Four cases with complex Philadelphia translocations, including one with appearance de novo of a "masked" Ph

Four cases of chronic myelogenous leukemia (CML) with complex Philadelphia (Ph) translocations are described. The first case was that of a 50-year-old woman in the chronic phase of CML. Her leukemic cells showed a complex Ph translocation involving chromosomes #9, #11, and #22 [i.e., t(9;9;22;11)(11qter----11q11::9q11----9q34:: 9p11----9pter;22qter----22q11::9q34?;11 pter----11q11::22q11----22qter)]. In addition to the complex Ph translocation, the leukemic cells contained del(10)(p13). The second case was that of a 21-year-old man whose leukemic cells contained a translocation involving chromosomes #5, #9, and #22 [i.e., t(5;22;9)(q31;q11;q34)], resulting in a "masked" Ph chromosome. The third case was that of a 37-year-old man whose leukemic cells had a complex Ph translocation involving chromosomes #8, #9, and #22 [i.e., t(8;9;22)(q13;q34;q11)]. The fourth patient was a 41-year-old woman diagnosed as having CML in myeloid blastic phase, at which time the first specimen was examined by us. This blood sample showed a karyotype of 45,XX, -9, -17, -22, +mar1, +mar2,9q+. No Ph chromosome was present. A standard Ph translocation was detected in the cells obtained from the spleen, when the patient underwent splenectomy for treatment of the blastic crisis. Subsequent specimens obtained from the blood and bone marrow showed that the leukemic cells contained three clones: 45,XX, -9, -17, -22, +mar1, +mar2,9q+/46,XX, -17, +mar1,t(9;22)(q34;q11)/46,XX,t(9;22)(q34;q11). Cells with the "masked" Ph chromosome were thought to have been derived from the clone with the standard Ph translocation. We postulate that some variant Ph translocations, including those with a "masked" Ph chromosome, may be generated by a stepwise process following the genesis of a standard Ph translocation.     

A fluorescence in situ hybridization study of complex t(9;22) in two chronic myelocytic leukemia cases with a masked Philadelphia chromosome

The t(9;22)(q34;q11) is evident in more than 90% of patients with chronic myelocytic leukemia (CML) and gives rise to the Philadelphia chromosome (Ph). Approximately 5%-10% of CML patients show variant translocations involving other chromosomes in addition to chromosomes 9 and 22. In some variant translocations, additional material is transferred on der(22), resulting in a masked Ph chromosome. In this paper, we report two apparently Ph-negative (Ph-) CML cases showing a t(7;9;22)(q22;q34;q11) and a t(8;9;22)(q12;q34;q11), respectively. A detailed molecular cytogenetic characterization was performed by fluorescence in situ hybridization (FISH), which disclosed the presence of the 5'BCR/3'ABL fusion gene on the der(7) and der(8) chromosomes, respectively. Derivative (22) appeared as a masked Ph chromosome in both cases. FISH analysis with appropriate BAC/PAC clones allowed us to precisely characterize the complex chromosomal rearrangements that were not detected by conventional cytogenetic analysis.     

Transposition of breakpoint cluster region (3' bcr) in CML cells with variant Philadelphia translocations

A probe derived from the 3' end of the CML breakpoint cluster region (bcr) was localized in chronic myelocytic leukemia (CML) cases with complex Philadelphia translocations, [t(8;9;22)(q13;q34;q11) and t(12;9;22)(p11;q34;q11)], and with "masked" Ph chromosomes, [t(9;5;22)(q34;q31;q11) and t(9;22)(q22;q34)], by a chromosomal in situ hybridization technique. In some cases, the 3' bcr rearrangements in the DNA were examined with Southern blot analysis. In each case, a significant accumulation of grains hybridized to the 3' bcr probe was observed at chromosomal segments derived from the long arm of a chromosome #22. In some cases, the accumulation of grains was detected on both translocated segments derived from the 22q and terminal portions of Ph chromosomes; Southern blot analysis revealed that breakage in chromosome #22 occurred within DNA sequences of the 3' bcr probe involved in the Ph translocations. In a CML cell line, K562, no accumulation of grains hybridized to the 3' bcr probe was detected, except at 22q11 of the normal chromosomes #22; Southern blot analysis of this cell line revealed that the 3' bcr sequences were missing. Thus, the data presented here suggest a complexity in the formation of "masked" Ph chromosomes.     

