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© 2001 American Society for Clinical Oncology BCR Rearrangement–Negative Chronic Myelogenous Leukemia RevisitedFrom the Departments of Leukemia, Bioimmunotherapy, Pathology, Biomathematics, and Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston, TX. Submitted August 11, 2000; accepted March 6, 2001.Address reprint requests to Razelle Kurzrock, MD, Department of Bioimmunotherapy, Box 302, 1515 Holcombe Blvd, Houston, TX 77030.
PURPOSE: To document the characteristics of patients with major breakpoint cluster region (M-bcr) rearrangement–negative chronic myelogenous leukemia (CML). PATIENTS AND METHODS: The hematopathologist, who was blinded to patients’ molecular status, reviewed the referral bone marrows and peripheral-blood smears from 26 patients with Philadelphia (Ph) translocation–negative CML who lacked Bcr rearrangement (and other evidence of a Bcr-Abl anomaly) and 14 patients (controls) with chronic-phase Ph-positive CML. Clinical data was ascertained by chart review. RESULTS: Among the 26 M-bcr rearrangement–negative CML patients, three pathologic subtypes emerged: (1) patients indistinguishable from classic CML (n = 9), (2) patients with atypical CML (n = 8), and (3) patients with chronic neutrophilic leukemia (n = 9). Among the 14 patients with Ph-positive CML who were included in the blinded review, 13 were classified as classic CML, and one was classified as atypical CML. The only statistically significant difference between M-bcr rearrangement–negative subgroups was in the proportion of patients having karyotypic abnormalities, an observation common only in patients with atypical CML (P = 0.008). However, the small number of patients in each subgroup limited our ability to differentiate between them. Interferon alfa induced complete hematologic remission in five of 14 patients; four of these remissions lasted more than 5 years. Only one of 26 patients developed blast crisis. The median survival of the 26 patients was 37 months. CONCLUSION: Patients with M-bcr rearrangement–negative CML fall into three morphologic subgroups. Disease evolution does not generally involve blastic transformation. Instead, patients show progressive organomegaly, leukocytosis, anemia, and thrombocytosis. Some patients in each subgroup can respond to interferon alfa.
THE HALLMARK OF chronic myelogenous leukemia (CML) is the Philadelphia (Ph) translocation t(9;22)(q34;q11), which, at the molecular level, results in the transfer of the 3' end of the ABL gene next to the 5' end of the disrupted BCR gene.1-5 The product is a chimeric BCR-ABL gene. In the majority of cases of CML, the BCR breakpoint occurs in the central, major breakpoint cluster region (M-bcr). Exon b2 or b3 of BCR is joined to exon a2 of ABL, resulting in a b2-a2 or b3-a2 junction. This transcript encodes a 210-kd protein (p210Bcr-Abl).4,5 The creation of this anomalous protein is believed to play a critical role in the pathogenesis and phenotypic manifestations of CML. Patients with Ph-positive CML have typical clinical and morphologic features: splenomegaly, neutrophilia, basophilia, frequent thrombocytosis, a low leukocyte alkaline phosphatase (LAP) score, and a hypercellular bone marrow with an increased myeloid:erythroid ratio and granulocytic hyperplasia. The disease course inevitably evolves from a chronic phase to a terminal blast transformation phase. A minority of patients (5% to 10%) do not have cytogenetic evidence of the Ph chromosome. Over the last few years, M-bcr rearrangement has been well documented in a subset of Ph-negative patients.6-11 It is now established that M-bcr rearrangement–positive CML patients have a molecular fingerprint (ie, the p210Bcr-Abl protein) and a clinical phenotype and outcome that is indistinguishable from that of their Ph-positive counterparts.6,7 The existence of a Ph-negative, M-bcr rearrangement–negative CML has, however, remained a matter of debate. Some investigators have suggested that these individuals represent part of the spectrum of chronic myelomonocytic leukemia (CMMoL).12 Others have reported that their features differ subtly, but recognizably, from classic CML, and that they should be designated atypical CML.13 A subset of these patients seems to have a disease highly reminiscent of CML.14,15 An entity called chronic neutrophilic leukemia has also been described and is characterized by a marked predominance of mature neutrophils (shift to the right, in contrast to the immature forms and shift to the left, which appear in the other subsets).16-22 In this article, we review the characteristics of 26 patients with CML who lack the Ph chromosome and M-bcr rearrangement. Our results suggest that these patients can be further subgrouped into three pathologic categories: (1) those with disease indistinguishable from CML, (2) those with disease classifiable as atypical CML,13 and (3) those with disease suggestive of chronic neutrophilic leukemia. The phenotypic characteristics, response to therapy, and outcome of these patients are reported.
