Serum erythropoietin in the evaluation of erythrocytosis: How much does it contribute to the diagnosis of polycythemia vera?

Ruchi Gupta; Manish Kumar Singh; Dinesh Chandra; Khaliqur Rahman; Ashwani Maddheshia; Akhilesh Sharma; Sanjeev Yadav; Rajesh Kashyap

doi:10.4103/joah.joah_77_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 14 | Issue : 2 | Page : 122-127

Serum erythropoietin in the evaluation of erythrocytosis: How much does it contribute to the diagnosis of polycythemia vera?

Ruchi Gupta, Manish Kumar Singh, Dinesh Chandra, Khaliqur Rahman, Ashwani Maddheshia, Akhilesh Sharma, Sanjeev Yadav, Rajesh Kashyap
Department of Hematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Date of Submission	27-Aug-2022
Date of Decision	25-Dec-2022
Date of Acceptance	03-May-2023
Date of Web Publication	27-Jul-2023

Correspondence Address:
Dr. Khaliqur Rahman
Department of Hematology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/joah.joah_77_22

Abstract

INTRODUCTION: Serum erythropoietin (S. EPO) continues to be a minor diagnostic criterion for discriminating polycythemia vera (PV) from other causes of erythrocytosis. However, in the current era of Janus kinase 2 (JAK2) mutation testing, its relevance for establishing the diagnosis of PV is controversial.
AIMS AND OBJECTIVES: The study aimed to assess the utility of S. EPO in patients with absolute erythrocytosis and ascertain its sensitivity and specificity for establishing the diagnosis of PV. Further, the clinicopathological features of PV and JAK2-EPO^low erythrocytosis were compared to discriminate the two disease states.
MATERIALS AND METHODS: A total of 286 samples had undergone concomitant testing for S. EPO levels and JAK2V617F mutation for various indications over a period of 60 months (January 2017–December 2021). Clinical details and laboratory parameters were retrieved from the electronic medical records.
RESULTS: Among 203/286 (70%) patients with erythrocytosis, JAK2 positivity was noted in 49 (24.1%) patients. A subnormal S. EPO level (<3.2 mIU/mL) was noted in only 27/43 (62.7%) cases of de novo PV, while the postphlebotomy PV patients (n = 6) had normal S. EPO levels. The sensitivity and specificity of low S. EPO for establishing the diagnosis of PV were 62.7% and 77.4%, respectively, while the sensitivity and specificity of high Hb levels (>16.5 g/dL) with low S. EPO and for diagnosing PV was 62.7% and 79.0%, respectively, while the sensitivity of high Hb levels with JAK2 positivity was 89.7%. In addition, 14.7% (30/203) of cases of high Hb/hematocrit (median: 18 g/dL and range: 16.3–23.8 g/dL) belonged to the JAK2^-EPO^low subgroup. The median age of these patients, red blood cell count, total leukocyte count, and platelet count were, however, significantly lower as compared to the PV (P < 0.05).
CONCLUSION: Overall, a low sensitivity and specificity of S. EPO were observed for diagnosing PV as an isolated investigation, thus questioning its diagnostic utility, though high levels had an excellent negative predictive value. However, this simple and inexpensive test remains an important screening tool for evaluating patients with absolute erythrocytosis.

Keywords: Erythrocytosis, JAK2, polycythemia vera, serum erythropoietin

How to cite this article:
Gupta R, Singh MK, Chandra D, Rahman K, Maddheshia A, Sharma A, Yadav S, Kashyap R. Serum erythropoietin in the evaluation of erythrocytosis: How much does it contribute to the diagnosis of polycythemia vera?. J Appl Hematol 2023;14:122-7

How to cite this URL:
Gupta R, Singh MK, Chandra D, Rahman K, Maddheshia A, Sharma A, Yadav S, Kashyap R. Serum erythropoietin in the evaluation of erythrocytosis: How much does it contribute to the diagnosis of polycythemia vera?. J Appl Hematol [serial online] 2023 [cited 2023 Sep 27];14:122-7. Available from: https://www.jahjournal.org/text.asp?2023/14/2/122/382416

