Incidence, Clinical Features, and Prognostic Impact of CALR Exon 9 Mutations in Essential Thrombocythemia and Primary Myelofibrosis: An Experience of a Single Tertiary Hospital in Korea
2015; 35(2): 233-237
Ann Lab Med 2018; 38(4): 296-305
Published online March 30, 2018 https://doi.org/10.3343/alm.2018.38.4.296
Copyright © Korean Society for Laboratory Medicine.
Naery Yang, M.D.1, Sholhui Park, M.D.1, Min-Sun Cho, M.D.2, Miae Lee, M.D.1, Ki-Sook Hong, M.D.1, Yeung Chul Mun, M.D.3, Chu-Myong Seong, M.D.3, Hee Jin Huh, M.D.4*, and Jungwon Huh, M.D.1*
1Department of Laboratory Medicine, College of Medicine, Ewha Womans University, Seoul, Korea.
2Department of Pathology, College of Medicine, Ewha Womans University, Seoul, Korea.
3Department of Internal Medicine, College of Medicine, Ewha Womans University, Seoul, Korea.
4Department of Laboratory Medicine, Dongguk University Ilsan Hospital, Goyang, Korea.
Correspondence to: Corresponding author: Jungwon Huh. Department of Laboratory Medicine, College of Medicine, Ewha Womans University, 1071 Anyangcheon-ro, Yangcheon-gu, Seoul 07985, Korea. Tel: +82-2-2650-5320, Fax: +82-2-2650-5091, JungWonH@ewha.ac.kr
Co-corresponding author: Hee Jin Huh. Department of Laboratory Medicine, Dongguk University Ilsan Hospital, 27 Dongguk-ro, Ilsandong-gu, Goyang 10326, Korea. Tel: +82-31-961-7893, Fax: +82-31-961-7902, hjhuh@duih.org
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This study aimed to determine GATA1 expression levels to better characterize subgroups in
This study enrolled 49 patients diagnosed as having
GATA1 expression decreased significantly in PMF compared with that in other subtypes, while no statistical difference was identified between ET and prePMF. GATA1 expression did not differ according to the mutation profiles or the allele burden of
Our results suggest that GATA1 expression is significantly low in PMF and decreases with progressive fibrosis and possibly with leukemic transformation, although our attempt to accurately distinguish between subgroups using GATA1 immunohistochemical approach did not achieve statistical significance. A large patient cohort with long term follow-up is required to evaluate the prognostic value of GATA1 expression.
Keywords: Polycythemia vera, Essential thrombocythemia, Prefibrotic primary myelofibrosis, Primary myelofibrosis, GATA1
The 2016 WHO classification system divided PMF into prePMF and overt PMF on the basis of the absence or presence of grade ≥2 bone marrow fibrosis [1].
The most commonly recognized mutation in
Even though the 2016 WHO classification has provided criteria for the diagnosis of prePMF [1], bone marrow pathology provides major clues for the differential diagnosis of prePMF from ET. For both ET and prePMF, the histological hallmarks lie in the megakaryocyte (MK) population: ET is marked by proliferation of enlarged and mature MKs with deeply lobed and hypersegmented nuclei, whereas prePMF is characterized by proliferation of markedly atypical MKs that often form clusters. Megakaryocytic atypia involves maturation defects along with atypical nuclear features such as hypolobulation, hyperchromicity, irregular folding, and dense clustering [15]. However, the morphological evaluation of bone marrow specimens is subjective, and the reproducibility has been questioned [8,9].
Therefore, we aimed to determine whether an immunohistochemical assay would allow for a more objective histopathological assessment of bone marrow MKs. Since each subtype of
To select study subjects, we re-evaluated bone marrow slides and reviewed clinical and laboratory data of patients with
Forty-nine patients who had
The control group for GATA1 stain comprised 40 patients, of which nine had
Electronic medical records were reviewed for retrospective collection of relevant clinical and laboratory data including the results of cytogenetics and
The follow-up period varied from nine to 167 months (median 86.5 months). No patient received
Cytogenetic analysis was performed by unstimulated 24- and/or 48-hour culture and conventional G-banding techniques using the fresh bone marrow aspirates obtained at diagnosis. For molecular assays, genomic DNA was extracted from fresh bone marrow aspirates or stored bone marrow cell pellets fixed for cytogenetic analysis using a DNA purification kit (Qiagen, Hilden, Germany). An allele-specific, real-time PCR assay (Real-Q
Quantification of
The allele burden of
The
The archived formalin-fixed and paraffin-embedded bone marrow biopsies were used for GATA1 immunohistochemical stain. Bone marrow biopsy samples obtained at diagnosis from 49 patients with
GATA1 expression was semi-quantitatively evaluated in terms of GATA1 score, which was obtained by multiplying the intensity score by the positive cell percentage score [23,24]. GATA1 immunostaining intensity was rated on a scale from 0 to 3: negative, 0; weak, 1; moderate, 2; and strong, 3 (Fig. 1). The percentage of positive MKs (the number of GATA1-stained MKs×100/total number of MKs) was graded from 0 to 4: less than 1%, 0; 1–20%, 1; 21–50%, 2; 50–80%, 3; and 81–100%, 4.
The differences in continuous variables (GATA1 score, age, hemoglobin level, white blood cell count, platelet count, lactate dehydrogenase, and
All statistical analyses were performed with MedCalc for Windows, version 16.4.3 (MedCalc Software, Ostend, Belgium).
