Neutrophilic-chronic myeloid leukemia (CML-N) has been described as a CML variant associated both with a distinctive molecular defect of the Philadelphia chromosome and with a more benign clinical course than classic CML. The translocation (9;22) in CML-N results in the transcription of an e19/a2 type BCR/ABL mRNA that codes for a 230-kD BCR/ABL protein (p230). The indolence of the clinical course of patients with CML-N has been disputed.
The objectives of this study were to quantify and correlate with clinical outcome the p230 mRNA and protein in patients with CML-N, to describe six new patients and the follow-up (with molecular analysis) of five previously reported patients with CML-N, and to review characteristics of all patients with CML-N and p230 BCR/ABL reported to date in the literature.
Quantitative polymerase chain reaction assays on specimens from the great majority of patients with CML-N revealed minimal numbers of molecules of p230 BCR/ABL transcripts per total RNA. This also was associated with a lack of detectable p230 BCR/ABL protein in patient specimens, even in one patient who was followed for 16 years after diagnosis. This may explain the milder leukemic phenotype in most patients with CML-N. A review of all 23 patients who had an e19/a2 type BCR/ABL translocation suggested that the low level of p230 BCR/ABL mRNA and the lack of detectable p230 BCR/ABL protein in patients with no additional cytogenetic abnormalities may predict their indolent clinical course.
Chronic myeloid leukemia (CML) is characterized by a specific fusion gene, BCR/ABL, and its product.1 The BCR/ABL gene is generated by the chromosomal translocation between the long arms of chromosomes 9(ABL gene) and 22 (BCR gene). This translocation is known as the Philadelphia chromosome (Ph). The Ph is associated with different leukemia phenotypes, a consequence of the molecular variability in the rearrangement between the BCR and ABL genes.2, 3 The breakpoints on chromosome 9 are almost always 5′ to ABL exon 2 or, occasionally, to ABL exon 3. The breakpoints on chromosome 22 differ in their position within the BCR gene. Five exons, exons 12–16, are located in major breakpoint cluster region (M-BCR), and the breaks frequently occur between exons 13 and 14 or exons 14 and 15. Translocations involving M-BCR result in BCR/ABL constructs that give rise to the production of a distinct fusion protein with a 210-kD molecular mass. The p210 BCR/ABL fusion protein underlies the pathogenesis of almost all CML and some acute leukemias. Another identified breakpoint area within the BCR gene falls within its first large intron, so that only the first exon of the BCR gene is joined to the ABL gene as a result of the chromosomal translocation. This region in the BCR gene is referred to as minor BCR (m-BCR), and translocations involving this region result in the production of a fusion protein with a 190-kD molecular mass. The p190 BCR/ABL has been found primarily in patients with Ph positive acute lymphoblastic leukemia and is seen only very rarely in patients with CML and acute myeloid leukemia.3 Due to the alternative splicing of primary hybrid transcript, limited amounts of the p190 protein also are detectable in CML associated with p210 protein.4–6
A new CML variant, neutrophilic-CML (CML-N), has been reported in association with a distinctive molecular defect in Ph, resulting in a novel BCR/ABL protein.7 The BCR breakpoint on chromosome 22 in this type of rearrangement occurs downstream from M-BCR,8 between exons 19 and 20, in a region designated as micro-BCR (μ-BCR). This translocation results in the transcription of an e19/a2 type BCR/ABL messenger RNA, which is translated into a 230-kD BCR/ABL protein. It was reported initially that CML-N was associated with a more benign course than classic CML.7 Such patients presented with lower white blood cell counts, minimal basophilia, milder anemia, and less prominent or absent splenomegaly. In the peripheral blood, most circulating myeloid cells were mature granulocytes, and blastic transformation did not occur. However, since the initial description of this entity in 5 patients,7, 8 11 additional patients with the e19/a2 translocation have been reported9–18 with different clinical pictures, questioning whether CML-N should be established as a separate clinicomolecular entity. In this report, we present six new patients with Ph positive myeloproliferative disease and an e19/a2 BCR/ABL gene junction, and we present an update on five patients who were diagnosed previously with CML with an e19/a2 BCR/ABL gene junction.7, 8, 17 The pertinent findings and relevance of these patients are discussed in relation to previously published reports on patients with p230 BCR/ABL Ph positive disease.
