|Year : 2022 | Volume
| Issue : 4 | Page : 879-885
|Evaluation of utility of immunohistochemistry markers as a tool for objective diagnosis of low-grade myelodysplastic syndrome in routine reporting: Prospective observational study
Richa Juneja, Haraprasad Pati, Prasad Dange, Gopila Gupta, Manoranjan Mahapatra, Seema Tyagi, Renu Saxena
Department of Hematology, All India Institute of Medical Science, Ansari Nagar, New Delhi, India
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|Date of Submission||06-Jan-2021|
|Date of Decision||12-May-2021|
|Date of Acceptance||15-May-2021|
|Date of Web Publication||02-Jun-2022|
| Abstract|| |
Purpose: Diagnosis of myelodysplastic syndrome (MDS) primarily relies on the detection of morphological dysplasia in bone marrow. It is subjective and many studies have reported lack of interobserver agreement in reporting. Biopsy is preferred specimen for megakaryocyte assessment. We studied 43 bone marrow biopsies from 40 suspected MDS patient having persistent undiagnosed cytopenia. Utility of immunohistochemistry (IHC) with CD61 and p53 in detecting low-grade MDS was analyzed over routine morphology. Method and Results: Total number of megakaryocytes and number of dysplastic megakaryocytes seen on CD61 IHC was significantly higher than that on H and E stain (P value < 0.05) Out of total 43 biopsies, 13 [30.2%] cases showed dysplastic megakaryocytes that were confirmed by interobserver agreement after IHC. From 30 cases with no significant dysplasia on morphology, 21/43 [48.8%] cases showed >10% dysplastic megakaryocytes on CD61 (P value 0.0001). Nine cases showed no significant dysmegakaryopoiesis with either H and E or CD61 IHC. Fourteen cases could meet higher cut off (30%) of dysmegakaryopoiesis with CD 61 IHC. Out of total 34 cases showing significant dysplasia 7 cases (20.6%) showed positivity for p53 on IHC, which is little less than that reported in low-grade MDS. Conclusion: CD61 IHC is helpful in making correct diagnosis of MDS in cases with minimal dysplasia and should be performed before excluding possibility of MDS on morphology in a patient with undiagnosed cytopenia. IHC is cost effective tool for MDS diagnosis in developing world where access to extensive flow cytometery and molecular testing is limited.
Keywords: CD61, immunohistochemistry, megakaryocytes, myelodysplastic syndrome
|How to cite this article:|
Juneja R, Pati H, Dange P, Gupta G, Mahapatra M, Tyagi S, Saxena R. Evaluation of utility of immunohistochemistry markers as a tool for objective diagnosis of low-grade myelodysplastic syndrome in routine reporting: Prospective observational study. Indian J Pathol Microbiol 2022;65:879-85
|How to cite this URL:|
Juneja R, Pati H, Dange P, Gupta G, Mahapatra M, Tyagi S, Saxena R. Evaluation of utility of immunohistochemistry markers as a tool for objective diagnosis of low-grade myelodysplastic syndrome in routine reporting: Prospective observational study. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Nov 30];65:879-85. Available from: https://www.ijpmonline.org/text.asp?2022/65/4/879/346510
| Introduction|| |
Myelodysplastic syndromes (MDS) are a group of clonal hematological neoplasms. MDS is characterized by ineffective hematopoiesis leading to cytopenia, morphological dysplasia, and risk of clonal evolution to Acute myeloid leukemia (AML). These disorders range from low-risk disease managed by supportive care and growth factors to high-risk disease requiring aggressive management like bone marrow transplant.
Diagnosis of MDS has evolved and requires persistent cytopenia/s in the presence of morphological dysplasia in ≥10% of one or more lineages, presence of excess blast/ring sideroblasts and/or MDS-defining cytogenetic abnormality. Clonal cytogenetic abnormality is detected in only about 50% of cases. Hence, the importance of detection of morphological dysplasia cannot be over emphasized. However, many non-neoplastic disorders may give rise to dyspoietic changes leading to over-diagnosis of MDS. On the contrary, in a few cases of MDS, morphological dyspoiesis may not be easily apparent leading to under-diagnosis. Thus, ancillary methods like immunohistochemistry are believed to be valuable in diagnosis of MDS especially the low-grade ones where the blasts are not increased, only when interpreted with other ancilliary test and in correct clinical context.
