Transformation of polycythemia vera to chronic myelogenous leukemia.
Abstract: Transformation of polycythemia vera to chronic myelogenous leukemia is a rare event. We report 2 women with long-standing polycythemia vera who developed chronic myelogenous leukemia. Both patients had no BCR/ABL1 fusion at the time of polycythemia vera diagnosis but were positive for the fusion at chronic myelogenous leukemia onset. Most patients with polycythemia vera have [JAK2.sup.V]617F mutation. Analysis of an archival bone marrow aspirate sample from 1 patient showed a heterozygous mutation status. The blood and bone marrow samples from the other patient showed the presence of homozygous [JAK2.sup.V617F] mutation and BCR/ABL1 fusion. The possible pathogenesis of such an event is discussed in the light of current literature.
Article Type: Case study
Subject: Polycythemia (Genetic aspects)
Gene mutations (Research)
Gene mutations (Physiological aspects)
Chronic myeloid leukemia (Genetic aspects)
Chronic myeloid leukemia (Diagnosis)
Chronic myeloid leukemia (Development and progression)
Authors: Mirza, Imran
Frantz, Christine
Clarke, Gwendolyn
Voth, Arnold J.
Turner, Robert
Pub Date: 11/01/2007
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2007 College of American Pathologists ISSN: 1543-2165
Issue: Date: Nov, 2007 Source Volume: 131 Source Issue: 11
Topic: Event Code: 310 Science & research
Accession Number: 230151923
Full Text: Polycythemia vera (PV) is one of the Philadelphia chromosome (Ph)-negative chronic myeloproliferative disorders (CMPDs). It is a clonal disorder of hematopoietic stem cell origin, associated with an acquired activating mutation of the JAK2 tyrosine kinase ([JAK2V.sup.617F]). (1-6) The disease has an incidence of at least 2 per 100 000. (7,8) The clinical course in most cases of PV ends with thrombosis or hemorrhage, but a significant number of patients transform to myelofibrosis with myeloid metaplasia, myelodysplasia, or acute myeloid leukemia. (9) A few cases of transformation of PV to chronic neutrophilic leukemia have been described. (10-13) A handful of cases of transformation of PV to chronic myelogenous leukemia (CML) have also been reported in the literature from 1964 to 2005. (14-21) However, convincing cytogenetic and molecular evidence of this transformation was only shown in a recent report. (21) We report 2 additional patients with PV who developed CML and document the simultaneous presence of JAK2 mutation ([JAK2.sup.V617F]) and Ph with molecular evidence of BCR/ABL1 fusion.

REPORT OF CASES

Case 1

An 82-year-old woman with a remote diagnosis of PV presented to her physician in the spring of 2005 with increasing shortness of breath, feeling unwell, and ankle edema. She had been diagnosed with PV in 1989, and the disease had been successfully managed for more than a decade with intermittent courses of radioactive phosphorus ([sup.32]P), hydroxyurea, and anagrelide. Her medical history was significant for chronic obstructive pulmonary disease, gout, hypertension, and congestive cardiac failure. Her presenting symptoms were attributed to cardiac failure, and she was treated with oxygen and intravenous furosemide. Physical examination revealed hepatomegaly and massive splenomegaly. The peripheral blood examination showed a moderate macrocytic anemia, thrombocytosis, and marked leukocytosis with neutrophilic left shift (Table 1). Examination of her peripheral blood smear and cytogenetic studies were performed, and a diagnosis of CML was made. Bone marrow examination was not performed. Treatment with increasing doses of hydroxyurea and imatinib mesylate (Gleevec; Novartis, Basel, Switzerland) was initiated with the goal to lower her white blood cell count and to decrease her splenomegaly. However, she became progressively more severely anemic and was transfusion dependent. Her cardiac status required continuous monitoring. She died of a cerebrovascular event 4 1/2 months after the diagnosis of CML.

Case 2

A 73-year-old woman presented with shortness of breath on exertion, fatigue, and an increase in abdominal distension with fullness and pain in the fall of 2005. The patient had a history of PV for the last 15 years and was referred for workup for splenectomy. She had been treated intermittently with phlebotomies and hydroxyurea, which had been discontinued for the last 6 months before presentation because of decreased white blood cell and platelet counts. Results of cytogenetic analysis performed on the peripheral blood sample 1 year prior to her presentation were normal. Her medical history was noncontributory. Physical examination showed massive splenomegaly (25 cm below costal margin) and hepatomegaly (17 cm below costal margin). Her complete blood count at presentation is listed in Table 1. Peripheral blood smear and bone marrow examinations were performed, and the patient was treated with imatinib mesylate for CML.