Patient Population All patients referred to the University of Texas M.D. Anderson Cancer Center with a tentative diagnosis of CML had a full work-up—including blood, bone marrow aspirate, and biopsy—and molecular studies, at our center. Data on all patients were recorded in the Leukemia Department computer database. Informed consent was obtained according to institutional guidelines.
Criteria for diagnosis of M-bcr rearrangement–negative chronic myelogenous leukemia were (1) a hypercellular bone marrow with granulocytic hyperplasia, (2) persistent, unexplained, peripheral granulocytic leukocytosis of
To rule out the presence of CMMoL, other myeloproliferative disorders (essential thrombocythemia, myelofibrosis, polycythemia vera), or leukemoid reaction, patients who had any of the following features were excluded: (1) monocytosis (bone marrow monocytes > 3%, peripheral blood monocytes > 6%), (2) polycythemia, (3) persistent thrombocytosis ( > 1,000 x 109/L) in the presence of only moderate leukocytosis (< 30 x 109/L), (4) significant bone marrow myelofibrosis or dysplasia, (5) bone marrow blasts > 5%, (6) coexisting significant thrombocytopenia and anemia (hemoglobin
Pathology Review
DNA Analysis by Southern Blot Probes used to determine M-bcr rearrangement status were a 3' bcr (1.2-Kb HindIII/BG/II) genomic probe (bcr[PR-1]) and a larger universal bcr probe encompassing most of the 5.8-kb M-Bcr (Phl/bcr-3) (Oncogene Science, Inc, Manhasset, Long Island, NY).
RNA Analysis by Northern Blot
Polymerase Chain Reaction Southern blot analysis and hybridization of all amplified samples were performed. Ten microliters of the amplified product was run on 3% Nusieve/1% Seakem (FMC, Rockland, ME) composite gels, transferred overnight to Genescreen Plus membrane (New England Nuclear, Boston, MA), and baked at 80°C for 2 hours. Oligonucleotide probes complementary to the junctional BCR-ABL sequences24 were 5' end labeled with phosphorus-32, and hybridization was performed overnight. The membranes were washed as recommended by the manufacturer and exposed to Kodak XAR film (Eastman Kodak Co, Rochester, NY) for 3 to 48 hours. To confirm the integrity of the cDNA and to confirm that the amplification procedure worked, all samples were also amplified for the normal ABL product using primers encompassing ABL exon 2 and ABL exon 1b and a probe that spans this region as previously described.27
Western Blot
Cytogenetic Analysis
Interphase Fluorescent In Situ Hybridization
Hypermetaphase Fluorescent In Situ Hybridization
Response Definition
Partial hematologic remission (PHR) required the WBC count to be
Statistical Analysis
Patient Characteristics A total of 26 patients meeting our criteria for M-bcr rearrangement–negative CML were identified in the Leukemia Department computer database (Table 1). The present database includes long-term follow-up on the 11 patients initially reported by us in 199014 and 15 new individuals. All consecutive patients who met the criteria were included.