Chromosomal in situ hybridization and Southern blot analyses using c-abl, c-sis, or bcr probe in chronic myelogenous leukemia cells with variant Philadelphia translocations

The Philadelphia (Ph) chromosome is a cytogenetic hallmark of chronic myelogenous leukemia (CML). Whereas the majority of Ph-positive CML patients show the standard Ph translocation involving chromosomes 9 and 22, t(9;22)(q34;q11), the minority of cases exhibit a variant type of Ph translocation involving these two and other chromosomes (complex type) or those involving #22 and chromosomes other than #9 (simple type). To get an insight into the nature of variant Ph translocations and the process of their formation, we examined the localization of the c-abl and c-sis oncogenes and the breakpoint cluster region (bcr) gene by chromosomal in situ hybridization in ten variant Ph translocations of CML including five simple and five complex ones as initially interpreted. In situ hybridization showed that c-abl localized to band 9q34 and c-sis localized to band 22q12-q13 were translocated on the Ph and on one of the rearranged chromosomes other than #9, respectively, in all the variant translocations examined. On the other hand, bcr localized to band 22q11 was translocated on various chromosomes but mostly on chromosome 9. Parallel Southern blot analyses on DNA from leukemic cells of five patients including two with simple translocations and three with complex ones revealed rearrangements of bcr with breakpoints occurring mostly in a 5' portion of 5.8-kb BamHI/BglII sequences, which are quite similar to those detected so far in CML cases with the standard Ph translocation. The present findings strongly suggest that variant Ph translocations of CML are all complex, and some of them are formed stepwisely from the standard translocation.     

Chromosome 22 breakpoints in variant Philadelphia translocations and Philadelphia-negative chronic myeloid leukemia

The standard t(9;22)(q34;q11) found in Philadelphia (Ph) chromosome positive chronic myeloid leukemia (CML) involves a highly restricted (5.8 kb) chromosome 22 breakpoint cluster region (bcr), which results in the formation of a chimeric gene comprising exons from the 5' end of bcr and protooncogene c-abl coding sequences from chromosome 9. In a survey of 21 patients with hematologic and clinical features of CML we detected rearrangement of the chromosome 22 bcr by gene probe analysis in all cases, including 16 with a standard t(9;22), two with variant Ph translocations [t(10;22)(q26;q11);t(11;22)(p15;q11)], one with a complex Ph translocation [t(9;11;22)(q34;q13;q11)], one with a complex translocation and a masked Ph[t(9;14;22) (q34;q24;q11)], and one Ph-negative case with a t(1;9)(p32;q34). These observations further substantiate the suggestion that, despite karyotypic heterogeneity, a common underlying molecular lesion, the bcr-abl gene chimera, is involved in the disease pathogenesis of CML.     

"Masked" Ph1 chromosome in a complex three-way translocation

A patient with myelofibrosis was found to have a 46,XX,del(1)(q24),del(11)(p11),-22,+mar karyotype in unstimulated peripheral blood (PB) and spleen cells. On detailed cytogenetic examination it was determined that this patient had an apparently "masked" Ph1 chromosome contained in a complex three-way translocation. Since phytohemagglutinin (PHA)-stimulated PB and spleen cells were essentially normal, the masked Ph1 chromosome was assumed to be an acquired cytogenetic abnormality. The portion missing from the masked Ph1 chromosome was apparently translocated onto del(1). Thus, the detailed karyotype was 46,XX,t(1;11;22)(q24;p11;q11 or q12),t(1;22)(q24;q11 or q12). This complex rearrangement was present primarily in cells belonging to the granulocyte-macrophage cell lineage, whereas E-rosetting cells, and presumably T lymphocytes, had normal karyotypes.     