Introduction

Erythrocytosis can be the consequence of numerous primary or secondary disorders and differentiating the two requires eliciting a detailed history, examination, and an array of investigations.^[1],[2] Polycythemia vera (PV) is the sole cause of clonal-acquired erythrocytosis resulting from a somatic mutation in the JAK2 gene. The World Health Organization (WHO) has laid down definite criteria for the diagnosis of PV, which include the following: hemoglobin (Hb) level of >16.5 and 16 g/dL in males and females, respectively, panmyelosis in the bone marrow, presence of JAK2 mutation, and/or low serum erythropoietin (S. EPO) levels. The nonclonal causes of erythrocytosis include disease states associated with tissue hypoxia, like renal/cardiac diseases or tumors producing EPO as a part of a paraneoplastic phenomenon like cerebellar hemangioblastomas. Usually, in these acquired states, the serum EPO levels are high. Congenital causes of erythrocytosis are rare, and the S. EPO levels in these conditions may be variable. After a comprehensive systemic examination, when likely cardiac and pulmonary causes have been ruled out, further investigations for identifying the underline etiology are facilitated by the determination of S. EPO^[3],[4],[5] which forms the backbone of numerous diagnostic algorithms, besides other routine investigations assessing cardiac and renal functions. S. EPO levels are largely tested by enzyme-linked immunosorbent assay (ELISA) or chemiluminescence-based assays, while a basic molecular laboratory setup is required for performing JAK2 mutation testing, to establish the diagnosis of PV. The more common JAK2V617F point mutation accounting for more than 95% of cases of PV can readily be detected by techniques such as restriction fragment length polymorphism and amplification-refractory mutation testing (ARMS-PCR), while sequencing is required for assessing the presence of JAK2 exon12 mutation.

The utility of S. EPO has been highlighted in older studies,^{[6],[7],[8],[9],[10]} and very low E. EPO levels have been highlighted to have high predictive value for establishing the diagnosis of PV. However, its utility as the sole diagnostic marker for PV has been questioned in recent literature and we have also observed numerous cases of low S. EPO to be negative for JAK2 mutation in routine practice, thereby compelling a formal evaluation of this laboratory investigation in establishing the diagnosis of PV in a real-world scenario at a tertiary care center. The study primarily aimed at assessing the sensitivity and specificity of S. EPO in establishing the diagnosis of PV in the Indian population. Further, the prevalence of PV among all cases of erythrocytosis was estimated. The laboratory profile of PV and JAK2-EPO^low erythrocytosis was also compared to identify subtle pointers to differentiate clonal from reactive/other hereditary causes of erythrocytosis.

Materials and Methods

This was a retrospective analysis of all the samples subjected to S. EPO testing in the department of hematology over a period of 5 years (January 2017–December 2021). The patient details were retrieved from the electronic medical records. The diagnosis of PV and essential thrombocythemia (ET) and primary myelofibrosis (PMF) had been established as per the criteria laid down by the WHO in 2016.^[1] All patients with Hb levels >16.5 having a concomitant JAK2V617F mutation testing and S. EPO levels analysis were evaluated. In addition, cases with low S. EPO levels and JAK2 positivity, wherein bone marrow examination was performed, were also re-assessed, independently by two pathologists to establish the diagnosis of PV. The term JAK2-EPO^low refers to cases that were negative for JAK2V617F mutation and had S. EPO levels <3.2 mIU/mL.

Serum erythropoietin measurement

A two-site ELISA-based measurement of S. EPO was carried out by a commercial kit (Tecan Pvt. Ltd., Germany). 400 μLserum was required for the assay. Briefly, the calibrator, controls, and patient sample are simultaneously incubated with the enzyme-labeled antibody and a biotin-coupled antibody in a streptavidin-coated microplate well. At the end of the assay incubation, the enzyme bound to the solid phase was incubated with the substrate, tetramethylbenzidine, and the intensity of the yellow color was directly proportional to the concentration of EPO in the sample. The normal reference range as mentioned by the kit (3.22–31.0 mIU/mL) was confirmed by evaluating 20 healthy individuals.

JAK2V67F mutation testing

Mutation analysis was performed using ARMS-PCR. It is a tetra-primer assay. Four sets of primers are used, as shown in [Table 1]. The PCR cycling conditions were as follows: denaturation at 94°C, 30 s, annealing at 58°C, 45 s, and extension at 72°C, 45 s, for up to 40 cycles. PCR products are resolved on 2% agarose gels. The assay had a sensitivity of 0.1%.

Table 1: Primers used for JAK2 mutation analysis

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Statistical analysis

The basic descriptive statistics were carried out using Microsoft Excel software (Windows 11) and SPSS version 16 (SPSS Inc. Chicago, IL, USA). Comparative analysis of the laboratory parameters between PV and JAK2-erythrocytosis was performed using Mann–Whitney U-test. P < 0.05 was considered statistically significant.

Results

A total of 483 samples were subjected to S. EPO level estimation over a period of 5 years (2017–December 2021) for various indications. Among these, 286 had undergone JAK2 mutation testing for various indications including erythrocytosis. All absolute erythrocytosis was noted in 203/286 patients, and a diagnosis of PV was established in 49 (24.1%) of these patients based on the peripheral blood/JAK2V617F mutation and/or bone marrow findings [Figure 1] as per the WHO 2016 criteria. There was a striking male preponderance in the cohort. The baseline demographics are provided in [Table 2].