The
The median value and the range of the allele burden of
The GATA1 score differed significantly between disease groups (
PV and prePMF showed a GATA1 score distribution similar to that of BHD. For PMF, the GATA1 score was significantly lower than that of PV, prePMF, or BHD. Further, 73% patients with PMF scored less than 6, whereas no patients had a GATA1 score less than 6 in the PV or prePMF groups (Fig. 2).
Patients with ET showed a heterogeneous GATA1 score distribution. Features that favored the diagnosis of prePMF, such as increased lactate dehydrogenase and/or megakaryocytic atypia, were present in four patients with ET who scored less than 6 at diagnosis. MF-1 fibrosis was present in one of these four patients, which was rare in ET. However, they were classified as ET because the patients either had normocellular marrow or the minor criterion for prePMF was not met. No statistically significant difference in GATA1 score was found between the ET and prePMF groups. GATA1 expression in the CML and lymphoma without bone marrow involvement groups was heterogeneous, as shown in Fig. 2.
GATA1 expression was compared between two groups in the
When GATA1 scores were compared according to mutation profile (
The median value of the GATA1 score tended to increase as
In the PV, ET, and prePMF groups, no patient had both
Figs. 3 and 4 depict GATA1 expression and clinical events in patients with follow-up bone marrow biopsies. In patients with PV, the GATA1 score on diagnosis was 9 or 12 and remained higher than 6 throughout the disease course until the development of overt myelofibrosis (MF≥2). GATA1 expression decreased to scores of 1–3 with progression of myelofibrosis, except for one patient (patient PV-4).
Among the five patients with ET, three (patients ET-4, ET-8, and ET-13) showed a steadily high GATA1 score (6 or higher). Two patients (ET-15 and ET-16) showed low GATA1 expression at diagnosis with a score of 4, but the scores increased to 6 or higher in the follow-up course. Patient ET-16 initially showed MF-1 myelofibrosis and low GATA1 score, but the fibrosis was reversed during the follow-up course along with increase in GATA1 score.
The GATA1 score decreased from 9 to 4 when the disease transformed to acute myeloid leukemia for one patient (prePMF-1). The GATA1 score of another patient (prePMF-2) decreased from 9 to 4 and the blasts increased in number (17% in bone marrow differential counts). However, in three patients with prePMF (prePMF-7, prePMF-8, and prePMF-9), the GATA1 scores remained high (score of 9–12) even when the myelofibrosis developed to MF-2.
In patients with PMF (PMF-4 and PMF-6), the GATA1 score was 1 or 2 at diagnosis, which remained unchanged during follow-up with leukemic transformation or extensive fibrosis. The GATA1 score of two patients (PMF-3 and PMF-7) was 2 at diagnosis, which increased during follow-up without improvement of fibrosis on bone marrow biopsy.
We demonstrated that GATA1 expression was significantly lower in PMF than in other subtypes and the allele burden of
The frequencies of
We assessed GATA1 expression in MKs to demonstrate its clinical relevance in distinguishing between
In the serial analysis, GATA1 expression was also low in patients with PV who developed post-PV myelofibrosis during follow-up. The GATA1 score was significantly lower in the
Our study has some limitations. The small patient population for each subtype of
In conclusion, although our attempt to accurately distinguish between subgroups using a GATA1 immunohistochemical approach did not achieve statistical significance, our results suggest that GATA1 expression decreases with progressive fibrosis and possibly with leukemic transformation.
No potential conflicts of interest relevant to this article were reported.
This study was supported by the Basic Science Research Program of the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology (NRF-2012R1A1A2044138), and the Ewha Womans University Scholarship of 2015.
Representative images of GATA1 immunoexpression in bone marrow biopsy samples according to the intensity of the reaction (×400): (A) negative, (B) weak, (C) moderate, and (D) strong. Megakaryocytes (arrows) are identified by CD61-stained (pink-colored) cytoplasm. GATA1-stained nuclei of megakaryocytes colored light to dark brown depending on the immunointensity. GATA1-stained nuclei are also seen in erythroid cells (arrowhead) without the CD61-positive cytoplasm.
Distribution of GATA1 expression according to disease groups (
Abbreviations: BHD, benign hematologic disease; CML, chronic myeloid leukemia; ET, essential thrombocythemia; LWOB, lymphoma without bone marrow involvement; PMF, primary myelofibrosis; prePMF, prefibrotic primary myelofibrosis; PV, polycythemia vera.
Serial analysis of GATA1 expression, the allele burden of
*
Abbreviations: ET, essential thrombocythemia; F/U, follow-up; mo, month; MF, myelofibrosis; NT, not tested; PV, polycythemia vera.
Serial analysis of GATA1 expression, the allele burden of
*
Abbreviations: AML, transformation to acute myeloid leukemia; F/U, follow-up; mo, month; MF, myelofibrosis; NT, not tested; PMF, primary myelofibrosis; prePMF, prefibrotic primary myelofibrosis.
Relationship of GATA1 expression in patients with
Variable | GATA1 score Median (range) | |
---|---|---|
Myelofibrosis grade† at diagnosis (N=49) | ||
MF<2 (N=38) | 9 (2–12) | |
MF≥2 (N=11) | 2 (1–9) | |
Myelofibrosis grade† at the first follow-up (N=18) | ||
MF<2 (N=14) | 9 (2–12) | |
MF≥2 (N=4) | 2 (1–9) | |
Mutation at diagnosis (N=35) | 0.436 | |
| 9 (1–12) | |
| 9 (1–12) | |
| N/A | |
Triple negative (N=9) | 6 (1–12) | |
0.428 | ||
1–20% (N = 1) | 6 (6) | |
21–50% (N = 10) | 12 (3–12) | |
51–80% (N = 8) | 12 (6–12) | |
80–100% (N = 6) | 12 (1–12) |