Utility of bone marrow biopsy in myeloproliferative neoplasm with special reference to megakaryocyte morphology

INTRODUCTION

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorder characterized by excessive production of one or more myeloid lineage cells.^[1] MPN can be divided into two major groups, BCR-ABL 1-positive chronic myeloid leukemia (CML) and the Philadelphia (Ph-1) chromosome-negative MPN, which include polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocythemia (ET).^[2] Philadelphia chromosome negative myeloproliferative neoplasms share common clinical and molecular features- most notably the Janus Kinase 2 V617F(JAK2V617F) mutation, which is detected in approximately 95% of polycythaemia vera cases and about 50% of essential thrombocythemia and primary myelofibrosis cases.^[3] They show significant differences in their prognosis, risk of leukemic transformation, and progression to myelofibrosis (MF).

Precise diagnosis of different MPN subtypes is essential to make use of novel targeted treatment strategies for MPN.^[4] Bone marrow (BM) histology was included into the diagnostic approach of MPN along with other clinical, molecular, and cytogenetic studies since the 2008 World Health Organization (WHO) classification system of hematological malignancies.^[5] Various parameters such as bone marrow cellularity, morphology, maturation, and topography of different cell lineage must be examined for reporting BM histology. Among them, morphological analysis of megakaryocytes has a major role in segregating the different MPN subtypes.^[6] Ph-1-negative MPN such as ET, early stages of PV, and PMF can present with increased platelet count in the peripheral smear. It is essential to differentiate ET from other MPNs with thrombocytosis as the overall prognosis and frequency of complications show accountable difference between them.^[7]

Bone marrow histology, apart from its role in diagnosing MPN subtypes, has a crucial role in prognosticating them by identifying their transformation into more severe conditions like blast crisis or MF. There is a paucity of literature highlighting the role of bone marrow histopathological examination in the diagnosis of MPN, especially in our population. We conducted this study regarding the role of bone marrow histology with special emphasis on megakaryocytic morphology in subclassification.

MATERIAL AND METHODS

This hospital-based retrospective study with prospective follow-up (ambispective cohort study) was performed in the department of Pathology with collaboration of the department of Medicine of a tertiary care center of the eastern region of India from May 2019 to May 2022. Institutional ethical committee approval was taken, and informed consents were obtained.

RESULTS

Morphology and number of megakaryocytes

Giant forms of megakaryocytes were identified in cases of both ET and PV. Megakaryocytes with hypersegmented staghorn nuclei, resembling “deer antler,” were only observed in ET. CML was characterized by the predominance of micromegakaryocytes. The pyknotic/dysmorphic forms of megakaryocytes were commonly found in cases of PMF with marked increase in reticulin fibers. Megakaryocyte clustering was another important finding, which was exclusively seen in cases of ET along with some cases of PV (25%) and CML (12.5%) showing reticulin fibers around them. Apart from this, ET was also found to have the highest number of megakaryocyte per low power field (60 ± 2.8/LPF), whereas MF had the lowest count (11.7 ± 4.6/LPF) [Table 3; Figures 1-4].

Table 3: Morphology of megakaryocytes.

Parameter	ET	PV	CML	MF	p-value
Mean megakaryocyte count/LPF	60±2.8	43±4.1	18.3±4.1	11.7±4.6	<0.0001
Types of megakaryocytes
Megakaryoblast	0	0	0	1	0.0095
Promegakaryocyte	0	0	1	0
Micro	0	0	6	3
Giant	2	2	0	0
Pyknotic/dysmorphic	0	0	0	3
Normal	0	2	1	0
Distribution
Diffuse	0	3	7	8	0.0113
Clustering	2	1	1	0

ET: Essential thrombocythemia, PV: Polycythemia vera, CML: Chronic myeloid leukemia, MF: Myelofibrosis, LFP: Low power field. p < 0.05 was considered statistically significant

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Figure 1: Bone marrow biopsy in a case of myelofibrosis (a) “Streaming effect” of fibrotic bands compressing the bone marrow cellularity (Hematoxylin and Eosin [H&E], ×40). (b) Reticulin staining showing increased reticular fibers (Reticulin stain, ×100). (c) Mature collagen fibers (Masson’s trichrome stain, ×100). (d) Characteristic pyknotic form of megakaryocyte (yellow arrow) (H&E, ×400).