Figure 1: Flowchart depicting the distribution of cases based on S. EPO levels. S. EPO: Serum erythropoietin

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Table 2: Baseline patient demographics of patients with erythrocytosis (n=203)

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JAK2V617F mutation and polycythemia vera

Overall, in the cohort of 286 patients, 65 patients were positive for JAK2V617F mutation. Morphologically, they were classified as PV (49/65, 77.7%), PMF (n = 7, 10.7%), ET (n = 8, 12.3%), and Refractory anemia with ring sideroblasts with Thrombocytosis (RARS-t) (n = 1) [Figure 2]. In patients with PV, a heterozygous mutation was noted in 35 cases (71.4%) while 14 cases showed the presence of a homozygous mutation.

Figure 2: Flow diagram depicting the distribution of cases based on JAK2 mutation profile

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Among the 49 patients of PV, 43 patients were treatment naïve, and only 27 (62.7%) of these patients had subnormal S. EPO levels (<3.22 mIU/mL). In the remaining six patients, the postphlebotomy sample had been evaluated, which revealed normalization of S. EPO levels with a median of 20.89 mIU/mL (17.7–29.1). There was no difference among the S. EPO levels of patients with homozygous JAK2-mutated PV (15/49) versus the heterozygous-mutated state.

Hemoglobin levels and serum erythropoietin in the diagnosis of polycythemia vera

Forty-four patients (44/49) of PV had Hb levels ≥16.5 g/dL and interestingly five cases with Hb levels <16.5 g/dL (median: 15.9 g/dL and range: 14.3–16.4). Although the Hb levels were <16.5 g/dL, bone marrow examination in all these five patients was markedly hypercellular with panmyelosis. The bone marrow aspirate smears were reviewed independently by two pathologists, RG and KR; the findings were correlated with JAK2 positivity for establishing the diagnosis of PV. There was no history of phlebotomy in these patients, and the S. EPO levels were subnormal.

The overall breakup of cases based on S. EPO levels is depicted in [Figure 2]. In patients with a confirmed diagnosis of PV (49), 23 patients had subnormal S. EPO levels, while 22 PV patients, including 6 postphlebotomy cases, had a normal S. EPO level. The overall sensitivity, specificity, positive predictive value, and negative predictive value of S. EPO levels in the diagnosis of PV were 62.7%, 79%, 45%, and 87.5%, respectively [Table 3]. A correlation of JAK2 mutation, Hb, and S. EPO levels is provided in [Figure 3].

Table 3: Depicting the sensitivity, specificity, predictive positive value, and negative predictive value of serum erythropoietin levels for the diagnosis of polycythemia vera

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Figure 3: Venn diagram depicting the correlation of JAK2 mutation profile, low S. EPO levels, and erythrocytosis. A total of 203 patients had erythrocytosis, with hemoglobin levels ≥16.5 g/dL. Sixty-five of these cases were JAK2 positive; 49 of which were diagnosed as PV (including 6 postphlebotomy cases), based on the WHO 2016 criteria. Overall, in this cohort, there were 56 patients, with a low serum EPO level; of which 27 cases were diagnosed as PV (the central domain). S. EPO: Serum erythropoietin, PV: Polycythemia vera, EPO: Erythropoietin

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Erythrocytosis with JAK2 negative and low serum erythropoietin (JAK2-erythropoietin^low) profile

A total of 14.7% (30/203) of cases of erythrocytosis (Hb ≥16.5 g/dL) belonged to the JAK2^-EPO^low subgroup. The median age of the patients was significantly younger than the PV group (36.5 years vs. 56 years, P < 0.05). Similarly, the other hematological parameters are also significantly lower, unlike those seen in cases with suspected myeloproliferative neoplasms (MPNs) [Table 2]. A bone marrow examination had been performed in 20 of these cases, wherein 18 (90%) cases showed a normocellular marrow with no evidence of a MPN, while two cases displayed bone marrow features suspicious of PV. These two cases aged 73 years and 45 years had S. EPO levels of 0.06 and 1.6 mIU/mL, respectively; likely, these patients were harboring JAK2 exon 12 mutations.

Discussion

The study was taken up to investigate many aspects of erythrocytosis, besides the role of S. EPO in the diagnosis of PV. The true prevalence of PV in the Indian subpopulation or among all cases of MPN is poorly reported. According to the Western literature, PV accounts for 4%–40% of all erythrocytosis in different studies,^[11] while the Indian data are sparse, variable, and skewed according to the experience of different tertiary care centers. In a recent study, Khurana et al. reported the prevalence of PV to be 20.4% in their cohort of 89 patients with erythrocytosis.^[12] Similarly, Singh et al. studied the clinical and molecular attributes of patients with BCR/ABL1-negative MPNs in India and observed the prevalence of PV to be approximately 14% (27/183 of their patients of classical MPN).^[13] In the present study, we observed PV to account for 24.1%, of all erythrocytosis and ~10% of all MPNs at our center. This is relatively higher than that reported by Varma et al. in their compilation of 231 patients of MPN.^[14]

