Quantification of IGF‐1 receptor is useful in the differential diagnosis of essential thrombocytosis from reactive thrombocytosis

Background: To make a definite diagnosis of essential thrombocytosis (ET) from reactive thrombocytosis (RT), the most reliable criteria are the presence of driver mutations, namely JAK2, CALR, or MPL gene mutations. In the absence of these driver mutations, so‐called triple‐negative ET, the differential diagnosis could be difficult. Although bone marrow biopsy could be helpful, it may be difficult in some cases, to do gene sequence analysis to identify other clonal marker gene mutations than the driver mutations, as only very few were found. Methods: IGF‐1R quantification by flow cytometry in mononuclear cells (MNC) from peripheral blood was performed in 33 patients with ET (untreated or off treatment with hydroxyurea), 28 patients with RT, and 16 normal volunteer controls. Results: We found IGF‐1R levels were significantly elevated in ET patients compared to RT patients or controls. A cutoff value of 253 was chosen from the logistic regression to predict each patient's group, a value ≥253 meant that a patient belonged to the ET group (sensitivity 96.4% and specificity 68.6%). Conclusion: We suggest that adding quantification of IGF‐1R in blood MNC by flow cytometry is useful in differentiating ET from RT.

Keywords: essential thrombocytosis (ET); insulin‐like receptor growth factor‐1 receptor (IGF‐1R); reactive thrombocytosis (RT)

The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia lists the criteria for diagnosing essential thrombocythemia (ET) as (a) platelet count more than 450 × 109/L; (b) bone marrow biopsy showing proliferation mainly of the megakaryocyte lineage with increased numbers of enlarged, mature megakaryocytes with hyperlobulated nuclei; no significant increase or left shift in neutrophil granulopoiesis or erythropoiesis; and very rarely a minor (grade 1) increase in reticulin fiber; (c) not meeting WHO criteria for CML, PV, PMF, myelodysplastic syndromes, or other myeloid neoplasms; and (d) the presence of JAK2, CALR, or MPL mutation. The minor criteria include the presence of a clonal marker or the absence of evidence of reactive thrombocytosis.[1]

Among these criteria, the most reliable criteria for diagnosing ET are the "presence of JAK2, CALR, or MPL mutation". These mutually exclusive "driver" mutations have respective incidences of 55%, 25%, and 3%, and the remaining of 17% are so‐called triple‐negative ET.[2] These 17% of cases will need other criteria to make a definite diagnosis as ET. The bone marrow criterion is helpful in some cases, but in many cases the differential diagnosis from reactive thrombocytosis (RT) could be misleading.[[3]] Because in RT, the megakaryocytes are also larger and increased in number and could be difficult to be differentiated from ET,[5] clustering of megakaryocytes and abnormal morphology is helpful.[6] So, a substantial proportion of triple‐negative patients with ET have evidence of polyclonal hematopoiesis and most likely do not have a true myeloproliferative neoplasm (MPN). The use of cytoreductive drugs in these patients will not be desirable.[4]

We recently published a paper, in which we used IGF‐1R quantification by flow cytometry; a substitution of performing EEC (endogenous erythroid colony) formation (2008 WHO criteria for PV[7]) could be helpful in differentiating polycythemia from secondary polycythemia.[8] Now, we found similarly that IGF‐1R determination is useful in differentiating between ET and RT. Thus, we propose that quantification of IGF‐1R by flow cytometry in the blood could also be a helpful addition in distinguishing ET from RT.

All MPN patients were diagnosed according to 2008 WHO criteria (peripheral blood was obtained from patients with written informed consent, the protocol for which was approved by the IRB of Brookdale University Hospital): 35 patients with ET (untreated or off treatment with hydroxyurea for at least 1 month), 28 with RT (5 sickle diseases, 8 arthritis, 8 iron deficiency, 6 with infection, and 1 with bleeding), and 16 normal volunteer controls. The clinical features of ET are listed in Table.

Clinical characteristics of 33 hydroxyurea untreated or off‐treated patients

• 2 Note
• 3 JAK2(+) denotes JAK2 V617F mutation positive, and % denotes percentage of JAK2 V617 mutation.