MATERIALS AND METHODS
The leukemia cell lines used included K562 cells (bearing b3/a2 BCR/ABL translocation coding for p210), KBM7 cells (bearing b2/a2 BCR/ABL translocation coding for p210), and 32DP230 cells (bearing e19/a2 translocation coding for p230).19–21 All cell lines were grown in RPMI medium containing 10% fetal bovine serum.
RNA Extraction and Reverse Transcription
Total RNA was isolated from each sample using TRIZOL reagent (Gibco, Gaithersburg, MD) following the manufacturer's directions. RNA was quantified, aliquoted, and stored at −70 °C. Nineteen microliters of RNA were heated for 10 minutes at 65 °C and then put on ice. Twenty-one microliters of reverse transcription reagents were added, including 95 mM Tris, pH 8.3; 142.5 mM KCl; 5.7 mM MgCl2; 19 mM dithiothreitol; 19 mM each of dATP, dCTP, dTTP, and dGTP; 200 μg/mL random hexamers pdN6; 1.4 × 104 units/mL Moloney-Murine leukemia virus (M-MLV) reverse transcriptase; and 1400 units/mL RNAsin (Pharmacia, Uppsala, Sweden). After incubation at 37 °C for 2 hours, the reaction was terminated by heating at 65 °C for 10 minutes; cDNA was stored at −20 °C until polymerase chain reaction (PCR) assays were conducted.
Construction of Competitor Template
The BCR/ABL competitor was kindly provided by Cross et al.22 PBKλ5 is 5.25 kB in size; thus, 1 ng equals 1.8 × 108 molecules. Dilutions were made in the range from 107 to 10 molecules per 2.5 μL with steps at every half order of magnitude on a logarithmic scale. Working dilutions of the competitor were stored at 4 °C.
Qualitative and Quantitative PCR Assay
Five microliters of cDNA were added to 45 μL of reagent solution, including 12 mM Tris, pH 6.3; 1.8 mM MgCl2; 60 mM KCl; 0.24 mM dNTP; 0.6 μM primers; and 0.6–1.0 units/25 μL Taq DNA polymerase. Primers were BCR exon 13 sense primer (5′-ACAGAATTCGCTGACCATCAATAAG-3′) and ABL exon 3 antisense primer (5′-TGTTGACTGGCGTCATGTAGTTGCTTGG-3′). The PCR cycle included 1 minute at 94 °C, 50 seconds at 64 °C, and 1 minute at 72 °C. After 35 cycles, the reaction was terminated with an extension period of 10 minutes at 72 °C. Multiplex PCR was performed following the method of Cross et al.23 Reaction products were electrophoresed on 1.5% agarose gels, and bands were visualized under ultraviolet light with ethidium bromide staining. Molecular sizes of the amplified products obtained were as follows: b3/a2, 385 base pairs (bp); b2/a2, 310 bp; e19/a2, 925 bp; and BCR, 808 bp. ABL mRNA was quantified in all samples as an internal standard to assess the amount of cDNA and the quality of RNA. Competitive nested PCR to amplify b2/a2, b3/a2, and e19/a2 BCR/ABL also was performed as described previously by Cross et al.22, 23 Due to the size difference between the competitor and BCR/ABL molecules, the number of molecules of competitor at the equivalence point was multiplied by 0.56 (559 bp/998 bp) for e19/a2, respectively, to derive the number of BCR/ABL molecules in the sample.