Fifty-four percent of the patients were men. Although their median age at diagnosis was 63 years, ages ranged widely; the youngest patient was 23 years old and the oldest patient was 88 years old. Most patients had few symptoms on presentation. A minority of the group presented with fever, weight loss, and night sweats (Table 1). One individual presented with biopsy-confirmed acute febrile neutrophilic dermatosis (Sweet’s syndrome).32,33 Physical examination was generally normal except for splenomegaly, which was discerned in 62% of patients at diagnosis.
The median WBC count at presentation was 36 x 109/L (range, 22 to 300 x 109/L); mature neutrophils constituted 45% to 90% of the total WBC count. By definition, patients had
Bone Marrow Pathology To ascertain the differences between the study patients and those with classic CML, bone marrows from 14 patients with chronic-phase Ph-positive CML were included in the review. The pathologist was blinded to the cytogenetic and molecular status of the patients. Thirteen of the 14 patients with Ph-positive CML were given a diagnosis of CML on pathologic review. However, one patient was given a diagnosis of atypical CML (based on criteria in Table 2).13,34-36
With regard to the 26 patients with M-bcr rearrangement–negative CML, three pathologic patterns emerged. First, there were nine patients whose bone marrow pathology was not distinguishable from that of classic Ph-positive CML (patients no. 3, 12, 14, 15, 16, 18, 21, 24, and 25)(Table 3 and Fig 1). However, two of these patients had abnormally large megakaryocytes, a feature that was believed to be unusual for classic CML (but has not been included in the criteria for diagnosis of atypical CML).13,34,35 The second category of patients could be classified as atypical CML according to previously established criteria (Table 2 and Fig 2).13,34,35 There were eight patients in this subgroup (patients no. 1, 2, 4, 6, 9, 11, 17, and 22). These patients generally showed granulocytic dysplasia in the bone marrow as well as increased erythroid cells. In the peripheral blood, there was a shift to the left but not as pronounced as that in classic CML. Basophilia was absent. However, within this group of eight patients, a spectrum existed from those clearly distinguishable from CML because of the presence of most of the above features in those in whom only very subtle distinguishing findings were present. For example, patient no. 6 was classified as atypical CML but only on the basis of lack of basophilia. The third group of patients could best be classified as chronic neutrophilic leukemia based on bone marrow and peripheral-blood pathology (n = 9; patients no. 5, 7, 8, 10, 13, 19, 20, 23, and 26).16-21 These patients had marked blood and bone marrow neutrophilia with a shift to the right (Figs 3 and 4).
Karyotype Cytogenetic studies were performed on all patients. Neither the Ph translocation t(9;22) nor its variants were seen in any patient. Indeed, no patient had an abnormality of chromosome 22. At the time of presentation, 15 patients (60%) had a normal diploid karyotype. One patient had no mitotic cells. The following karyotypic abnormalities were seen in the other 10 patients: trisomy 21 (patients no. 2 and 4), trisomy 8 (patients no. 2 and 9), loss of the long arm of chromosome 20 (patients no. 7 and 22), loss of the long arm of chromosome 13 or translocation involving this region (patients no. 16 and 17), trisomy 19 (patient no. 2), t(9;14) (p13;q32) (patient no. 8), loss of chromosome 17 (patient no. 11), and trisomy 14 (patient no. 6). Most of these patients had cytogenetic anomalies in a minority of the metaphases with the majority of metaphases remaining diploid. In addition, some of the above patients had several distinct clonal anomalies that coexisted. For instance, patient no. 2 showed 22 diploid metaphases, one metaphase with trisomy 8, one metaphase with trisomy 21, and one metaphase with trisomy 19. Eighteen patients also had follow-up karyotype analysis performed one or more times after the baseline test. Three patients showed clonal evolution (patients no. 9, 10, and 11). The abnormal karyotypes that emerged with time in these individuals included a translocation between the short arm of chromosome 1 and the long arm of chromosome 6, loss of the long arm of chromosome 20, and trisomy 21 combined with loss of the long arm of chromosome 15.