Mononuclear cells were isolated from human peripheral blood by gradient centrifugation with Ficoll‐Paque PLUS (GE Healthcare). Isolated cells were washed with cell wash buffer (phosphate‐buffered saline containing 2 mmol/L EDTA and 0.5% fetal bovine serum) and stained with PE‐conjugated control IgG or anti‐IGF1R IgG (R&D Systems, mouse monoclonal IgG1, Clone #33 255) according to the recommendation of the manufacturer. Briefly, one million mononuclear cells were resuspended in 100 µL wash buffer and incubated with 10 µL of PE‐conjugated control or anti‐IGF1R IgG for 30 minutes. Fluorescent intensity was detected by a flow cytometry (BD Accuri C6, BD Biosciences) and analyzed using FlowJo software. The final value of mean fluorescence intensity (MFI) of each sample was the difference between the reading of anti‐IGF1R and control IgG.

Data were summarized by mean ± SE. Statistical analyses were performed with unpaired Student's t test and one‐way ANOVA for comparison of groups. Results are given as mean with standard error and 95% confidence intervals (CIs).

To define a cutoff value of IGF‐1R to predict a patient's diagnosis as ET or RT, logistic regression was used to model the data, and the cutoff was chosen based on the value that provided the best classification.

IGF‐1R data for both ET and RT patients are normally distributed, and the P‐values are >.05 for both Shapiro‐Wilk and Kolmogorov‐Smirnov methods. The representative histogram for ET, RT, and controls is shown in Figure. IGF‐1R was measured by median fluorescence intensity and expressed as mean ± SE. As shown in Figure , untreated or off‐treated ET patients had significantly higher IGF‐1R (361.1 ± 39.5) than RT patients (132.2 ± 13.0), (P < .05) and normal controls (165.6 ± 10.0), (P < .05), while no statistical difference was found between RT and controls. Patients with ET treated with hydroxyurea were found to have a significantly lower IGF‐1R expression with mean ± SE of 249.6 ± 37.06 (Figure S2), as we found previously in PV patients treated with hydroxyurea.[8] A Logistic regression shows ET is a statistically significant (P < .001) predictor of patient's groups (ET or RT). A cutoff value of 253 was chosen from the Logistic regression to predict patient's group, a value <=253 means that a patient belongs to RT group, with sensitivity of 96.4%. and specificity of 68.6%. The PPV (positive predicative values) is 71.0%, and NPV (negative predicative values) is 96.0%. The NPV is very high, indicating that if IGF‐1R value is greater than 253 (cutoff value), then we can predict the diagnosis of ET in patients with very high confidence.

In hydroxyurea untreated or off‐treatment ET patients, (a) no correlation between IGF‐1R expression levels and white blood cells, hemoglobin, platelet counts, or degree of splenomegaly was found; (b) JAK2 V617F(+) was found in 26 patients and 17 (65%) had elevated levels; it appears that IGF‐1R expression does not have any correlation with the allele burden levels (allele burden information was not available for many patients); (c) six patients had a CALR(+) mutation, 3 out of 6 (50%) had elevated IGF‐1R expression, and (d) one patient with MPL gene mutation with elevated IGF‐1R expression. More cases in a larger cohort will be needed to draw definite conclusions about the correlation between levels of IGF‐1R levels and different driver mutations.

Two patients with triple‐negative driver mutations were found to have elevated IGF‐1R. The first patient was a 70‐year‐old, African American woman who had 5‐year history of thrombocytosis with platelet counts ranging from 500 to 700 × 109/L with no history of thrombosis or splenomegaly or carcinoma. There was no evidence of reactive thrombocytosis or other myeloid neoplasms, and no clonal markers were identified. Bone marrow (Figure S1, Figure S2) showed increased megakaryocytes with no dysplastic features or clustering and hence was non‐diagnostic. We believe that she has essential thrombocytosis because her IGF‐1R levels were measured to be 602. The second patient who was triple‐negative patient was a 47‐year‐old Haitian woman with platelet counts of more than 1 million, first identified in 2016. She did not have any history of thrombosis or carcinoma or any other secondary etiology nor had any evidence of splenomegaly. Bone marrow showed marked megakaryocytic hyperplasia. She was diagnosed with triple‐negative ET mainly based on bone marrow findings. She was treated with hydroxyurea and anagrelide. The IGF‐1R was measured to be 360.4, which we think had further supported her diagnosis as ET.