Real-Time Reverse Transcriptase-PCR
In Patients 2 and 6–11 (Table 1), the amount of p230 encoding mRNA was measured by real-time reverse transcriptase (RT)-PCR. One microgram of total RNA extracted was prewarmed for 10 minutes at 60 °C and incubated for 10 minutes at 25 °C and then for 30 minutes at 48 °C in a 20-μL reaction mixture containing 10 mM Tris HCl, pH 8.3; 50 mM KCl; 5.5 mM MgCl2; 0.5 mM of each deoxyribonucleotide; 20 units of RNAsin; 2.5 mM random examers (Pharmacia); and 100 units of M-MLV reverse transcriptase (BRL, Bethesda, MD). The p230 BCR/ABL sequences were amplified in a 2-μL aliquot of cDNA solution using the ABI 7700 instrument (Perkin Elmer Applied BioSystems, Foster City, CA) in a 50-μL final volume of a mixture containing 10 mM Tris HCl, pH 8.3; 50 mM KCl; 5.5 mM MgCl2; 0.2 mM of each deoxyribonucleotide (Perkin Elmer); 2.5 units of Taq Gold™ (Perkin Elmer); 0.1 mM of the probe; and 0.9 mM of the sense and antisense primers. Primers were BCR exon 19 sense primer (5′ TGA AGG CAG CCT TCG ACG 3′), ABL exon 2 antisense primer (5′ TCC AAC GAG CGG CTT CAC 3′), and BCR/ABL probe in ABL exon 2 (5′ CAG TAG CAT CTG ACT TTG AGC CTC AGG GTC T 3′). PCR was performed as follows: 50 °C for 2 minutes, 95 °C for 10 minutes, then 45 cycles at 95 °C for 15 seconds and 60 °C for 1 minute. The CT of each sample was used to interpolate a standard curve generated by assaying, in parallel with the samples, 1:10 serial dilutions (from 106 to 102) of p230 synthetic DNA diluted in a 100-ng E. coli RNA solution. The levels of glyceraldehyde 3-phosphate dehydrogenase were quantified in parallel with BCR/ABL, by real-time PCR assay, to evaluate the integrity and the amount of sample RNA.
Table 1. Characteristics of Patients Reported in the Literature with BCR/ABL Coding for p230 Transcripts
Age at diagnosis (yrs)
WBC (× 109/L)
Plt (× 109/L)
Additional CG abnormalities
Survival (cause of death)
WBC: white blood cells; Hb: hemoglobin; Plt: platelets; Ph: Philadelphia chromosome; CG: cytogenetic; F: female; ND: not done; NR: not reported; M: male; BMT: bone marrow transplantation.
Western blot analyses were performed on 1–10 × 106 cells from peripheral blood or bone marrow. KBM7 and 32DP230 cells were used as a positive control. Cell extracts were lysed in boiling sodium dodecyl sulfate (SDS) sample buffer for 5 minutes, separated by 6.5% SDS-polyacrylamide gel electrophoresis, transferred, and then treated with a 1:13,000 dilution of 8E9 anti-ABL antibody.22 Immunoreactive bands were visualized by reacting with the substrate of the enhanced chemiluminescence western blotting detection system (ECL Plus; Amersham, Arlington Heights, IL) and were subjected to autoradiography.
Medical histories, including the results of molecular studies, from 11 patients with CML-N who were referred to the investigators are presented. All peripheral blood and bone marrow samples were obtained under approved protocols and with informed consent.
Patient 1 (Table 1) is a woman age 33 years who was listed in the table of a recent letter (Patient 11).17 The patient presented in February 1999 with a complete blood count (CBC) that showed a hemoglobin (Hb) level of 13.7 g/dL, a white blood cell (WBC) count of 10.8 × 109/L with a normal differential, and a platelet count (plt) of 418 × 109/L. In March 1999, a bone marrow evaluation showed 100% cellularity and granulocytic and megakaryocytic hyperplasia with 3% blasts. Cytogenetic analysis showed 20 of 20 Ph positive cells. The patient was diagnosed with CML. Upon referral to The University of Texas M. D. Anderson Cancer Center (UTMDACC) in June 1999, she was asymptomatic, and the physical examination was unremarkable. There was no palpable splenomegaly. Bone marrow examination was repeated, demonstrating 100% cellularity, megakaryocytic hyperplasia, mild eosinophilia, and 3% blasts. Cytogenetic analysis showed t(9;22)(q34;q11) in 30 of 30 metaphases. The patient's blood samples were examined for the presence of BCR/ABL fusion transcripts and protein; all tests were performed at least twice on samples that were obtained at two different times 5 months apart (the patient was not treated). A band corresponding to the BCR/ABL fusion transcripts coding for p230 protein was detected after RT-PCR (Fig. 1). Quantitative/competitive nested PCR revealed 2403 molecules of p230 transcripts per μg total RNA (Fig. 2, top), whereas PCR for ABL transcripts showed 5.88 × 105 molecules per μg total RNA (data not shown). In the second sample obtained 5 months later (Fig. 2. bottom), nested PCR showed 49 molecules of p230 transcript per μg total RNA, whereas PCR for ABL transcripts showed 1.2 × 105 molecules per μg total RNA (data not shown). The p230 BCR/ABL protein was not detected in peripheral blood cells by Western blot analysis (Fig. 3). The patient subsequently was lost to follow-up.