Breakpoint cluster region rearrangements in chronic myelogenous leukemia with a masked Philadelphia chromosome

Cytogenetic and molecular analyses were performed in a case of chronic myelogenous leukemia. The cytogenetic study revealed that the leukemic cells of this patient contained a three-way translocation involving chromosomes #5, #9, and #22, resulting in a masked Philadelphia chromosome; the karyotype of the leukemic cells was 46,XY,t(5;22;9)(q31;q11;q34). Southern blot analysis of leukemic cell DNA was performed using a 1.1 kb HindIII-EcoRI breakpoint cluster region (bcr) probe. The results showed that BglII digested DNA showed two abnormal bcr fragments (i.e., 5.2 kb and 2.7 kb) in contrast to the results with DNA from two patients with a standard Ph chromosome [t(9;22)(q34;q11)] who showed one normal 5.0-kb bcr fragment in addition to altered fragments of about 4.3 kb or 4.0 kb. Bam HI digests of DNA from the leukemic cells of the patient with the masked Ph chromosome showed two bands (3.3 kb and 6.5 kb), whereas, DNA from the two patients with standard Ph translocations showed only a 3.3-kb bcr fragment. The results indicate that the molecular events in a masked Ph affect the bcr locus in a manner similar to that seen in standard Ph chromosomes.     

High resolution banding of chronic myeloid leukemia chromosomes

Bone marrow aspirates from 29 patients with chronic myeloid leukemia were studied using the methotrexate synchronization culture method. Successful cytogenetic preparations exhibiting long and well banded chromosomes were obtained from all of them. The standard t(9;22) was seen in 23 patients, four had variant translocations, and two were Ph-negative. Of the four patients with variant translocations, one had a simple translocation in which the missing segment of chromosome #22 was translocated onto the short arm of chromosome #9. The remaining three patients had complex translocations. The first involved chromosomes #11, #19, and #22, the second involved chromosomes #9, #11, and #22, and the third involved chromosomes #9, #14, and #22. Karyotypic abnormalities in addition to the Ph chromosome were seen in four patients: in three these changes developed during the chronic phase and in one during the blastic phase. Using Q-, R-, and G-banding techniques, we found that the breakpoint on chromosome #22 is just below the centromere, namely in band 22q11.2 and on chromosome #9 in band 9q34.1. The standard translocation, therefore, can be written as t(9;22)(q34.1;q11.2). Furthermore, the breakpoint on 22q appeared to be identical in all cases with standard as well as the variant translocations. Our results show that the methotrexate synchronization method permits consistent high resolution banding of CML chromosomes, and support the concept that there is no difference in the amount of material translocated from 22q in different patients.     

The karyotype of Philadelphia chromosome-negative, bcr rearrangement-positive chronic myeloid leukemia

Philadelphia (Ph) chromosome negative chronic myeloid leukemia (CML) can be distinguished from clinically similar disorders on the basis of the presence of rearrangement of the breakpoint cluster region (bcr) of chromosome 22. We have identified six patients with Ph-negative CML, each with bcr rearrangement. Apparently normal karyotypes were observed in two cases, and a third contained a rearrangement that did not appear to involve chromosomes 9 or 22. The other three cases had translocations involving chromosome band 9q34 but no case contained the common derivative chromosome 9pter----9q34::22q11----22qter. One case appeared to contain either a deletion of an unrearranged bcr locus in approximately 50% of cells or duplication of rearranged bcr, both 5' and 3' of the chromosome 22 breakpoint. Considerable complexity exists in the types of genetic changes that can juxtapose bcr and the c-abl oncogene in CML. Based on the molecular and cytogenetic analyses of these and other cases described in the literature, we conclude that most cases of true Ph-negative CML arise from submicroscopic genetic exchanges rather than masking of simple t(9;22)(q34;q11) translocations by secondary rearrangements.     