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Figure 2: Bone marrow biopsy in case of CML. (a) Intertrabecular spaces packed with 100% cellularity (Hematoxylin and Eosin [H&E], ×40). (b) A cluster of immature cells surrounded by large number of granulocytes and few megakaryocytes (dwarf forms, yellow arrow head) (H&E, ×400). (c) Metamorphosis to myelofibrosis is characterized by fibrotic bands (yellow arrow) (H&E, ×100). (d) Immature cells comprising more than two-thirds of an IT space, suggesting metamorphosis to blast crisis (yellow two-headed arrow) (H&E, ×100).

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Figure 3: Bone marrow biopsy in a case of polycythemia vera. (a) Increased bone marrow cellularity (Hematoxylin and Eosin [H&E], ×40). (b) Mixture of pleomorphic megakaryocytes with different cell sizes (small, medium, and large) (H&E, ×100). (c) Characteristic megakaryocyte showing nuclear hyperlobation (yellow arrow head) (H&E, ×400). (d) Bone marrow showing fibrotic bands suggesting metamorphosis to myelofibrosis (H&E, ×100).

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Figure 4: Bone marrow biopsy in a case of essential thrombocythemia. (a) Increased bone marrow cellularity, predominantly affecting megakaryocyte lineage (Hematoxylin and Eosin [H&E], ×100). (b) Megakaryocytes accumulated in “paratrabecular” space (H&E, ×400). (c) Characteristic megakaryocytes exhibit multilobulated nuclei resembling “deer antlers (H&E, ×400). (d) Large population of immature cells, suggesting metamorphosis to blast phase (H&E, ×400).

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DISCUSSION

Dramatic advancement in our understanding of the molecular basis of MPN led to the development of novel targeted treatment modalities such as JAK2 inhibitors.^[11] The 2008 WHO classification system for hematological malignancies included bone marrow histology in the diagnostic approach of MPN. Even though the role of bone marrow biopsy is limited in the diagnosis of MPN, it also plays a crucial role in the prognosis of the disease. In developing countries and in low-resource settings, bone marrow histology still has an immense role in both diagnosing and subclassification of MPN.

Classic MPN is an uncommon disorder.^[12] According to data available from a population-based study conducted in the United States, incidence rates (IR) were highest for PV (IR = 10.9) followed by ET (IR = 9.6) for the 2001–2012 duration,^[13] but there is a paucity of literatures regarding the incidence of MPN subtypes and its metamorphosis in our population. Single literature published on North Indian population 120 cases of MPN over 12 years’ time span CML was the most common variant (81.7%), followed by PV (5%), ET (4.2%), and then PMF (0.8%).^[14] Similarly, in our study, CML was the most common MPN diagnosed during the study period (38.09%). But on the contrary, one large Asian population-based study showed an upward trend in nonCML-MPN, whereas CML revealed marginal elevation.^[15]

Derivation of megakaryocytes from hematopoietic stem cell is a stepwise process controlled by various transcription factors such as RUNX1, GATA1, and GF11B. Thrombopoietin also plays a crucial role in this process. Following the acquisition of an MPN driver mutation, these megakaryocytes exhibit abnormal maturation, reduced expression of GATA1 and increased release of various cytokines and chemokines (transforming growth factor-β, interleukin 8, interleukin 6, CXCL4, etc.) which contribute to the development of fibrosis, bone formation, and myeloproliferation.^[16] Crucial evaluation of different histopathological parameters is useful for segregation of MPN from other non-neoplastic disorders as well as distinguishing of CML from non-CML MPN. Qualitative and quantitative changes in bone marrow megakaryocytes are the most important parameters among them. Reactive thrombocytosis can develop as a physiological response to conditions such as acute or chronic inflammatory and infectious disorders, acute blood loss, hemolytic anemia, and iron deficiency. Understanding the morphological features of normal megakaryocyte at different stages of its maturation is crucial to avoid misdiagnosing such conditions. Immature megakaryocytes show horse shoe-shaped nucleus having significant chromatin condensation, scanty basophilic cytoplasm with few granulations. On the other hand, mature megakaryocytes have pleomorphic nucleus, coarsely granular chromatin pattern, abundant pale to pink cytoplasm with fine clusters of granules.^[8] Bone marrow findings tend to change over the time in MPN, in some cases may get transformed to MF or acute leukemia.^[17]

PV usually has an indolent clinical course with signs and symptoms related to excessive hematopoiesis, coagulopathy, and mild hepatosplenomegaly with a possible metamorphosis to osteosclerosis or other MPN, myelodysplastic syndrome (MDS), and/or acute leukemia.^[18] Bone marrow is hypercellular with classic trilineage proliferation, with predominance of pleomorphic megakaryocytes (small, medium, and large).^[19] During the study period, we had four cases of PV, which were showing trilineage proliferation with giant forms of megakaryocytes. In PV, megakaryocytes are distributed dispersedly or in dense clusters at intertrabecular/para-trabecular spaces. Increase in reticular fibers can also be seen in some cases.