Patient 2 (Table 1) is a woman age 59 years who was evaluated in October 1999 for leukocytosis. This patient was also listed in the table of a recent letter (Patient 9).17 Her CBC showed an Hb level of 12.7 g/dL, a WBC count of 50.0 × 109/L, a normal differential, and a plt count of 750 × 109/L. Bone marrow evaluation showed myeloid hyperplasia without blasts. Cytogenetic analysis showed 100% of metaphases with t(9;22)(q34;q11). RT-PCR analysis on a bone marrow sample was positive for BCR/ABL transcript, consistent with the e19/a2 BCR/ABL junction; real-time RT-PCR showed 7694 p230 molecules per μg total RNA (data not shown). The patient was treated with interferon α (IFNα) 3 MU three times per week. At her last follow-up in January 2001, the patient was in complete hematologic remission, and cytogenetic analysis was not informative due to lack of mitoses.
Patient 3 (Table 1) is a women age 55 years who developed malaise, lower back/pelvic pain, and headache in June 1999. Upon evaluation, she had an abnormal CBC (Hb, 14.1 g/dL; WBC count, 31.2 × 109/L with left shift; and plt count, 564 × 109/L). A bone marrow evaluation performed in November 1999 showed hypercellularity with granulocytic and megakaryocytic hyperplasia, eosinophilia, and basophilia. Cytogenetic analysis showed 19 of 20 Ph positive cells, and she was diagnosed with CML. In December 1999, she was seen at UTMDACC for an opinion. Her WBC count was 38.5 × 109/L with left shift and 6% blasts, the Hb level was 12.8 g/dL, and plt count was 535 × 109/L. On examination, no spleen was palpable. Ph was found in all 20 examined cells. Nested PCR analysis of a peripheral blood sample demonstrated 560 molecules of p230 and 663 molecules of p210 per μg total RNA; PCR for ABL transcripts showed 0.76 × 105 molecules per μg total RNA. Neither p230 nor p210 protein was detected by Western blot analysis (data not shown). The patient was treated with pegilated IFNα and cytarabine but had to discontinue the treatment after 6 months due to toxicity. Since then, she has been on intermittent hydroxyurea for control of her WBC count.
Patient 4 (Table 1) is woman age 58 years who was diagnosed with CML in May 1995 after a routine CBC (Hb, 13.2 g/dL; WBC count, 24.4 × 109/L with left shift; and plt count, 837 × 109/L). Bone marrow was hypercellular with trilineage proliferation, and cytogenetic analysis showed 19 of 20 Ph positive cells. The patient was treated initially with daily IFNα injections; however, because of severe toxicity and lack of cytogenetic response, treatment was changed after 6 months to hydroxyurea. Approximately 2.5 years later, the treatment was changed to anagrelide (due to thrombocytosis) and then to busulphan (due to a high WBC count). The patient was seen at UTMDACC for the first time in December 1999 while she was receiving busulphan for 6 months. Physical examination was unremarkable. Her CBC showed a WBC count of 15.2 × 109/L with left shift and 1% blasts, an Hb level of 13.7 g/dL, and a plt count of 606 × 109/L. Bone marrow was markedly hypercellularity and fibrotic, with myeloid predominance (5% basophils and 6% blasts) and megakaryocytic hyperplasia. Cytogenetic study showed 20 of 20 Ph positive cells. Nested PCR analysis of a peripheral blood sample demonstrated 705 molecules of p230 and 151 molecules of p210 per μg total RNA; PCR for ABL transcripts showed 0.52 × 105 molecules per μg total RNA. Again, neither p230 nor p210 protein was detected by Western blot analysis (data not shown). The patient started treatment with STI571 in January 2000.
