INTRODUCTION:

Myelodysplastic syndrome (MDS) is a clonal stem cell disorders characterized by single or multilineage dysplasia, cytopenia, ineffective hematopoiesis, and intact maturation with a variable increase in blasts¹.

There is an ineffective attempt of the bone marrow to successfully commit its physiological role of sustained hematopoiesis¹. The peripheral blood cytopenia result from ineffective hematopoiesis and increased apoptosis as evidenced by an accompanying hyperplasia in bone marrow. These entities evolve progressively and may transform into acute leukemia.

INCIDENCE AND EPIDEMIOLOGY:

If we question ourselves, how much is the disease load? The figures stating about the incidence of MDS in India is still buried and there is a limited epidemiological data elsewhere. In the United states a crude annual incidence rate of 4.1 per 100,000 is reported with 30,000 to 40,000 new cases per year². As per the data available on MDS cases in the western world, it is commonly seen in the older age group with advanced cases being detected in >=65years and has a male predominance³. Beside the treatment induced MDS, it is uncommonly seen in <50years of age⁴ and the risk of MDS increases with age⁵.

MATERIALS AND METHODS:

Study design: Prospective and Retrospective

Study period: 5 years [1stMarch 2011 – 9thApril 2016]

Sample size: 30

Study place: Department of Pathology, Kasturba Medical College, Manipal University, Manipal.

Study Method:

Data of patients diagnosed as MDS at KMC, Manipal between 1st March 2011 and 9th April 2016, were collected from hospital records and Lab information system. Evaluation of peripheral smear, bone marrow aspirate and bone marrow biopsy slides were done. In each case frequency of all morphological abnormalities in erythroid, myeloid and megakaryocytic lineage were recorded. The bone marrow biopsies were evaluated for cellularity, cytomorphology, presence of ALIP (Abnormal localization of immature precursors) and fibrosis if any. These patients were classified according to 2008 WHO criteria as well as 2016 WHO classification. The frequency of each parameter was assessed using appropriate statistical methods.

Follow up of the cases when available was done and any transformation to leukemia was recorded.

Inclusion criteria:

All the patients irrespective of age and gender diagnosed as MDS according to 2008 WHO classification on bone marrow examination and cytogenetic analysis whenever available.

RESULTS:

The 30 cases studied were classified according to WHO 2008 and WHO 2016 classification. The cases were discussed according to WHO 2008 classification. There were 2 cases of RARS, 7 cases of RCMD, 6 cases of RAEB-1 and 15 cases of RAEB-2.

The patients belonged to a broad age group of 9 to 76 years. The mean age and the median age of presentation was 54.67 and 59 years respectively. MDS was commonly seen in the 6^th decade of life (8/30, 27% cases). There was a male predilection with male to female ratio of 3.3:1. The common presenting complains were anaemia (26/30, 86.6% of cases) >Fever (12/30, 40% of cases) >Bleeding (5/30, 16% of cases).

The most common physical examination finding was splenomegaly (8/30, 26.7%) >hepatomegaly (7/30, 23.4% cases) > lymphadenopathy (3/30,10% cases).

Cytopenias:

In all the cases studied pancytopenia (12/30, 40% cases) >unilineage cytopenia (anemia) (10/30, 33% cases)>bicytopenia(8/30, 26.67%cases) was seen.

Bone marrow findings:

Cellularity (on bone marrow biopsy)

Cellularity was assessed in all 30 caseson bone marrow biopsy. We noticedhypercellular (19/30, 63.3% cases)> cellular (8/30, 26.7% cases)>hypocellular (1/30, 3.3% cases) particles. The cellularity wascommonly increased for age (21/30, 70% cases)> decreased for age(8/30, 26.7%).

Erythroid lineage:

In the present study, megaloblastoid cell change was the most common maturation pattern seenamong the erythroid cells (12/30, 40% cases). Among the nuclear dysplastic features, karyorrhexis(23/30,76.6% cases) >nuclear budding (15/30,50%) >multinucleation (11/30, 36.67%) were seen(Figure1: Image C and Image A). Among cytoplasmic dysplastic features patchy hemoglobinization (12/30, 40%) was the most common feature identified(Table 1).

Figure 1:

Image A: Bone marrow aspirate – Dysplastic erythroid precursor with multinucleation(Leishman40 x)

Image B: Bone marrow aspirate - Dysplastic erythroid precursor with nuclear bridging (Leishman100 x).