Micromegakaryocytes are persistently shown to be one of the important hallmark features of dysmegakaryopoiesis. These small monolobate megakaryocytes are likely to be missed on hematoxylin and eosin (H and E) stained sections. IHC with CD61 and other megakaryocytic markers like CD41, vWF antigen is known to enhance their detection in known cases of MDS.
P53 is a tumor suppressor protein encoded by TP53 gene on chromosome 17p13.1 only the mutated p53 protein can be easily detected by IHC because of its prolonged half-life. Selective over expression of p53 in MDS marrow versus nonclonal cytopenia is documented in literature.,, However, over time this marker was more utilized for its prognostic value. In last few years investigators have revisited its utility to differentiate aplastic anemia from hypoplastic MDS marrows.
Despite all the efforts, detection of dysplasia is subjective and suffers interobserver variability. With this background we intend to study utility of 2 IHC marker CD61 and p53 in enhancing the diagnosis of low-grade MDS, especially in cases that lack significant dysplasia on bone marrow aspirate and biopsy. The access to extensive flow cytometry panels and costly molecular techniques for MDS is limited in developing world. We intended to evaluate utility of widely available cost-effective technique like IHC in routine reporting of low-grade MDS cases by hematopathologist.
| Material and Methods|| |
This was an observational study conducted in the tertiary care center. Study was approved by institutional ethics committee. From January 2017 till March 2019, cases with persistent cytopenias (minimum 6 months) were selected both retrospectively from records and also prospectively. The cytopenia was defined as per WHO definition that is, hemoglobin <100 g/L, absolute neutrophil count <1.8 × 109/L and platelet count <100 × 109/L. Jenner-Giemsa stained bone marrow aspirate and bone marrow biopsy stained with H and E stain were evaluated for evidence of dyspoiesis in all cases. Ancillary investigations like karyotyping, PNH by flow cytometry, auto immune work up, etc., were also done. Results were obtained from patients' medical records.
Forty patients of suspected MDS, in which no secondary cause of cytopenia like auto-immunity, nutritional deficiency, viral infection, chronic kidney diseases, drugs, or obvious hematological cause like aplastic anemia, NHL infiltration, etc., could be identified and where bone marrow biopsies were available were included in this study. Because the study aimed at using IHC to detect morphologically inapparent dyspoiesis, cases with morphological apparent findings viz. MDS with excess blasts (detected by morphology or CD34 immunohistochemistry) and MDS with multilineage dysplasia were excluded from the study.
IHC using monoclonal mouse CD61 antibody obtained from Master diagnostics [clone 2F2] and monoclonal mouse p53 antibody obtained from Dako [Clone: DO-7; Isotype: Ig G2b, Kappa] was performed on 43 formalin fixed paraffin embedded sections of bone marrow biopsies of all 40 patients, along with positive controls (p53 positive serous ovarian carcinoma) and negative controls (bone marrow biopsies from patients with immune thrombocytopenia). Antigen retrieval was done by (HIER method–heat induced epitope retrieval). IHC was done using super sensitive polymer-HRP ready to use kit (Lab vision) following standard protocol.
Two independent hematopathologist observers examined both H and E-stained slides and IHC slides. Counting was done on high power [400X] for CD61 and H and E-stained slides whereas p53 positive cells were counted under oil immersion. The following parameters on H and E stain and CD61 stain were analyzed:
- Total number of megakaryocytes
- Megakaryocytes with normal morphology
- Micro megakaryocytes—Mononucleated megakaryocytes with a diameter of 7–10 μm ( size comparable to that of a promyelocyte or less) lacking features of a blast cell
- Hypo lobate megakaryocytes—Megakaryocytes of normal size with a large non-lobulated nucleus and a mature granular cytoplasm.
- Multinucleate megakaryocytes—Megakaryocytes of normal size with multiple (at least three) round separated nuclei
Total dysmegakaryopoiesis in % on H and E and CD61 = c + d + e/a X100 [As counted on H and E and CD61, respectively]. [Figure 1]a, [Figure 1]b, [Figure 1]c As per WHO 2016 definition, more than 10% of dysplasia was considered significant. In case of discrepancy between the observers, the slides were reviewed together and a consensus was made. That was considered for statistical analysis.