Patient 5 (Table 1) is a man age 67 years who was referred to the UTMDACC in July 2000. Four months earlier, he had presented with fevers, sweats, and fatigue. His CBC at that time showed an Hb level of 13.1 g/dL, a WBC count of 136.5 × 109/L with 19% blasts, and a plt count of 255 × 109/L. Blasts displayed the following markers: CD13 (95%), CD33 (94%), and CD34 (70%). A bone marrow evaluation showed 100% cellularity with fibrosis, left-shifted myeloid maturation with elevated number of blasts, and increased numbers of small megakaryocytes. Cytogenetic analysis showed 25 of 25 Ph positive cells. However, 20 of 25 of cells also contained additional chromosomal abnormalities, trisomy 8, and an isochromosome of the 17q-arm. Physical examination showed no palpable splenomegaly. The patient was diagnosed with CML in transformation. He underwent induction chemotherapy with daunomycin and cytarabine but did not achieve remission. Upon referral to the UTMDACC in July 2000, CBC showed an Hb level of 10.6 g/dL, a WBC count of 34.3 × 109/L with 5% blasts, and a plt count of 5 × 109/L. Bone marrow examination was repeated and demonstrated 35% cellularity with 6% blasts, marked fibrosis, granulocytic and megakaryocytic hyperplasia, and mild basophilia. Cytogenetic analysis showed t(9;22)(q34;q11) in 19 of 19 metaphases; 4 of the cells also contained +8; and 10 of the cells also contained iso(17) and +8. The patient's blood samples were examined for the presence of BCR/ABL fusion transcripts and protein. Quantitative/competitive nested PCR revealed 933 molecules of p230 and 200 molecules of p210 transcripts per μg total RNA. PCR for ABL transcripts showed 7.53 × 105 molecules per μg total RNA. Neither p230 nor p210 BCR/ABL protein was detected in the peripheral blood cells from this patient by Western blot analysis. The patient currently is receiving salvage therapy for advanced-stage CML.
Patient 6 (Table 1) is man age 57 years who was evaluated, as reported previously,7 in 1985 for leukocytosis (Hb, 15.8 g/dL; WBC count, 43.0 × 109/L with 88% neutrophils; and plt count, 191 × 109/L). Bone marrow evaluation showed myeloid hyperplasia without left shift; no mitoses were available for cytogenetic analysis. The spleen was not enlarged. In 1988, the WBC count was 43.7 × 109/L, and the spleen became palpable 4 cm below the costal margin. Cytogenetic study showed 9 of 36 cells (25%) Ph. IFNα was started (3 MU per day) with resultant reduction in spleen size and a reduction in WBC count to 20–30 × 109/L. IFNα was discontinued in 1991 due to poor compliance, and intermittent low-dose hydroxyurea was started. This patient was reported in 1996.7 Since then, the patient has been monitored closely. The patient did not receive any treatment for the last 4 years. Western blot analysis was performed on the patient's peripheral blood sample in January 1997 and showed no evidence of p230 protein. Western blot analysis was repeated on the patient's bone marrow sample in April 1997, again without evidence of p230 protein. In October 1999, real-time RT-PCR showed < 1000 molecules of p230 BCR/ABL transcripts per μg total RNA. At his last follow-up 16 years later, in January 2001, the patient continued in excellent condition, and his WBC count was 32 × 109/L.
Patient 7 (Table 1) is woman age 35 years who was evaluated, as reported,7 for the first time in 1988 due to an abnormal CBC (Hb, 12.7 g/dL; WBC count, 45.5 × 109/L; and plt count, 1240 × 109/L). Bone marrow analysis showed a myeloid:erythroid ratio of 18:1 without left shift. Cytogenetic analysis showed Ph positive metaphases in 60% of cells. The spleen was not enlarged. Hydroxyurea was given for 7 months. In 1992, the WBC count was 21 × 109/L, and the plt count was 700 × 109/L. The spleen was palpable at 3 cm below the costal margin. The patient was started on IFNα 3 MU three times per week. This patient was reported in 19967 and, since then, has been monitored closely. In April 1997, Western blot analysis of a peripheral blood sample showed no evidence of p230 protein. At her last follow-up in January 2001, she was in complete hematologic and cytogenetic remission, was continuing on IFNα treatment, and her clinical status was excellent 13 years after the diagnosis.