According to WHO criteria, the histology of bone marrow in ET was characterized by the presence of either single or clustered large to giant mature megakaryocytes with deeply lobulated staghorn‐like nuclei, normal or slightly increased cellularity, normal granulopoiesis, normal‐sized erythroid foci, and lack of fibrosis.[1] Many recent publications have discussed the differential diagnosis of bone marrow histology between PV (especially masked PV) and ET.[[9]] In PV, bone marrow biopsy shows an increase in all three cell lineages (panmyelosis) and high‐power view reveals conspicuous variability in megakaryocyte cell size (pleomorphism). Also, many papers have discussed differential diagnoses in bone marrow between prefibrotic myelofibrosis and ET,[[12]] with prefibrotic myelofibrosis having more abnormal megakaryocytes, high clustering index, and lack of reticulin fibrosis in ET. To date, no recent papers have discussed the differential diagnosis between ET and RT based on bone marrow biopsy. In the paper by Thiele et al,[5] a strikingly proportionate increase in early megakaryocytes occurred in all ET and RT patients but not in controls, although a predominance of large and giant elements with large, highly lobulated nuclei was more evident in ET patients. However, in many cases, the differential diagnosis may not be so evident, according to Putti et al,[4] who found that ET and RT were misdiagnosed. In such cases of RT, using chemotherapy with hydroxyurea will not be desirable.

The minor criteria for ET by WHO 2016 include the presence of a clonal marker. Clonal dominance genes, such as TET2 or DNMT3A, are associated with low frequency (12% in ET and 18% in PMF) [16]; the true meaning of these two gene mutations is unclear, because these genes are associated with aging process, so‐called clonal hematopoietic indeterminate potential (CHIP).[15] All the other mutations with epigenetic regulators or spliceosome are found almost exclusively in PMF. Moreover, CALR is the first mutation in nearly all these cases, and additional mutations are all secondary in ET.[[16]] In an analysis of 69 ET cases using next‐generation sequence (NGS) studies by Lundberg et al,[18] 13% of cases were negative for any somatic mutations; therefore, only very few cases were found to have clonal marker mutations after discovering the driver mutations with CALR or JAK2 V617 or MPL, and most of the secondary mutations were found in association with the driver mutations. In other 55 triple‐negative ET patients studied by whole‐exome sequencing [19] and in 26 cases studied by NGS,[20] only a few cases of atypical MPL and JAK2 gene mutations were found. So, in triple‐negative ET patients, clonal marker mutations were found by further sequence studies in only a few cases after driver mutations.

IGF‐1R levels were not correlated to JAK2 V617F mutation status in ET patients (Table) in our small series; 17 out of 26 (65%) JAK2 mutation‐positive patients had elevated IGF‐1R, and the IGF‐1R expression levels did not correlate to the levels of JAK2 allele burden of mutation. One patient with MPL W515 mutation had elevated IGF‐1R, and three patients (50%) with CALR gene mutation had elevated IGF‐1R levels.

IGF‐1R quantification to diagnose ET yielded a sensitivity of 96.4%. and specificity of 68.6%. None of the RT patients had values more than 270. Elevated levels of IGF‐1R quantification related to more than 2 standard deviation, 300, can establish a definite diagnosis of ET. The two triple‐negative patients demonstrated that IGF‐1R can be added to the differential diagnosis of ET, besides bone marrow examinations and can be as supplemented to the definite diagnosis. Therefore, we propose that IGF‐1R, a simple non‐invasive and more objective test, can be supplemented to bone marrow examinations for definite diagnosis of triple‐negative ET. A larger cohort will be needed to further explore the value of IGF‐1R level in helping the definitive diagnosis of ET and to be included as minor criteria if further large cohort studies confirm our findings.

The authors thank Dr Ruqin Chen and Dr Shuguang Chen for assistance with the statistical analyses.

JCW received grants from Celgene Corp. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The author declares no conflicting interests with the commercial funder relating to using products, consultancy, patents, product development, or market products. The other authors declare no competing financial interests.

JCW designed and analyzed the data, and wrote the manuscript; GS performed the research, and collected and analyzed the data; CW collected and analyzed the data; VG collected and analyzed the data; PR collected blood samples, analyzed the data, and carried out English editing; HC collected the specimens, analyzed the data, and contributed to the manuscript.