MATERIALS AND METHODS

Histologic Examination

Bone marrow biopsies were fixed in B-5 solution, decalcified, and processed. Wright-Giemsa-stained peripheral blood and bone marrow aspirate smears and hematoxylin-eosin-stained sections of bone marrow biopsy were reviewed. The biopsy sample in case 2 was also evaluated for reticulin fibrosis (Gordon and Sweet reticulin), and the degree of fibrosis was graded according to recent recommendations. (22)

Cytogenetic and Fluorescence In Situ Hybridization Analysis

Conventional G-band karyotyping was performed on metaphase cells from the unstimulated peripheral blood sample or bone marrow aspirate specimen cultured for 48 hours, using conventional procedures. The results were reported using the International System for Human Cytogenetics Nomenclature. (23) For the detection of the BCR/ABL1 fusion gene, cells were also analyzed by the fluorescence in situ hybridization (FISH) method using the LSI BCR/ABL dual-color, dual-fusion translocation probe (Vysis, Downers Grove, Ill). The probe was hybridized to chromosomes according to the manufacturer's protocol. Images of 4 metaphase cells in case 1 and 200 interphase nuclei in both cases were captured using a fluorescence microscope and analyzed with Cytovision (Applied Imaging, San Jose, Calif).

Molecular Analysis

BCR/ABL1 Analysis by Reverse Transcription Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction.--Patient total RNA was prepared from peripheral blood, bone marrow, or bone marrow smears using either the Purescript RNA Isolation kit (Gentra Systems, Minneapolis, Minn) or the QIAamp RNA Blood Mini kit (Qiagen, Valencia, Calif). Extracted RNA was suspended in diethylpyrocarbonate-treated water when extracted using the Gentra kit or in kit-supplied ultrapure water when extracted using the Qiagen kit. Total RNA was archived at -80[degrees]C.

BCR/ABL1 analysis by 2-step reverse transcription polymerase chain reaction (RT-PCR) was carried out, with minor modifications, as previously described. (24) Reverse transcription was performed using the Perkin Elmer GeneAmp RNA PCR Core kit (Perkin Elmer, Wellesley, Mass) essentially as described. Priming was by random hexamer and a total of 3 [micro]L of total RNA was incorporated into each reaction (approximately 150 to 1000 ng). Following PCR amplification of cDNA, BCR/ABL1 amplicons were separated by 6% Tris-borate-EDTA (TBE) polyacrylamide gel and visualized using ethidium bromide. BCR/ABL1 analysis by quantitative PCR was performed on the Roche LightCycler v2.0 using LightCycler software v4.0 and Roche t(9;22) Quantification Kit chemistry (Roche Applied Science, Penzberg, Germany) as described in the instruction manual. Both BCR/ABL1 and glucose-6-phosphate dehydrogenase transcripts were quantitated, generating the ratio of BCR-ABL-glucose-6-phosphate dehydrogenase, which is expressed as percent BCR/ABL1 transcript present. Polyacrylamide gel electrophoresis (PAGE) was performed on recovered amplicons. To visualize the amplicons, the gel was stained for 15 minutes with SYBR green (Molecular Probes, Eugene, Ore) diluted 1:10000 in TBE buffer, pH 8.0. The gel was imaged using the appropriate filter for SYBR green on the PerkinElmer Alpha Imager 2200 (PerkinElmer).

[JAK2.sup.V617F] Mutation Analysis by PAGE and Real-Time PCR.-- For the patient samples and in-house positive and negative controls, JAK2 analysis was carried out using total RNA isolated using either the Purescript or the QIAamp chemistries as described previously. Reverse transcription was performed using the Applied Biosystems RNA PCR Core kit (Applied Biosystems, Foster City, Calif) essentially as described. Priming was by random hexamers and a total of 3 [micro]L of total RNA was incorporated into each reaction. The RT-minus reactions were identical except for the replacement of reverse transcriptase with water. Following RT, cDNA was used as the template for PCR using the JAK2 Activating Mutation Assay for Gel Detection (InvivoScribe Technologies, San Diego, Calif), performed as described by the manufacturer. For each reaction or control size ladder PCR, 5 [micro]L of the appropriate cDNA or kit-supplied control DNA was added to 45 [micro]L of reaction or control size ladder master mix. Following PCR, 25 [micro]L of each reaction was digested using BsaXI restriction enzyme (New England Biolabs, Ipswich, Mass) for 16 hours at 37[degrees]C. Both digested and undigested JAK2 amplicons, as well as undigested control size ladder amplicons, were frozen at -30[degrees]C until analysis by PAGE.