Patient 8 (Table 1) was a woman age 76 years who was evaluated, as reported,8 for the first time in 1985. Her CBC showed a WBC count of 28 × 109/L and a plt count of 1020 × 109/L. Cytogenetic analysis showed Ph positive metaphases without additional abnormalities. On physical examination, the spleen was enlarged. Busulphan was prescribed, and she achieved hematologic remission. After 5 years, she was lost to follow-up. This patient was reported in 1990.8 The original bone marrow biopsy has been recovered from the archive. Real-time RT-PCR showed 28,219 molecules of p230 BCR/ABL transcripts per μg total RNA. The patient died in 1995 of heart failure.
Patient 9 (Table 1) was woman age 57 years with a history of breast carcinoma (treated with surgery, chemotherapy, and radiotherapy during 1990–1991) who was evaluated in January 1996 for leukocytosis. Her CBC showed a WBC count of 39 × 109/L, an Hb level of 13.5 g/dL, and a plt count of 450 × 109/L. Bone marrow biopsy revealed findings consistent with CML. Cytogenetic analysis showed Ph positive metaphases in 100% of cells without additional abnormalities. The spleen was not enlarged. Real-time RT-PCR analysis of a bone marrow sample showed 304,598 molecules of p230 BCR/ABL transcripts per μg total RNA. The patient was treated with IFNα 5 MU/m2 subcutaneously (SQ) daily and cytarabine 20 mg SQ for 10 days each month; however, after 1 year of therapy, she achieved only hematologic remission. The patient then presented with enlarging lymph nodes in February 1997 and, after evaluation, was diagnosed with CML in lymphoid blast crisis. After unsuccessful therapy for advanced-stage CML, she expired in January 1998.
Patient 10 (Table 1) is man age 61 years who was diagnosed with CML in October 1997. His CBC showed a WBC count of 27 × 109/L, an Hb level of 15.4 g/dL, and a plt count of 278 × 109/L. Bone marrow biopsy revealed findings consistent with CML, but cytogenetic analysis showed Ph positive metaphases in only 4 of 11 evaluable mitoses. The spleen was not enlarged. Real-time RT-PCR analysis of a bone marrow sample showed 3243 molecules of p230 BCR/ABL transcripts per μg total RNA. He was treated with IFNα 5 MU/m2 SQ daily and cytarabine 20 mg SQ for 10 days each month (only for 5 courses due to persistent leukopenia). After 1 year, he achieved a complete cytogenetic response. Therapy was then stopped secondary to mild intolerance. Two years later, in October 2000, 1 of 14 cells examined on the cytogenetic analysis showed the Ph chromosome, and therapy with IFNα was resumed at 3 MU/m2 SQ daily. Currently, the patient is in complete cytogenetic remission, continues on IFNα treatment, and his clinical status is excellent.
Patient 11 (Table 1) is a women age 49 years who was diagnosed with CML in September 1999. Her CBC showed a WBC count of 64 × 109/L, an Hb level of 13.5 g/dL, and a plt count of 840 × 109/L. Bone marrow biopsy revealed myeloid hyperplasia with moderate increase in reticulin. There were 1% blasts. Cytogenetic analysis showed that all cells were Ph positive. The spleen was not enlarged. Real-time RT-PCR analysis of a bone marrow sample showed 297,041 molecules of p230 BCR/ABL transcripts per μg total RNA. The patient was treated with IFNα 5 MU/m2 SQ daily for 6 months. Due to a lack of hematologic and cytogenetic response, cytarabine 20 mg SQ for 10 days each month was added. The patient was in hematologic remission, but not in cytogenetic remission, as of January 2001.
CML-N was described in 1996 as a clinical entity characterized by primary, chronic, nonprogressive leukocytosis.7 The original description required the following criteria: 1) moderate neutrophilic leukocytosis; 2) rare, circulating, immature myeloid cells without a myelocyte peak; 3) excess bone marrow mature myeloid cells; and 4) absent or minimal splenomegaly.7 The Ph carrying e19/a2 BCR/ABL gene fusion abnormality added a molecular diagnostic confirmation of the CML-N entity. Although the cytogenetically visible abnormality is the same as in classic CML, the different molecular lesion may be responsible for the more benign clinical course.