Molecular Studies
Treatment
Fourteen patients were treated with interferon alfa (IFN- Other management strategies were used in only a very small number of patients. Three patients were treated with busulphan; they achieved short-lived PHRs (3, 7, and 12 months). Two patients had splenectomy, and neither improved. One patient received low-dose cytarabine with no response, and another was given high-dose cytarabine combined with cisplatin with no response. The one individual who received fludarabine attained a PHR that lasted 4 months. One of the two patients who underwent allogeneic bone marrow transplantation responded; this patient achieved a 6-month PHR.
Disease Evolution
Breakdown of Phenotypic Characteristics and Course by Subcategory
Patients with typical CML. As mentioned earlier, nine patients (four men and five women) had disease indistinguishable from typical CML by bone marrow and peripheral-blood pathology review. The median age was 60 years. Two patients (22%) presented with "B" symptoms. Most (78%) had splenomegaly at the time of diagnosis. The median initial WBC count was 57 x 109/L; hemoglobin, 13.2 g/dL; and platelet count, 263 x 109/L. Mature neutrophils comprised a median of 58% of WBCs (range, 45% to 90%). Only two of the eight patients with available LAP scores had low values, while three had elevated values. All of the patients but one had a diploid karyotype. The one exception had a translocation between the short arm of chromosome 1 and the long arm of chromosome 13.
With regard to treatment, five of the patients received IFN- Patients with atypical CML. On pathologic review, eight patients (seven men and one woman) were classified as having atypical CML (Table 2).13,34 The median age was 60 years. Three patients had B symptoms on presentation. Most (75%) had splenomegaly. The median WBC count at diagnosis was 36 x 109/L; hemoglobin, 11.7 g/dL; platelet count, 270 x 109/L. Mature neutrophils comprised a median of 77% of WBCs (range, 56% to 86%). Four patients had low LAP scores at diagnosis, while two had very high LAP scores. (Scores were not available in two patients.) All except one patient had one or more karyotypic abnormalities: trisomy 21 (n = 2), trisomy 8 (n = 2), trisomy 14 (n = 1), monosomy 17 (n = 1), 13q- (n = 1), and trisomy 19 (n = 1).
Only one patient (14%) of seven treated with IFN- Patients with chronic neutrophilic leukemia. Nine patients (three men and six women) were characterized as having chronic neutrophilic leukemia based on blood and bone marrow pathology review. The median age was 74 years. However, the range of ages was wide; the youngest patient was 23 years old and the oldest patient was 88 years old. Two patients presented with B symptoms. Three patients (33%) presented with splenomegaly, and one had Sweet’s syndrome at the time of diagnosis. The median WBC count was 36 x 109/L; hemoglobin, 11.0 g/dL; and platelet count, 188 x 109/L. Mature peripheral-blood neutrophils comprised a median of 72% of WBCs (range, 59% to 90%). Six patients had low LAP scores. (LAP score was elevated in one patient, not performed in one patient, and normal in one patient). Six of the patients had a diploid karyotype; one had insufficient metaphase for analysis, one had a deletion of the long arm of chromosome 20, and one had a t(9;14) anomaly.
Both patients treated with IFN-
Survival
Although patients in the subgroup with atypical CML had shorter median survivals compared with those in the other subgroups, there was no statistically significant difference in survival among the three patient subgroups (P = .7, log-rank test) (Fig 6 and Table 4). The 3-year survival rate was 53% (95% CI, 18% to 80%) for the patients who were indistinguishable from classic CML, 50% (95% CI, 15% to 77%) for those with atypical CML, and 53% (95% CI, 18% to 80%) for those with chronic neutrophilic leukemia. The 5-year survival rates were 36% (95% CI, 6% to 68%), 13% (95% CI, 1% to 42%), and 40% (95% CI, 10% to 70%), respectively (Fig 6).