All control size ladder, digested and undigested JAK2 amplicons for all samples and controls were analyzed by 6% TBE PAGE. For each sample analyzed, 16 [micro]L of amplicon or digest was mixed with 4 [micro]L of 5X nucleic acid sample buffer (BioRad, Hercules, Calif), and the entire 20-[micro]L volume was loaded and separated by PAGE and visualized after staining with SYBR green (as described previously).

To perform [JAK2.sup.V617F] mutation analysis by real-time PCR, the remaining undigested JAK2 amplicons for each sample were cleaned using the QIAquick PCR Purification kit (Qiagen) according to the kit handbook. The cleaned amplicons were eluted in 40 [micro]L of sterile ultralow EDTA TE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0) and quantitated using the Fluoroskan Ascent FL fluorometer (Thermo Electron Corporation, Vantaa, Finland) and PicoGreen quantitation reagent (Molecular Probes, Invitrogen, Burlington, Ontario). The cleaned amplicons were analyzed for [JAK2.sup.V617F] mutation by melting curve analysis at the Mayo Clinic (Rochester, Minn). (25) The results were reported as positive or negative for the [JAK2.sup.V617F] mutation.

RESULTS

Case 1

Rare blast cells and nucleated red blood cells were identified on the peripheral blood smear. Mild monocytosis, eosinophilia, and basophilia were also present. It was noted that the white blood cell count had steadily increased during a period of 3 years to the current level. The leukoerythroblastic blood picture and, in particular, basophilia were suspected to herald a transformation to CML (Figure 1, A). Cytogenetic analysis of unstimulated peripheral blood cells showed a Ph due to translocation (9; 22)(q34;q11.2) in 4 of 4 metaphases examined (Figure 1, B). Fluorescence in situ hybridization analysis of metaphase chromosomes using a probe to detect BCR/ABL1 fusion confirmed this translocation. Additionally, analysis of interphase FISH of 200 cells from unstimulated blood was also positive for the presence of BCR/ABL1 fusion in 120 (60%) of 200 cells (Figure 1, C). In the absence of more recent material for analysis, DNA isolated from archival bone marrow aspirate smears showed a heterozygous JAK2 mutational status by both methods described (Figure 2).

Case 2

Peripheral blood smear examination showed a leukoerythroblastic blood picture with marked anisopoikilocytosis of erythrocytes. Basophils with eosinophilic granules were readily identified. Leukocyte alkaline phosphatase score was 125. A bone marrow aspirate and biopsy examination showed a hypercellular marrow (100%) with marked myeloid hyperplasia. Megakaryocytic dysplasia was noted, and cells with hyperchromatic nuclei, nuclear hypolobation, and atypical nuclei were seen. Intrasinusoidal hematopoiesis was observed, and the reticulin stain showed increased myelofibrosis, which was graded as MF-2 (Figure 1, D). Given a combination of features of myelodysplastic syndrome and a CMPD, a preliminary diagnosis of chronic myelomonocytic leukemia 2 with eosinophilia was favored. However, cytogenetic analysis on the bone marrow showed a Ph due to translocation (9;22)(q34; q11.2) in 8 of 20 metaphases examined (Figure 1, E). The quantitative PCR of RNA extracted from bone marrow cells showed BCR/ABL1 fusion transcripts quantitated at 5.50% (Figure 1, F). A revised diagnosis of CML, accelerated phase, was made. Retrospectively, metaphase FISH analysis on the peripheral blood sample from 2004 did not show any evidence of a Ph. JAK2 mutation analysis revealed a homozygous mutation status by both methods described (Figure 2). Treatment with 400 mg of imatinib mesylate every third day was initiated but later discontinued because of development of pleural effusion, edema, and significant thrombocytopenia. The most recent complete blood count showed a hemoglobin of 11.9 g/dL, mean corpuscular volume of 78.7 [micro][m.sup.3], platelets of 69 X [10.sup.3]/[micro]L, and white blood cell count of 63.8 X [10.sup.3]/[micro]L. The massive splenomegaly has been a source of much discomfort and consternation for the patient and she is now awaiting splenectomy.