|Figure 1: (a) Cellular bone marrow showing few dysplastic megakaryocytes [H and E stain 400X] b, c. Significantly increased dysplastic megakaryocytes highlighted with CD61 IHC like monolobate megakaryocytes with moderate cytoplasm highlighted with arrow (b) micromegakaryocytes – small megakaryocytes of the size of myeloid precursor with scant cytoplasm and identified with Cd61 positivity highlighted with lines (b), Dysplastic multinucleated megakaryocyte with 4 separated nuclear lobes (c) [CD61 IHC 1000X]|
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Ten cases with cytopenias due to non-neoplastic conditions (like PRCA, anemia due to CKD, hemolytic Paroxysmal nocturnal hemoglobinuria (PNH)) were taken as control cases for p53 IHC stain. None of these cases did show any positivity for p53. Hence, as per literature,any number of clear nuclear positivity of p53 was taken as positive. [Figure 2] All 43 biopsies were stained with p53 IHC and reported as positive or negative by two independent observers.
|Figure 2: Many mononuclear cells showing nuclear positivity for p53 IHC [1000X]|
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The data were analyzed for statistical significance. Categorical variables were presented in number and percentage [%] and continuous variables were presented as mean ± SD and median. Normality of data was tested by Kolmogorov-Smirnov test. If the normality was rejected then non-parametric test was used.
Quantitative variables were compared using Wilcoxon signed rank test (as the data sets were not normally distributed) between CD61 and H and E. Qualitative variables were correlated using Chi-Square test. McNamer test and Bhapkar test was used to find out the difference in measurement between dysmegakaryopoiesis on H and E and on CD61. A P value of < 0.05 was considered statistically significant. The data were analyzed using Statistical Package for Social Sciences [SPSS] version 21.0.
| Results|| |
Demographics and basic lab parameters
A total of 43 bone marrow biopsies from 40 patients with suspected MDS were studied. The age group of the study population ranged from 1.5 to 85 years with a mean age of 46.6 years. There was equal sex distribution in the study group with 21 males and 19 females (male to female ratio of 1.1:1).
Most common presentation was weakness and fatigue in 39 (97.5%) patients. Bleeding was reported in 9 (22.5%) of patients. On examination pallor was the most common finding being present in 39 (97.5%) patients. Mild splenomegaly was noticed in 4 (10%) of the patients.
Totally, 13 (32.5%) patients had only single cytopenia, whereas 13 (32.5%) and 14 (35%) patients had bicytopenia and pancytopenia, respectively. Anemia was the most common cytopenia in 97.5% followed by thrombocytopenia in 25 [62.5%] patients of the study group. Neutropenia was present in 16 (40%) of the patients.
Small PNH clone was detected on granulocytes and monocytes by multi-parametric flow cytometric immunophenotyping in 3 (7.5%) patients.
Out of 43 bone marrow biopsies analyzed 17 (39.5%) were normocellular for age whereas 16 (37.2%) and 10 (23.2%) biopsies were hyper cellular and hypocellular for age, respectively. Three patients underwent biopsy twice, 2 of them showed dyspoiesis on both biopsies with IHC, and one patient didn't show dyspoiesis on either of biopsy.
Comparison of H and E and CD61 IHC to detect dysmegakaryopoiesis
Out of 43 biopsies 13 had >10% dysplastic megakaryocytes on reexamination of H and E stained slide but lacking interobserver agreement. All these cases confirmed to have dysplasia on CD61 IHC. Twenty-one cases failed to show dysmegakaryopoiesis on H and E but detected on CD61 IHC. Nine cases had no significant dysmegakaryopoiesis with either staining. IHC with CD61 demonstrated >10% dysplastic megakaryocyte in 48.8% cases having <10% dysplasia on H and E and facilitated diagnosis in these cases. CD61 provided interobserver agreement and higher yield of dysplasia in 30.2% cases. [Figure 3]
|Figure 3: X axis shows 2 subset of study group one with <10% dysplasia (red with H and E and blue after performing IHC) and other group with >10% dysplasia (red with H and E and blue after performing IHC). Y axis shows total 43 biopsies depicted as 100 percentage 20.9% of total cases did not show dysmegakaryopoiesis even after IHC. All cases with >10% dysplasia on H and E but lacking agreement were confirmed on IHC with additional 48.8% cases getting detected by IHC|
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Mean number of megakaryocytes seen on CD61 (mean, 104.7) were significantly higher than those on H and E (mean, 66.4) with P value of < 0.0001. Twelve cases showed less than 30 megakaryocytes on H and E. Totally, 5 [41.6%] out of these 12 cases crossed this adequacy threshold of 30 megakaryocytes on CD61 as per WHO 2016. [Table 1]
|Table 1: Comparative findings of total number of megakaryocytes and dysplastic megakaryocytes on H and E and CD 61 IHC in bone marrow biopsies of suspected MDS patients (n=43)|
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Out of three types of dysplastic megakaryocytes, detection of micro megakaryocytes and hypo lobate megakaryocytes is significantly increased with use of CD61 IHC as compared to H and E. (P value < 0.0001). However, multinucleate megakaryocytes detected on H and E and IHC are not statistically different (P value-0.184).