CML is a generic designation given to specific clinical features. The best-studied form of CML has the classic Ph-positive genotype and accounts for more than 90% of cases. A marked granulocytic proliferation is its phenotypic hallmark, and the BCR-ABL chimeric gene is its molecular fingerprint. Patients with a Ph-negative CML have been described for many years. However, the earliest cases predated the emergence of molecular techniques that eventually revealed that some of these patients still harbored the aberrant BCR-ABL hybrid gene. Their disease was therefore identical to Ph-positive CML at the molecular (as well as the phenotypic) level.6-11 Hence, Ph-negative and Ph-positive CML have had to be redefined as M-bcr–negative CML versus M-bcr–positive CML. The percentage of patients with M-bcr rearrangement among Ph-negative CML patients has varied from less than 10% to more than 50% in different series.12,15,19-22,38,39 The classification of the remaining patients is less clear-cut.36 Some investigators claim that at least a portion of these patients are reminiscent of those with M-bcr–positive disease,14,15,39 whereas other investigators have described clear distinctions.12,13,34 To some extent, the variation in both the rates of M-bcr rearrangement positivity and the phenotypic descriptions of the patients may be due to the initial selection criteria; some studies included patients who could have been diagnosed with CMMoL, whereas others had more strict criteria. The situation is further complicated by the fact that M-bcr rearrangement–negative CML, Ph-positive CML, CMMoL, M-bcr–negative CML, essential thrombocytosis, myelofibrosis, and polycythemia vera seem to be nosologically related disorders. Nevertheless, the exuberant marrow fibrosis, expanded RBC mass, florid megakaryocytic hyperplasia, or significant monocytosis that are the hallmark of the non-CML myeloproliferative disorders and CMMoL were not seen in our patients, an observation supporting the distinctness of the M-bcr rearrangement–negative subgrouping of patients with a CML phenotype. Furthermore, pathologic review of our patients’ bone marrow and peripheral-blood findings indicated that three morphologic subgroups existed: (1) those with disease indistinguishable from CML, (2) those with atypical CML (Table 2),13,34-36 and (3) those with chronic neutrophilic leukemia. With rare exceptions, nearly all cases of Ph-positive CML have a p210-encoding BCR-ABL gene in which the breakpoint occurs in the central M-bcr. The M-bcr encompasses five exons termed b1 to b5 that correspond to the 12th to 16th exon of the BCR gene.3 The resultant transcript contains either a b2a2 (BCR exon 13 [or M-bcr exon b2] joined to ABL exon 2) or a b3a2 junction (BCR exon 14 [or M-bcr exon b2] joined to ABL exon 2) or both. A small number of patients have been described who have a Ph chromosome–positive CML, albeit with a very indolent course, and lack M-bcr rearrangement (Southern blot of DNA) or the classic b2-a2 or b3-a2 junction (PCR of cDNA). These patients have been found to have yet another junction—c3-a2 (otherwise known as e19-a2)—that joins the 19th exon of BCR to ABL exon 2 and is translated into a p230Bcr-Abl.25,37,40-44 Rare anecdotal reports of Ph-positive CML with e1-a2 junctions leading to the production of p190Bcr-Abl(in the absence of p210Bcr-Abl) have also been reported3,40,45-48 and may be associated with a phenotype intermediate between CML and CMMoL.45 Other junctions occur as well, but they are extremely rare.40,49,50-54 Because alternate breakpoints exist, it could be argued that tests for M-bcr rearrangement alone are inadequate to rule out these unusual junctions. However, it should be noted that, almost without exception,40,49 alternate junctions were found in patients bearing a Ph translocation (rather than in Ph-negative patients). Furthermore, 14 of our patients had one or more additional molecular tests performed (beyond Southern blot). These tests included Western blot, FISH, polymerase chain reaction, and Northern blot of mRNA, which ruled out the presence of Bcr-Abl proteins, subchromosomal translocations between chromosomes 9 and 22, alternate junctions, and aberrant BCR or ABL transcripts, respectively (Table 3). Importantly, six of the nine patients who had disease indistinguishable on a pathologic basis from classic Ph-positive CML had molecular tests (Western blot, n = 6; FISH, n = 3) in addition to Southern blot (Table 3). These tests eliminated the possibility that a BCR-ABL configuration existed and resulted in production of a Bcr-Abl protein but was missed by Southern blot because of a deletion within BCR or an alternative BCR-ABL junction. Furthermore, three of the patients with pathology consistent with chronic neutrophilic leukemia had Western blot that ruled out the presence of a p230Bcr-Abl as well as polymerase chain reaction (n = 2) that ruled out an e19-a2 junction. The latter abnormalities have been associated with a chronic neutrophilic leukemia phenotype by some investigators,25 but not by others.42