In this report, we describe six new patients and update (with molecular analysis) five previously reported patients with Ph positive myeloproliferative disorders and the e19/a2 BCR/ABL abnormality, resulting in p230 mRNA production. A review of all 23 patients with Ph positive p230 CML (Table 1) showed that their presentation was somewhat atypical compared with classic CML: Sixteen patients were females, 16 patients had no palpable splenomegaly, and only 3 patients had WBC counts > 100 × 109/L. Five patients presented with very high plt counts (> 1000 × 109/L). Although some authors have suggested that these patients had Ph positive essential thrombocytosis,9, 11 the diagnostic criteria for that disorder specifically exclude patients with BCR/ABL gene rearrangement.24 Most patients described previously with Ph positive essential thrombocytosis, when studied by RT-PCR analysis, had the classic p210 junction.25 Among patients with CML and thrombocytosis, at least four different BCR/ABL junction variants (including e19/a2) have been detected, albeit with various frequencies.26
There is some confusion regarding the relation between CML-N and chronic neutrophilic leukemia. In chronic neutrophilic leukemia, clonality of the cells has not been reported consistently; thus, there is uncertainty about whether all reported patients had leukemia.27 You and Weisbrot28 reported that the syndrome of chronic neutrophilic leukemia was comprised of severe, sustained, mature, neutrophilic leukocytosis; hepatosplenomegaly; elevated leukocyte alkaline phosphatase; elevated serum vitamin B12; elevated serum uric acid; the absence of an underlying disease provoking a reactive leukemoid reaction; anatomic evidence of organ infiltration by granulocytes; and myeloid metaplasia. Although the clinical criteria for the diagnosis of chronic neutrophilic leukemia and CML-N overlap to some extent, CML-N is defined by Ph and the presence of the p230 BCR/ABL fusion gene.2
In their original report, Pane et al.7 suggested that patients with CML-N may have a more benign clinical course than patients with classic CML. This was questioned later as new patients with p230 disease and an adverse clinical course were described.9–18 A review of all reported patients (Table 1) showed that 10 patients were well and alive ≥ 3 years after the diagnosis (the longest follow-up is > 16 years). Six patients have died: Patient 8 was treated with busulphan, achieved a hematologic response, and died of an unrelated cause 10 years after the diagnosis; Patient 12 was never treated for CML-N and died of myocardial infarction 3 years after the diagnosis; Patient 17 died of complications 3 months after undergoing bone marrow transplantation (BMT) while in complete remission; and Patients 9, 15, 20, and 23 died in the blastic phase of CML. Three of four patients who died of leukemia presented with extrachromosomal abnormalities in addition to Ph. Overall, eight patients presented with additional chromosomal abnormalities. Patient 5 presented with accelerated-phase CML and has failed induction chemotherapy. Patient 14 had no response to IFNα treatment and was awaiting BMT. Patient 17 (death from complications of BMT) had failed IFNα treatment and was in the accelerated phase of CML prior to BMT. Patient 19 presented with significant basophilia and a high proportion of circulating, immature granulocytes. Patient 21 had failed IFNα treatment and was in the accelerated phase of CML prior to BMT. Thus, as suggested recently,17 chromosomal abnormalities in addition to Ph appear to be associated with a more malignant course of the disease.
The presence of Ph (which has the p230 fusion gene) as a single abnormality, however, does not necessarily confer an indolent course, as evidenced by Patient 9, who died in the blastic phase of CML. Still, the reason for the association of p230 disease with a milder leukemia phenotype in the majority of patients with CML-N (those without additional cytogenetic abnormalities) has not been clear to date. It has been reported that the three forms of BCR/ABL have different intrinsic leukemogenic activity when expressed in a hematopoietic progenitor cell.21, 29 The characterization of the tyrosine kinase activity of the three proteins revealed that p230 was less potent than p190 or p210.29 Recent in vitro experiments have shown that p230 had transforming ability similar to that of p190 or p210 when expressed in 32D myeloid cells or Rat1 fibroblasts.30 However, in mouse primary bone marrow cultures, p230 was less transforming, so that the cells required exogenous hematopoietic growth factors for optimal growth, whereas cells that expressed p190 and p210 grew independently of growth factors.21 Persistence of a pool of Ph negative progenitors in some patients with CML-N years after diagnosis, and in the absence of treatment (Patients 6 and 7), may suggest a limited proliferative advantage of the Ph-abnormal clone.