Of total 43 bone marrow biopsies 30 showed < 10% dysplastic megakaryocytes on H and E. In these 30 biopsies 5 (11.5%) showed more than 30% dysplastic megakaryocytes on CD61 IHC.
Out of 10 cases with >10% but <30% dysmegakaryopoiesis on H and E, 3 cases had >30% dysmegakaryopoiesis on CD 61 and 6 cases had >40% dysmegakaryopoiesis on CD61. [Table 2]
|Table 2: Gain in percentage of dysplastic megakaryocytes with CD61 over H and E stain in bone marrow biopsies of suspected MDS patients [n=43]|
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P53 expression in study cohort
Out of total 43 cases, 8 (18.6%) were positive for p53. Out of these 8 cases 7 are found in group with significant dysmegakaryopoiesis.
Out of 40 patients CTG findings were available in 36 subjects. Twenty-three patients had normal cytogenetics and 4 had MDS-defining karyotype abnormality. All the cases with MDS-defining cytogenetic abnormalities showed significant megakaryocytic dysplasia on CD61.
9 patients had cytogenetic abnormality, but it was not MDS defining. Only 6 out of these patients had significant dysplasia on CD61. [Table 3] [Table 4] summarize comparative findings between 2 groups that is one with cytogenetic abnormality (MDS defining or not defining n = 13) versus rest 27 patients.
|Table 3: Cytogenetic study* in suspected MDS patients (n=36) (Normal cytogenetics in 23 patients and abnormal in 13 patients)|
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|Table 4: Comparative findings between 2 groups i.e., one with cytogenetic abnormality (MDS defining or not defining n=13) versus rest 27 patients|
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| Discussion|| |
We studied total 43 bone marrow biopsies from 40 patients with suspected MDS. Patients of all age groups were included with no sex predilection in study cohort.
In cases with persistent unexplained cytopenias where neither the blasts are increased nor the MDS-defining chromosomal abnormality is present, diagnosis of MDS relies primarily on detection of morphological dysplasia. Almost One fifth of MDS cases remain undiagnosed especially those with fibrosis, hypocellular marrow, subtle dysplasia, and paucity of blast. In our study group, 13 [32.5%] cases had cytogenetic abnormality only 4 being MDS defining. These cases with cytogenetic abnormality indicate clonal nature of disease process. Four cases with MDS-defining abnormality can serve as gold standard to confirm findings of IHC. High grade MDS cases like those with excess blasts and multilineage dysplasia were excluded from cohort. Our study cohort of suspected MDS constitutes some low-grade MDS cases which are missed on routine morphology evaluation and ICUS/CCUS cases. This explains lower yield of CTG abnormality.
Small PNH clone was detected in 3 (7.5%) of the patients. Small PNH clone can be detected in 50 to 60% of aplastic anemia, 5.5 to 8% of MDS cases and even isolated cytopenia.
With these baseline characteristics 43 bone marrow biopsies from 40 patients were analyzed. H and E stained biopsies were reviewed and 13 patients had >10% dysplasia without interobserver agreement on H and E which was confirmed on CD61 immunostaining. In remaining 30 bone marrow biopsies the total number of megakaryocytes detected on CD61 was significantly higher than that on H%E (P value-<0.0001]. Similarly, normal megakaryocytes detected on CD61 were higher than that on H and E. These findings are concordant with Thiele et al. and Das et al. However, the study group in these two paper was known case of MDS and morphometry was used for analyzing slides. We wanted to analyze practical utility of CD61 for detection of dysmegakayopoiesis for routine reporting in suspected MDS cases.
Out of three main dysplastic forms of megakaryocytes, micro megakaryocytes are most difficult to detect on H and E staining. We found CD61 significantly increases detection rate of micro megakaryocytes and hypo lobate megakaryocytes as compared to H and E stain with P value of < 0.001. Das et al. and Thiele et al., also reported similar findings in known case of MDS patients with obvious dysplasia and using morphometry. P Valent et al. recommends that in the absence of obvious morphological dysplasia, excess blast, or MDS-defining CTG abnormality—IHC with CD61 and CD34 showing features of dysplasia, aberrant immunophenotype on flow cytometry, and multiple somatic mutations on NGS help to make provisional diagnosis of MDS. However, large size of multinucleate megakaryocytes helps in detecting them easily without requirement of IHC. Our study supports utilization of CD61 in routine reporting without morphometry to evaluate megakaryocytes for dysplasia.