COMMENT

Our 2 cases illustrate the transformation of PV to CML. Chronic myeloproliferative disorders are known to undergo transformation from one form to another in a minority of cases. Of the 8 previously described cases with transformation from PV to CML (Table 2), lack of a marker for the Ph-negative diseases made it difficult to document the coexistence of the 2 CMPDs. Fortunately, recent discovery of the acquired activating V617F mutation of the JAK2 tyrosine kinase in a significant number of Ph-negative CMPDs offered us the opportunity to test the validity of the morphologically and cytogenetically apparent PV to CML transformation. (1,3,4,6) The [JAK2.sup.V617F] mutation is seen in up to 80% of cases of PV and has resulted in some suggestion that the detection of this mutation should be the first line of diagnostic workup in patients with erythrocytosis. (26) Of significance, this mutation has not been seen in chronic phase CML; neither has it been detected in transformation of CML to blast crisis. (27,28) The exclusivity of this mutation allowed us to confirm the historical diagnosis of PV in both cases. In case 1, the Wright-Giemsa-stained bone marrow aspirate smears were available from 1989, and no specimen was available from her recent diagnosis of CML in 2005. Analysis of the archival material revealed heterozygous mutation status for [JAK2.sup.V617F]. In case 2, both recent peripheral blood and bone marrow aspirate samples were available, but there was no archival sample from the time of her diagnosis with PV. Nonetheless, molecular studies revealed that the patient was homozygous for [JAK2.sup.V617F] mutation, consistent with her past diagnosis of PV. The morphologic observation of bone marrow fibrosis likely correlates with her homozygous status. (29) Additionally, both patients not only had demonstrable BCR/ABL1 fusion either by FISH or PCR but were also Ph-positive by routine cytogenetic analysis. Of other Ph-negative CMPD, 2 cases of essential thrombocythemia have also been reported to undergo transformation to CML. (30,31) Subsequent analysis in 1 case showed that the patient did also harbor [JAK2.sup.V617F], presumably related to the preexisting essential thrombocythemia. (32)

Whether this transformation of PV to CML is because of clonal evolution or secondary to myelosuppressive treatment is at best speculative. Chronic myelogenous leukemia has been reported to arise in the setting of ionizing radiation or alkylating agent therapy for varied conditions such as Hodgkin lymphoma; acute myeloid leukemia; myelodysplastic syndrome; and breast, esophageal, and small cell lung cancer. (33) However, in a detailed review of the therapy-related CML, no definitive evidence for increased risk of developing CML was observed in individuals undergoing chemotherapy or radiation therapy compared with the general population. (33) Given that both of our patients received intermittent therapy with hydroxyurea, and were selectively offered either radioactive phosphorus or phlebotomy, the likelihood of a therapy-related CML appears remote in both cases.

There are 2 possible models to explain the transformation of PV to CML. First, that the transformation occurred in a normal or abnormal stem cell independent of PV clone, or second, that it occurred in a PV clone. Although there is no clear evidence to support one model instead of the other, it is intriguing to note that coexisting Ph-negative clonal disorders are revealed in a subset of CML patients who have been treated with imatinib. (34-36) Conversely, low levels of BCR/ABL1 transcripts have been detected in some essential thrombocythemia patients without Ph rearrangement at the cytogenetic level. (37,38) This and the chronology of reported transformations of PV or essential thrombocythemia to CML suggest that the emergence of CML is likely a secondary event that results in expansion of a clone with greater proliferative advantage. It also appears to be consistent with the epidemiologic data that has shown that pathogenesis of chronic phase CML is a result of 2 or more genetic events rather than a single hit. (39) Based on these observations, Mauro et al (40) have proposed the notion of field carcinogenesis or inherent genomic instability in patients with coexisting Ph-positive and Ph-negative disorders. In the 2 cases presented in this report, we could not determine whether the transformation of PV to CML occurred in the same clone or was due to emergence of a second coexisting clone.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

In conclusion, we report 2 cases with transformation of Ph-negative to Ph-positive CMPD and furnish molecular evidence of such an event. The [JAK2.sup.V617F] and BCR/ABL1 fusion transcripts were the molecular signatures that allowed us to document the transformation of PV to CML and their simultaneous coexistence. Although there is no clear explanation for this phenomenon, we anticipate that advances in the knowledge of molecular mechanisms involved in the pathogenesis of CMPD will shed further light on the possible association between these entities.

We thank Rebecca McClure, MD, for her assistance in confirming the JAK2 mutation results. We also thank Tom Turner for creating the composite figures.

References

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Imran Mirza, MD; Christine Frantz, MS; Gwendolyn Clarke, MD; Arnold J. Voth, MD; Robert Turner, MD

Accepted for publication April 23, 2007.