The WHO still includes subnormal levels as minor diagnostic criteria for PV diagnosis.^[1] Various studies have been carried out in the past to assess the sensitivity and specificity of S. EPO for PV, and the results have been variable. In a study in 2002, Messinezy et al. evaluated the role of S. EPO in 125 patients with erythrocytosis and observed the sensitivity and specificity of low S. EPO to be 64% and 92%, respectively, in diagnosing PV.^[15] However, Mossuz et al. reported a sensitivity of 97% and a predictive value of 97.8% of low S. EPO levels for the diagnosis of PV in their cohort of 241 patients of erythrocytosis which included 116 patients of PV.^[16] Further, using receiver operating characteristic curve analysis, they defined new cutoffs for strengthening the diagnostic power of S. EPO level to 100% specificity and a 100% predictive value for the direct diagnosis of PV (<1.4 mIU/L) and secondary erythrocytosis (>13.7 mIU/L). A similar controversial trend has been observed in recent studies assessing the utility of S. EPO as a diagnostic tool for PV. In 2014, Ancochea et al. studied 99 patients of PV and observed that JAK2 V617F quantification displayed an excellent diagnostic accuracy for PV in patients with hematocrit >52% (males) or >48% (females) and reported that a sensitivity and specificity were 79% and 97%, respectively, and interestingly, the addition of low S. EPO did not improve the diagnostic accuracy.^[8] Similarly, in a recent large single-center study over a period of 11 years of 577 patients, Davila-Gonzalez et al. observed that the area under the curve of JAK2 V617 (0.8970) was statistically larger than that of EPO test (0.6765). Further, they showed that S. EPO levels <2 mIU/mL had >99% specificity but only 12% sensitivity to predict PV.^[17] In another study, Maslah et al. assessed the diagnostic performances of S. EPO and JAK2 mutations in 1090 patients with suspected polycythemia. Among patients with high Ht/Hb levels, a low EPO level predicted true polycythemia with a 92% predictive positive value (PPV) while a JAK2 mutation had a 90% PPV. In their study, a very-low EPO level (≤1.99 mUI/mL) had a PPV of 100% for PV diagnosis.^[18] In the present study, however, we observed a relatively lower sensitivity and specificity of low S. EPO for diagnosing PV, indicating that a significant population with low S. EPO levels and erythrocytosis did not have PV in our cohort. Further, the cutoffs proposed by Mossuz et al. or Maslah et al. were also applied in our study population, but they failed to increase the sensitivity of S. EPO; 11/31 patients in the JAK2^-EPO^low had levels below 1.4 mIU/mL. However, we also observed that high Hb levels (>16.5 g/dL) and JAK2 positivity had a much higher PPV (89.7%) for the diagnosis of PV than high Hb and low S. EPO alone (62.7%). All these studies, including ours, highlight the variable sensitivity and specificity of S. EPO for establishing the diagnosis of PV. Moreover, it is evident that a high hematocrit (Hct)/Hb and JAK2 have better predictive values for PV than high Hct/Hb and low S. EPO levels.

An additional observation of the study was that a significant number of patients with erythrocytosis and subnormal EPO levels (30/203, 14.7%) were negative for JAK2V617F mutation. The cases in this JAK2^-EPO^low subgroup were younger and had normal or near-normal leukocyte and platelet counts when compared to the PV group. These subtle observations can also assist in discriminating PV from other causes of erythrocytosis, especially in states where EPO is low. After excluding the possibility of JAK2 exon 12 mutations, it may be worthwhile to subject these samples to next-generation sequencing. They may detect novel or variant mutations in JAK2 genes in these JAK2V617F-negative subgroups or help in the diagnosis of rare congenital erythrocytosis, wherein mutations in the EPOR pathway can lead to abnormally low S. EPO levels.^[19]

Conclusion

The diagnosis of PV largely relies on high Hb levels and JAK2 mutation status. S. EPO levels are supplementary and assist in diagnosis, but the specificity of low S. EPO for diagnosing PV is low. It, however, continues to be an important test parameter in the diagnostic algorithm for differentiating PV from other secondary causes of erythrocytosis, since the normal or high EPO has a high negative predictive value for excluding PV. In addition, it is worth noticing that low serum EPO may be observed in other unusual/rare causes of erythrocytosis, wherein there is the absence of leukocytosis and thrombocytosis or other features suggestive of a MPN. Thus, an integrated approach combining the history, physical examination, complete blood counts, S. EPO levels, and JAK2 mutational workup are essential for an accurate diagnosis in a case with erythrocytosis.

Ethical approval

A retrospective study and all the procedures performed were part of the routine care.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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