Image C: Bone marrow aspirate - Dysplastic erythroid precursor with nuclear budding (Leishman100 x).

Image D: Bone marrow aspirate – Dysplastic erythroid precursor with cytoplasmic vacuolation(Leishman40 x).

Image E: Bone Marrow Aspirate –Ring sideroblasts(Perl’s Prussian blue100x).

Table 2: Myeloid dysplasia in subtypes

2008 Classification	2016 Classification	Morphology (Increase in precursor forms)	Giant precursors	Nuclear hypolobation (Pseudo pelgerhuet)	Irregular hypersegmentation	Hypogranular forms	Auer rods	Pseudo Chediak-Higashi granules	Myeloblasts (% of cases)
RARS	MDS-RS-SLD (n= 1)	0	0	0	0	0	0	0	0, 0
RARS	MDS-RS-MLD (n= 1)	0	0	0	0	0	0	0	0, 0
RCMD	MDS-MLD (n= 7)	2(6.7%)	3(10%)	1(3.3%)	0	0	0	0	0, 0
RAEB-1	MDS-EB-1 (n= 5)	4(13.3%)	2(6.7%)	2(6.7%)	0	0	0	0	4 (13.3%)
RAEB-2	MDS-EB-2(n= 15)	14(46.7%)	4(13.3%)	1(3.3%)	1(3.3%)	1(3.3%)	0	0	13(43.3%)

Table 4: Cytogenetics in MDS subtypes

2008 Classi-fication	2016 Classification	46XY	46XX	Trisomy 8	Del (20q)	Complex karyotype (del 5 plus trisomy 8)	Chromosome 9 polymorphic variant	Not available
MDS (all subtypes) (n=30)		4(13.3%)	3(10%)	1(3.3%)	3(10%)	1(3.3%)	1(3.3%)	17(56.7%)
RARS	MDS-RS-SLD (n= 1)	0	0	0	0	0	0	1(3.3%)
RARS	MDS-RS-MLD(n= 1)	1(3.3%)	0	0	0	0	0	0
RCMD	MDS-MLD(n= 7)	0	1(3.3%)	0	1(3.3%)	0	1(3.3%)	4(13.3%)
RAEB-1	MDS-EB-1 (n= 6)	2(6.7%)	1(3.3%)	0	0	1(3.3%)	0	2(6.7%)
RAEB-2	MDS-EB-2 (n= 15)	1(3.3%)	1(3.3%)	1(3.3%)	2(6.7%)	0	0	10(33.3%)

Abnormal Localization of Immature Precursors (ALIP):

In the present study 11/30(36.7%) cases showed ALIP (Figure 4: Image O). Maximum cases seen were of RAEB-2 type (8/15,53.3% cases). CD34 in ALIP: CD 34 was done in 5/30(16.7%) cases and showed positivity in all the 5 cases (Figure 3: Image P). Out of these, ALIP was seen in 3/5 cases.

Figure 4:

Image O: Bone marrow biopsy–Abnormal localization of immature precursors (H and E 40 x)

Image P: Immunohistochemistry on bone marrow biopsy – CD34 positive cells(H and E 40 x).

Paratrabecular localization of megakaryocytes:

It was seen in 9/29 cases (31%). Paratrabecular localization was slightly more common in high-risk MDS (2/30,6.7%) as compared to low risk MDS. One case was of RAEB-1 and the other was RAEB-2.

CD 34 positive megakaryocytes:

One out of 30 cases (3.3%) with CD34 positive megakaryocytes was of RCMD. The megakaryocytes seen in this case were bizarre forms, micromegakaryocytes, and hypolobated forms. Dysplasia in >50% of the megakaryocytes was seen in this case.

Cytogenetics:

Cytogenetics was assessed in 13/30(43.3%) cases. Ten cases were males and three cases were females. Among all cases, 4/30(13.3%) had 46XY karyotype, 3/30(0.1%) cases had del (20q), 1/30(3.3%) cases had trisomy 8, 1/30(3.3%) cases had chromosome 9 polymorphic variant and 1/30(3.3%) had a complex karyotype (trisomy 8 with Del(5). All the female cases had normal karyotype. Refer to table 4 for cytogenetics in MDS subtypes.