ET is considered a diagnosis of exclusion, which mainly affects megakaryocytic lineage with prominent thrombocytosis in the peripheral blood. Platelet count >4.5 lac/Cumm with atypical bone marrow histology and exclusion of all other WHO-defined diseases such as CML, PV, PMF, and MDS are necessary for diagnosis of the disease.^[20] JAK2, calreticulin, or tetmethylcytosine dioxygenase 2 genes identification is necessary to differentiate ET from other reactive causes of thrombocytosis.^[21] Bone marrow biopsy is typically hypercellular with predominance of megakaryocytic lineage. Megakaryocytes in ET are large or gigantic with abnormal chromatin clumping and abundant basophilic cytoplasm, mostly distributed singly or in loose clusters.^[22] Emperipolesis can also be appreciated in some cases.^[23] One of the characteristic features that megakaryocytes exhibit in ET is hypersegmented staghorn nuclei that resemble “deer antlers” as seen in one of our cases. Grade 0 or 1 fibrosis can be seen in ET; diagnosis of ET should be kept out if marked fibrosis is noted in bone marrow.

PMF is characterized by a gradual evolution of prefibrotic hypercellular initial face to an overt fibrotic phase often accompanied by osteosclerosis. Clinically, it is indistinguishable from PV or ET transformation to MF. About 30–50% of cases are diagnosed in pre-fibrotic phase.^[24]This phase is characterized by bone marrow hypercellularity with granulocytic or megakaryocytic proliferation. Minimal reticulin fibrosis (grade 0 or grade 1) is seen in this stage. Megakaryocytic morphology and topography are the key to diagnose pre-fibrotic phase of PMF. Characteristically, megakaryocytes show increased nuclear: cytoplasmic ratio, abnormal chromatin density, and a “cloud-”like nuclei. Megakaryocytes are more atypical in pre-PMF stage compared to other MPN, showing accentuated dysplasia. PrePMF stage also shows increased angiogenesis and sinusoidal dilatation, demonstrated by CD34 staining. More than 50% cases of PMF are diagnosed in the overt fibrosis stage^[25]. Bone marrow in this stage is usually normocellular or hypocellular in nature. Fibrosis compresses the bone marrow cellularity and produces a “streaming effect.” One of the characteristic features of overt fibrotic stage of PMF is grade 2 or 3 level reticulin fibers (type III collagen) or collagen fibers (type 1). Megakaryocytes show dense aggregate formation with dysplastic features.

Bone marrow histopathology has a limited role in the diagnosis of CML. Even though the BCR-ABL-positive CML is the most common subtype of MPN, very less proportion of patients undergo bone marrow biopsy examination. In CML, bone marrow shows marked increase in cellularity with a myeloid: erythroid ratio higher than 10:1. Megakaryocytes are usually small in size (dwarf forms) with intermediate nuclear: cytoplasmic ratio, normal chromatin pattern. Hypolobulated megakaryocyte is a characteristic feature of CML. Macrophages resembling Gaucher cells (pseudo-Gaucher cells) can be seen in almost 70% of the CML cases.

Histopathological examination of bone marrow should go hand in hand with cytogenetic and molecular studies for diagnosing and prognosticating a case of MPN. Over the years, the significance of cytogenetic in MPN has both revolutionized the discipline and, at the same time, proven the inherent value of classical BM histopathological examination. Bone marrow is typically hypercellular in all subtypes of MPN representing clonal excess hematopoeisis.^[26]Megakaryocyte morphology and its distribution in the bone marrow are one of the major findings to look for the exact categorization of MPN subtypes. Apart from this, assessment of reticulin fibers also plays a crucial role in prognosticating patients.

There is a lack of cytogenetic studies in our institution. Study conducted in a larger population, for a longer duration of period, is necessary for further confirmation of the results.

CONCLUSION

Bone marrow biopsy remains an indispensable tool in the diagnosis and prognostication of myeloproliferative neoplasms. Detailed assessment of megakaryocyte morphology, number, and topographic distribution provides critical histomorphological clues for accurate subclassification of MPNs, particularly in Philadelphia chromosome negative entities.