Attempts by our group7, 8 and by others9 to verify the expression of p230 protein in patients bearing the e19/a2 type BCR/ABL gene have been unsuccessful. Patient 6 had undetectable p230 protein in both peripheral blood and bone marrow 12 years after diagnosis and 6 years after discontinuation of IFNα. In the only reported patient with detectable p230 protein (Patient 20 in Table 1), most of the leukemic cells showed a duplication of the Ph.15 This may suggest that a greater degree of p230 protein production may have conferred a more malignant course, although other chromosomal abnormalities were found in addition to Ph. In this report, using two different techniques, we have shown that the transcription of the p230 gene in the great majority of patients with CML-N was extremely low, resulting in undetectable levels of p230 protein. Samples from Patient 6 contained minimal numbers of molecules of p230 transcripts per total RNA. This may explain the milder leukemic phenotype in most patients with CML-N. Although the level of p210 BCR/ABL transcription in newly diagnosed patients with classic CML is in the range of ≈35,000 p210 molecules per μg total RNA, the order of magnitude of p230 BCR/ABL transcription in most patients with untreated CML-N is similar to the order of magnitude of p210 BCR/ABL transcription in patients with classic CML patients who achieved complete cytogenetic remission after IFNα treatment.31, 32 Although transcription of the p230 gene in most patients with CML-N was very low, we did find patients with very high p230 mRNA expression. Those patients presented with more aggressive disease: Patient 9 died in blastic phase CML, whereas Patient 11 had a poor response to IFNα and cytarabine therapy.
Why there is a difference in individual p230 gene expression remains unknown. The transcription of p230 gene may be low because of the presence of at least 3 transcription silencer elements within the intronic sequences at the 3′ of exon 14 of the M-BCR region.33 These elements are lost in p190 disease, are partly retained in p210 disease, and are fully retained in p230 disease. Sequence analysis also revealed that at least one of the GAP-homology domains of the BCR gene was retained within the p230 gene.7 In addition, p230 expression may be low because of the occurrence of multiple alternative splicing of the original messenger RNA responsible for the production of out-of-frame transcripts that produce truncated p230 protein.10
It is interesting to note that, in three patients with newly diagnosed CML-N (Patients 3, 4, and 5), we detected p210 transcripts in addition to p230 transcripts. Similar to the classic p210-associated CML, in which p190 transcripts are detectable along with p210 transcripts,4–6 this finding is likely due to the alternative splicing of primary p230 mRNA. Thus, resultant numbers of both p230 and p210 transcripts are minimal. Unlike classic CML, however, in which p190 protein is detectable along with p210 protein4 and may or may not have clinical significance,5, 6, 34 in our three patients with CML-N, neither p230 nor p210 proteins were detectable.
It should be emphasized that patients with p230 positive CML (CML-N) can be overlooked easily if appropriate molecular studies are not performed. In standard practice, molecular studies are not requested if cytogenetic studies already have shown t(9;22). The detection of p230 positive patients occurs when simultaneous cytogenetic and molecular studies are done (e.g., in a research setting), which may show a paradoxical situation of the presence of t(9;22) but absence of M-BCR and m-BCR rearrangements by conventional Southern blot analysis. In addition, RT-PCR positivity for P230 BCR/ABL mRNA may be overlooked unless primers are used that cover the e19/a2 breakpoint. The presence of t(9;22) and RT-PCR negativity for M-BCR and m-BCR then stimulates additional studies to detect p230 disease at the mRNA level.
In conclusion, CML-N may have an indolent course in the absence of chromosomal abnormalities other than Ph because of the low expression of p230. This supports the need to conduct additional molecular studies, even if cytogenetic studies already have shown t(9;22), because of the prognostic importance of the molecular findings.