IPSS risk category:

Very low score- There was only one case of RCMD in this category. It had an IPSS-R score of 1.5. This case also had CD34 (+) megakaryocytes on bone marrow biopsy. Though IPSS-R score was very low, CD 34(+) megakaryocytes indicated a poor prognosis¹².

Low score – There were 2 cases in this category. One case had RCMD and the other had RARS. Both the cases had a low score of 2 in each.

Intermediate-There were 3 cases in this category. Out of these 3 cases, 2 were of RAEB-1 and one was of RAEB-2 and had a score of 4.5 in each case.

High –There were 5 cases in this category. Of all the 5 cases, 1 case was of RCMD, 2 cases were of RAEB-1, and 2 were of RAEB-2. Each of these cases had a score of 5 except one case of RAEB-2 where the score was 6.

Very high –There were 2 cases in this category. Both these cases were of RAEB-2 and had a score of 7 and 7.5 respectively.

DISCUSSION:

Age and gender distribution:

Porta,et al⁶, Germing et al⁷, Xiaomei et al⁸, Smith et al⁹ and Sekeres et al¹⁰ has documented the median age of MDS in the eighth decade with male predominance.

In the present study, the median age of presentation was lower and was seen in the sixth decade.

Cytopenias:

Xiong et al¹¹ documented pancytopenia and bicytopenia in a higher number of cases as compared to the present study.

Bone marrow findings:

Erythroid dysplasia:

Morphology: Germing et al¹² observed megaloblastoid changes in 30 to 40% cases. This observation was in concordance with the present study.

Xiong etal¹¹ documented megaloblastoid changes in more number of cases than in the present study.

The megaloblastoid changes were seen in >5% of the cells.

The erythroid dysplastic features in descending order of occurrence, besides the pattern of maturation, as observed by following authors and in the present study are:

1. Xiong et al¹¹ – Hyperlobation> Multinucleation > Internuclear bridging > Karyorrhexis, cytoplasmic vacuolation.

2. Germing et al¹²- Multinucleation > Cytoplasmic changes > Internuclear bridging.

3. R.Head et al¹³- Internuclear bridging > Nuclear budding.

4. Present study- Karyorrhexis > Nuclear budding > Patchy hemoglobinization > Multinucleation > Cytoplasmic vacuolation > Internuclear bridging, cytoplasmic bridging.

The variable predominance of various nuclear and cytoplasmic dysplastic features was observed in other studies and in the present study. Megaloblastoid maturation pattern had a common occurrence in the present study and in studies done by Xiong et al¹¹ and Germing et al¹².

Myeloid dysplasia:

Overall left shift was less often observed in the low-risk MDS as compared to high-risk MDS.

Myeloblasts: Xiong, et al¹¹ and Germing, et al¹² document median blast percent of < 5%. This finding was in concordance with the present study.

Pseudo PelgerHuet neutrophils was the most common myeloid dysplastic feature observed in the present study as well as by Germing et al¹².

Megakaryocytic dysplasia:

Hypercellularity of megakaryocytes and micromegakaryocytes were the most consistent finding observed in all MDS cases in the present study except for the 2 cases of RARS where no megakaryocytic dysplasia was seen. These findings were not documented in the study done by Germing et al¹².

Cytogenetics:

Hasse et al¹⁴ and Solé, et al¹⁵documented normal karyotype being more commonly observed than abnormal karyotype. This finding was in concordance with the present study.

Hasse et al¹⁴ and Solé, et al¹⁵ documented complex karyotype as the most common cytogenetical abnormality among other abnormal karyotypes.

The present study had del 20(q), trisomy 8, complex karyotype and polymorphic 9 variant seen in one case each. A polymorphic variant of chromosome 9 was not documented by other authors.

Solé, et al¹⁵ noticed that normal karyotype (66% in RARS and 41% in RAEB) was more common than abnormal karyotype. Abnormal karyotype was seen in 0.6 to 7.5% of RARS cases and 0.4 to 47.7% of RAEB cases. Trisomy 8 was more commonly seen than Del (20q) in both the subtypes.

The present study had a single case of RARS with a normal karyotype. Del (20q) was more commonly observed (9.5%) than trisomy 8 in RAEB. Complex karyotype was present in a case of RAEB, that is del (5) along with trisomy 8. A single case of RCMD with chromosome 9 polymorphic variant was seen in the present study.