From the Departments of Laboratory Medicine and Pathology (Drs Mirza and Clark and Ms Frantz) and Medicine (Drs Voth and Turner), University of Alberta, Edmonton.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Imran Mirza, MD, 4B4.31 WMC, Department of Laboratory Medicine and Pathology, University of Alberta, 8440 1 12th St, Edmonton, Alberta, Canada T6M 2E7 (e-mail: imirza@cha.ab.ca).
Table 1. Complete Blood Counts at the Time of Chronic Myelogenous
Leukemia Diagnosis *

Test                                             Case 1       Case 2

Hemoglobin, g/dL                                 9.8         10.2
RBC count, X [10.sup.6]/[micro]l                 2.76         ...
Hematocrit, %                                   29            ...
Mean corpuscular volume, [micro][m.sup.3]      104            ...
Mean corpuscular hemoglobin concentration, %    34.3          ...
RBC distribution width, %                       21.1          ...
Platelet count, X [10.sup.3]/[micro]L          605           65
WBC count, X [10.sup.3]/[micro]L                67.7        214.4

Absolute counts, X [10.sup.3]/[micro]L (%)
  Neutrophils                                   58.1 (86)    60.1 (28)
  Bands                                          1.4 (2)     25.7 (12)
  Myeloids                                       2.7 (4)     47.2 (22)
  Lymphocytes                                    0.7 (1)     15.0 (7)
  Monocytes                                      2.0 (3)     10.7 (5)
  Eosinophils                                    1.4 (2)     10.7 (5)
  Basophils                                      1.4 (2)     19.3 (9)
  Blasts                                         0           25.7 (12)

Test                                           Reference Range

Hemoglobin, g/dL                                  12.0-16.0
RBC count, X [10.sup.6]/[micro]l                  3.80-5.20
Hematocrit, %                                     0.36-0.46
Mean corpuscular volume, [micro][m.sup.3]           80-100
Mean corpuscular hemoglobin concentration, %      32.0-36.0
RBC distribution width, %                           <15.6
Platelet count, X [10.sup.3]/[micro]L              140-450
WBC count, X [10.sup.3]/[micro]L                   1.8-7.5

Absolute counts, X [10.sup.3]/[micro]L (%)
  Neutrophils                                      4.0-11.0
  Bands                                              ...
  Myeloids                                            0
  Lymphocytes                                      1.0-4.5
  Monocytes                                        0.0-1.1
  Eosinophils                                      0.0-0.7
  Basophils                                        0.0-0.3
  Blasts                                             ...

* RBC indicates red blood cell; WBC, white blood cell; and ...,
not available.

Table 2. Cases With Transformation of Polycythemia Vera (PV) to
Chronic Myelogenous Leukemia (CML) *

                              Cytogenetics
Case   Age,    PV Duration,
No.    y/Sex        y         PV       CML

 1     39/M         3         ...      Ph+
 2     71/F         4         ...      Ph+
 3     80/M         9         ...      Ph+
 4     50/M         8         ...      Ph+
 5     68/M        1.5        ...      Ph+
 6     54/F         8         46,XX    Ph+
 7     58/M         2         ...      Ph+
 8     77/F         8         46,XX    Ph+
 9     82/F         16        ...      Ph+
10     73/F         15        46,XX    Ph+

             Molecular Testing

Case   PV BCR/ABL1            CML
No.    and [JAK2.sup.V617F]   BCR/ABL1

 1     ...                    ...
 2     ...                    ...
 3     ...                    ...
 4     ...                    Neg (SB)
 5     ...                    ...
 6     ...                    Pos (SB)
 7     BCR/ABL1 Neg           ...
 8     BCR/ABL1 Neg           Pos (RT-PCR)
 9     BCR/ABL1 Neg           Pos (FISH)
         (RT-PCR) JAK2 Pos    ...
 10    BCR/ABL1 Neg (FISH)    Pos (Q-PCR)
         JAK2 Pos

Case
No.    Source, y

 1     Kemp et al, (14) 1964
 2     Koulischer et al, (15) 1967
 3     Hoppin and Lewis, (16) 1975
 4     Haq, (17) 1990
 5     Jantunen and Nousiainen, (18) 1991
 6     Roth et al, (19) 1993
 7     Ganti et al, (20) 2003
 8     Saviola et al, (21) 2005
 9     Present report
 10    Present report

* Ellipses (...) indicate not done; Ph+, Philadelphia
chromosome positive; Neg, negative; SB, Southern blot
hybridization; Pos, positive; RT-PCR, reverse transcription
polymerase chain reaction; FISH, fluorescence in situ
hybridization; and Q-PCR, quantitative real-time polymerase chain
reaction. Adapted with permission from Blackwell Publishing from
Saviola et al. (21)
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