevalence and impact of chronic health problems among the elderly, it is important to take necessary steps so as to maximize both the quantity and quality of life for the elderly. Normocytic normochromic (NC/NC) anemia was the predominant type. As the chronic diseases including diabetes mellitus and hypertension are highly prevalent, such a pattern seen is consistent with the reported literature. Anemia, which can be screened by hemoglobin levels, gives direction for further evaluation when used in conjunction with red cell parameters.
An incidence of 2.8% MDS was observed in the present study. As only the elderly population was included, incidence may be higher in general population. MDS are diseases of the elderly, with a median age at diagnosis of more than 70 years and with >10% of the patients being younger than 50 years of age. There are no known ethnic differences in the incidence of MDS, but MDS in Asian populations tend to occur at an earlier age, more often have a hypocellular marrow and present less often with isolated 5q deletion (5q-syndrome), while trisomy 8 seems to be more frequent than in Western populations. In another Indian study, where 30 cases of MDS were studied over a period of 8 years, the mean age at presentation was 55 years. One of the findings in the study was the patients were symptomatic for a prolonged period (average: 358.8 days) before the diagnosis was made  which reinforces the need to consider MDS high in the differential diagnosis in elderly patients with chronic health problems/persistent cytopenia. The median age of the MDS patients in this study was 65.9 years consistent with the literature.
The clonal chromosome abnormalities can be observed in 30% to >80% of patients depending on the MDS subtype and whether the disease is de novo or chemo- or radiotherapy-induced. In the remaining 20%–70% of patients with a normal karyotype, there is growing evidence that submicroscopic alterations such as point mutations, micro-deletions, micro-amplifications, epigenetic changes, or copy-number neutral loss of genetic information as by uniparental disomic provide the genetic basis for the disease. Karyotype also has the highest prognostic weight of all parameters in the IPSS-R. Among the 15 patients, normal karyotype was observed in eight patients. According to the ISCN, an abnormal clone is defined by at least two metaphases with the same supernumerary chromosome or structural change, or at least three metaphases with loss of the same chromosome. Complex abnormalities are defined as three or more independent abnormalities in at least 2 metaphases. The single cell abnormalities observed in the rest of the patients were considered as normal for IPSS scoring. In patients presented with cytopenia, the cytogenetic study was not optimal as observed in one patient. In a prospective study from Kerala, cytogenetics study was done in 52 patients among 60 patients diagnosed with MDS. The common abnormality seen was 5q del.
The IPSS is a vital standard for assessing the prognosis of primary untreated adult patients with MDSs. The impact of age was a major prognostic parameter for overall survival, although not for AML evolution. IPSS-R scoring system applied in this study risk stratified the patients which correlated with clinical outcome. The absent or low expression of S6K1 and 4EBP1 may be the reason for the cytopenia observed in the patients. It is also possible as no high-risk patients were in the study, the expression of S6K1 and 4EBP1 was low as observed by Follo et al. Stimulatory studies may be required to confirm the low levels as done by Al-Shouli et al. In their study, mTOR protein level in MDS was reduced and did not change in response to complement receptor stimulation. Lack of response to complement related co-stimulation and increase in C5aR2 expression suggested a potential mechanism for T reg expansion in MDS. The expansion of regulatory T cells (T regs) is one of the important factors in the progression of intermediate/high-risk MDS to acute myeloid leukemia.
| Conclusions|| |
An incidence of 2.8% MDS was observed in the present study. The median age reported is consistent with Asian literature. IPSS-R scoring with the inclusion of cytogenetic analysis effectively risk stratified the patients for the management. Although no definite conclusions can be drawn on the expression of S6K1 and 4EBP1 of mTOR pathway due to limited sample size, their role in the cytopenia cannot be ruled out.
The study was supported by financial grant from Nitte University, Mangalore after the approval of Institutional scientific committee and ethics committee (Ref.NURG/STF/06/11/2014).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Fenaux P, Haase D, Sanz GF, Santini V, Buske C; ESMO Guidelines Working Group, et al.
Myelodysplastic syndromes: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol 2014;25 Suppl 3:iii57-69.
Solomon PR, Munirajan AK, Tsuchida N, Muthukumarasamy K, Rathinavel A, Selvam GS, et al.
Promoter hypermethylation analysis in myelodysplastic syndromes: Diagnostic & prognostic implication. Indian J Med Res 2008;127:52-7.
] [Full text]
Greenberg PL, Tuechler H, Schanz J, Sanz G, Garcia-Manero G, Solé F, et al.
Revised international prognostic scoring system for myelodysplastic syndromes. Blood 2012;120:2454-65.
Chen G, Zeng W, Miyazato A, Billings E, Maciejewski JP, Kajigaya S, et al.
Distinctive gene expression profiles of CD34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities. Blood 2004;104:4210-8.
Pellagatti A, Esoof N, Watkins F, Langford CF, Vetrie D, Campbell LJ, et al.
Gene expression profiling in the myelodysplastic syndromes using cDNA microarray technology. Br J Haematol 2004;125:576-83.
Laplante M, Sabatini DM. MTOR signaling at a glance. J Cell Sci 2009;122:3589-94.
Follo MY, Mongiorgi S, Bosi C, Cappellini A, Finelli C, Chiarini F, et al.
The Akt/mammalian target of rapamycin signal transduction pathway is activated in high-risk myelodysplastic syndromes and influences cell survival and proliferation. Cancer Res 2007;67:4287-94.
Rooney DE, Czepulkowski BH, editors. Tissue culture methods in human cytogenetics. In: Human Cytogenetics: A Practical Approach. Oxford, United Kingdom: IRL Press; 1986. p. 1-37.
Shaffer LG, McGowan-Jordan J, Schmid M. An International System for Human Cytogenetic Nomenclature. 1st
ed. Basel: S. Karger Publishers; 2013. p. 1-140.
Tettamanti M, Lucca U, Gandini F, Recchia A, Mosconi P, Apolone G, et al.
Prevalence, incidence and types of mild anemia in the elderly: The “Health and anemia” population-based study. Haematologica 2010;95:1849-56.
Varma N, Varma S. Proliferative indices, cytogenetics, immunophenotye and other prognostic parameters in myelodysplastic syndromes. Indian J Pathol Microbiol 2008;51:97-101.
] [Full text]
Shah NM, Prajapati SG, Adesara RP, Patel AP. An analysis of 30 cases of myelodysplastic syndrome. Indian J Pathol Microbiol 2009;52:206-9.
] [Full text]
Gupta R, Rahman K, Singh MK, Kumari S, Yadav G, Nityanand S, et al.
Clinico-pathological spectrum and novel karyotypic findings in myelodysplastic syndrome: Experience of tertiary care center in India. Mediterr J Hematol Infect Dis 2017;9:e2017048.
Kennedy JA, Ebert BL. Clinical implications of genetic mutations in myelodysplastic syndrome. J Clin Oncol 2017;35:968-74.
Narayanan S. Clinical, hematological, and cytogenetic profile of adult myelodysplastic syndrome in a tertiary care center. J Blood Med 2017;8:21-7.
Al-Shouli S, Perezabellan P, Toulza F, Kordasti S, Mufti G. Reduced levels of mTOR and TGF- β signaling pathway-associated proteins in patients with high risk MDS. Blood 2016;128:5501-5.
Hofmann WK, de Vos S, Komor M, Hoelzer D, Wachsman W, Koeffler HP, et al.
Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow. Blood 2002;100:3553-60.
[Figure 1], [Figure 2]