There is debate about cut off for significant dysmegakaryopoiesis. WHO 2016 and Della porta et al. suggest dysmegakaryopoiesis detected with cut off of 30/40% will increase the specificity. Hence, we tried to analyze whether IHC helps in detecting dysplastic megakaryocytes with higher cut off (30%). Out of 30 biopsies with <10% dysplastic megakaryocytes on H and E 5 (11.5%) had >30% dysplastic megakaryocytes on CD61.
Out of 10 cases with >10% but <30% dysmegakaryopoiesis on H and E, 3 cases had >30% dysmegakaryopoiesis on CD61 and 6 cases had >40% dysmegakaryopoiesis on CD61. Hereby suggesting IHC will assist in detecting dysmegakaryopoiesis even at higher cut off and with more confidence. However, interpretation of these IHC findings should always be done with caution after all possible causes of secondary dysplasia like nutritional, auto immune, secondary to NHL infiltrate have been excluded by extensive work up with battery of investigations. This should prevent us from overdiagnosis of MDS in such scenarios.
Mutant p53 protein is detected with IHC because of long half-life and has good concordance with molecular detection of mutation. P53 has been shown to differentially over express in bone marrow biopsy of MDS patients versus other cytopenia and ranges from 35 to 70%.
Recently, Cha et al. and Park et al. showed utility of P53 in differentiating aplastic anemia from refractory cytopenia of childhood., We also noticed p53 expression in 7/34 (20.6%) of our patients with significant dysplasia. Park et al. reported 11/14 (78.6%) patients with RCC had p53 expression whereas none of their Aplastic anemia case showed p53 expression. Elghetany et al. also reported higher number of p53 positive cases in MDS group (57% in hypocellular MDS and 79% in cellular MDS) versus non-MDS cytopenia.
Possible explanation for lower p53 positivity rate can be attributed to difference in study design. They assessed p53 in patients with RCC/MDS; however, our study cohort is patients with minimal dysplasia many a times picked up on IHC only.
One of our cases showed p53 expression in the absence of significant dyspoiesis on BMA, H and E, and IHC. Patient's cytogenetic study showed hyperdiploidy in 3 metaphases suggesting clonal nature of underlying disease. Patient may develop obvious dyspoiesis on follow up; however, we didn't receive repeat bone marrow for evaluation. We simply evaluated pattern of expression of P 53 in our cohort of cytopenic patients. It can be mutated and expressed in variety of neoplasm. Hence, p53 is not the diagnostic marker for dysplasia/MDS.
| Summary and Conclusion|| |
Diagnosis of MDS primarily relies on detection of morphological dysplasia as only 50% of MDS cases have MDS-defining cytogenetic abnormalities.
Our study suggests that IHC with CD 61 facilitate better assessment of megakaryocytes and detection of megakaryocyte dysplasia than routine H and E stain. It also helped us to attain interobserver agreement in assessment of dysplasia. With current movement towards increasing cut off for significant dysmegakaryopoiesis to 30/40%, our study provides evidence that IHC helps to meet these newer cut off over H and E stain. Morphometry was intentionally avoided as intention of the study was to assess the practical utility of IHC in picking up subtle dysplasia in routine reporting. P53 positivity was noted in small section of our patients however when positive alarmed of underlying clonal process. Due to cost constraints our patients were not tested for sequencing of gene panel associated with myeloid panel. However, availability of such data would have provided supportive evidence to underlying MDS as per International MDS working group criteria.
In conclusion, CD61 helps in detecting dysplastic megakaryocytes and make correct diagnosis of MDS in cases with minimal dysplasia and should be performed before excluding possibility of MDS on morphology in a patient with undiagnosed cytopenia. MDS is the disease which often suffers discrepancy in opinions and poses challenges in correct diagnosis. Studying IHC panel including CD34, glycophorin, CD117, CD61, and p53 in large patient cohort may help us to utilize IHC better for MDS diagnosis.
Dr. Aastha Gupta and Mrs. Monica Tiwari
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| Supplementary Files|| |
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404 Type IV Quarters, AIIMS Campus, MIHAN, Nagpur – 441 108, Maharashtra
Source of Support: None, Conflict of Interest: None
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[Table 1], [Table 2], [Table 3], [Table 4]
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