Correlation with survival status and time:

J. Neukirchen et al¹⁶ observed high death rate in very high-risk as compared to very low-risk MDS.

The present study had death in 1/1 (100%) case with very low-risk, 2/3 cases (66.6%) with intermediate risk, 5/5 cases (100%) with high and 2/2 cases (100%) with very high-risk. We presume that CD 34 (+) megakaryocytes in the very low risk category added to the poor prognosis, but this finding needs to be further proved.

Voso et al¹⁷ noted survival time of 14 months in very high-risk, 12.9 months in high-risk, 34.4 months in intermediate risk and 75.1 months in low-risk MDS. Cases with very low-risk MDS were alive.

The present study has a median survival time of 14 months in very high-risk, 4 months in high-risk, 30 months in intermediate risk,12.2 months in low-risk and 12 months in very low-risk.

The present study does not have an information on survival time for 4 cases.

CONCLUSION:

In the present study we found an increase in the severity of anemia and thrombocytopenia from low-risk to high-risk MDS. High-risk MDS showed more myeloid dysplastic feature than low-risk MDS. The Iron stores were low in high-risk MDS than in low-risk MDS in the present study. Among all 30 cases, 11 cases had ALIP and 90.9% (10/11) cases died. Likewise, among all the 30 cases, 9 has paratrabecular localization of megakaryocytes and 88.8% (8/9) cases died. Both these factors need further research to label them as poor prognostic indicators. We started this study with a motive of observing anything unusual in MDS cases in terms of cytomorphology and cytogenetics¹⁸. We got 3 uncommon findings of polymorphic variant of chromosome 9, CD 34 positive megakaryocytes in bone marrow biopsy and eosinophils with basophilic granules in 3 separate cases with myelodysplastic syndrome. We conclude this study with the thought that this should not be the end and further studies on this can bring out points to raise an early alarm, especially with the use of ayurvedic medicines¹⁹ and thus add to better patient care¹⁸^,²⁰^-²⁵.

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Received on 30.03.2023 Modified on 12.07.2023

Research J. Pharm. and Tech 2024; 17(2):673-678.

DOI: 10.52711/0974-360X.2024.00104

2008 Classification	2016 Classification	Cellularity			Normal morphology	Micro megakaryocytes	Nuclear hypolobation	Polylobation	Multinucleation
2008 Classification	2016 Classification	Increased	Normal	Decreased	Normal morphology	Micro megakaryocytes	Nuclear hypolobation	Polylobation	Multinucleation
RARS	MDS-RS-SLD (n= 1)	0	0	1 (3.3%)	1 3.3%)	0	0	0	0
RARS	MDS-RS-MLD (n= 1)	1 (3.3%)	0	0	1 (3.3%)	0	0	0	0
RCMD	MDS-MLD(n= 7)	4 (13.3%)	1 (3.3%)	2 (6.7%)	2 (6.7%)	6 (20%)	2 (6.7%)	1 (3.3%)	1 (3.3%)
RAEB-1	MDS-EB-1 (n= 6)	3(10%)	2 (6.7%)	1 (3.3%)	0	6(20%)	0	1 (3.3%)	2 (6.7%)
RAEB-2	MDS-EB-2 (n= 15)	8 (26.7%)	6 (20%)	1(3.3%)	0	13(43.3%)	3 (10%)	3 (10%)	6 (20%)

2008 Classification	2016 Classification	Multinuclearity	Nuclear budding	Internuclear bridging	Karyorrhexis	Cytoplasmic vacuolation	Cytoplasmic bridging	Patchy hemoglobinization
RARS	MDS-RS-SLD (n= 1)	1 (3.3%)	0	0	1 (3.3%)	1(3.3%)	0	0
RARS	MDS-RS-MLD (n= 1)	1 (3.3%)	0	0	1 (3.3%)	0	0	1 (3.3%)
RCMD	MDS-MLD (n= 7)	3(10%)	4(13.3%)	0	7 (23.3%)	2(6.7%)	1(3.3%)	4(13.3%)
RAEB-1	MDS-EB-1 (n= 5)	1 (3.3%)	3(10%)	0	3 (10%)	1(3.3%)	0	2 (6.7%)
RAEB-2	MDS-EB-2 (n= 15)	5(16.6%)	8(26.7%)	3(10%)	11(36.7%)	0	2(6.7%)	5(16.7%)