Eight M-Bcr–negative patients were given a diagnosis of atypical CML in accordance with the French-American-British (FAB) Cooperative Group guidelines.13 Interestingly, one of the 14 patients with Ph-positive CML examined by the pathologist in the blinded review also received this diagnosis. Therefore, the FAB guidelines13 (with an emphasis on peripheral-blood findings) as used by us (albeit with an emphasis on bone marrow aspirate and biopsy findings in addition to peripheral-blood morphology) confirm the uniqueness of atypical CML but are not absolute in their ability to differentiate CML from atypical CML and its variants. Furthermore, within the subgroup of atypical CML, a spectrum of changes was noted, with some individuals showing all the features delineated in Table 2, while others demonstrated only very subtle changes. One patient was put in this category on the basis of absent basophils alone. The FAB guidelines for atypical CML were published in 1994.13 Not surprisingly, therefore, some of the patients previously published by our group in 1990 as typical CML14 were now reclassified as atypical CML. M-bcr–negative atypical CML patients were distinguished clinically from the other M-bcr–negative categories by a predominance of men, frequent cytogenetic abnormalities, and relatively lower response rates to IFN-
Chronic neutrophilic leukemia has been previously published in case reports by other investigators.16-21 Most describe this entity as characterized by neutrophilia with a shift to the right (in contrast to CML in which the granulocytic series show a shift to the left), hepatosplenomegaly, and elevated LAP scores. The diagnosis of chronic neutrophilic leukemia should be made with considerable caution and only after all possibilities of a leukemoid reaction and other myeloproliferative disorders have been eliminated. Clinical, cytogenetic, and molecular studies are critical in this differentiation. Our nine patients with this disorder all had marked, right-shifted, peripheral-blood, and bone marrow neutrophilia. However, only one third had splenomegaly at diagnosis, and only one patient had an elevated LAP score (Table 4). A previous report described successful treatment of two such patients with IFN- There were several areas of similarities and differences between the three subcategories (Table 4). However, these distinctions did not reach statistical significance with the exception of the karyotype differences (P = .008) (chromosomal abnormalities seen predominantly in atypical CML), perhaps because of the small number of patients in each category.
In conclusion, we report that M-bcr rearrangement–negative patients with CML may be pathologically indistinguishable from classic Ph-positive CML or may present as atypical CML13 or as chronic neutrophilic leukemia.16-21 Most patients have a diploid karyotype except in the subgroup with atypical CML. With rare exception, M-bcr–negative CML patients do not progress to blast crisis. Rather, in most patients, progression is manifested by increasing leukocytosis and organomegaly, as well as worsening anemia and thrombocytopenia. Some patients in all three subcategories respond to IFN-
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
20 x 109 cells/L, (3) absence of the Ph chromosome, and (4) no M-bcr rearrangement. Additional molecular tests for BCR-ABL, as outlined below, were also performed on selected patients and proved negative in all cases. 
11 g/dL coexisting with platelets 
2 test or Fisher’s exact test as indicated, and continuous data were compared using the Mann-Whitney test or Kruskal-Wallis as appropriate. 
; dose range, 3 x 106 units/d to 9 x 106 units/d). Six of these patients (43%) responded (five CHRs and one PHR). The durations of the CHRs were 1, 63, 84, 101, and 100+ months. 