Two apparent Philadelphia chromosomes arising from translocations with different chromosomes in a patient with CML: 46,XY,t(7;22)(p22;q11),t(9;22)(q34;q11)

Chromosome studies on bone marrow cells and unstimulated peripheral lymphocytes from a patient with chronic myelogenous leukemia revealed the presence in all cells of two apparent Philadelphia chromosomes: one resulting from the classical translocation with a chromosome #9, and the other arising from a translocation between chromosomes #22 and #7. There was no normal chromosome #22. Some of the cells also had an i(17q), indicative of blast crisis. Repeated chromosome studies at different times during the course of the disease revealed the evolution of additional karyotypic changes. All cells from later samples had an extra #8; some of these cells had a third Philadelphia chromosome, whereas, others had a second Y chromosome. Although a few normal cells were seen in PHA-stimulated lymphocyte cultures, indicating that the patient has a normal constitutional karyotype, most of the cells had a karyotype identical to that found in unstimulated cultures. This unusual karyotype, 46,XY,t(7;22)(p22;q11),t(9;22)(q34;q11), represents the first case in which two apparent Philadelphia chromosomes are present in the leukemic cells from a patient in the absence of a normal #22 chromosome.     

A unique, complex variant philadelphia chromosome translocation in a patient with typical chronic myelogenous leukemia

The Philadelphia (Ph) chromosome [der(22) t(9;22)(q34;q11)] is the characteristic chromosomal abnormality found in chronic myelogenous leukemia (CML). This chromosome has been reported in patients with other chromosomal abnormalities. In this study, we describe a patient with hematologically typical chronic-phase CML with an unusual and complex translocation involving chromosomes 9, 11, and 22. These complex translocations were identified by G-banded conventional cytogenetics and confirmed by fluorescence in situ hybridization (FISH) using whole chromosome painting probes (wcp). To the best of our knowledge, these are unique translocations involving the short and the long arms of chromosome 9 in 4 different translocations with the short arm of chromosome 11 and the long arm of chromosome 22.     

Philadelphia chromosome (Ph) positive chronic myelocytic leukemia (CML): frequency of additional findings

This article documents the cytogenetic findings in 79 patients with typical Ph-positive chronic myelocytic leukemia (CML). Direct preparations of bone marrow and/or peripheral blood of 46 males and 33 females were studied with different banding techniques. Seventy patients were studied during chronic phase. Three (4.3%) had unusual or complex translocations: t(6;22)(p21;q11), t(8;12;9;22)(p21;q21;q34;q11), and t(9;11;22)(q34;q13;q11). One (1.4%) had a +Ph, 1 (1.4%) had a +8, 1 (1.4%) had a del(3)(p13,p23), and 4 of 30 males (13.3%) showed loss of Y chromosome. Five of 8 cases studied during blast crisis had additional abnormalities. The +8 occurred in 4 cases, +10 and +19 each in 3 cases, +6, + 9q+, and +13 each in 2 cases, and +5, +11, +14, +21, +Ph, i(17q), dic(1;9), and structural abnormalities of chromosomes #1, #5, #12, and #13 each in 1 case. Two cases studied in blast crisis alone had complex translocations leading to the Ph. Because it cannot be ruled out that these translocations are secondary, they were not included in the calculation of the frequency of atypical translocations.     

Chromosome abnormalities in leukemia and lymphoma

Nonrandom chromosome changes have been identified in a number of malignant human tumors. The leukemias are among the best studied malignant cells and they provide the largest body of relevant cytogenetic data. In chronic myeloid leukemia, a reasonably consistent translocation [t(9;22) (q34;q11)] is observed in 93 percent of all Ph1 positive patients. In the other patients, translocations are either two-way, involving No. 22 with some other chromosome or complex translocations involving Nos. 9 and 22 and another chromosome. In acute nonlymphocytic leukemia, two translocations are each specifically associated with leukemic cells arrested at two different stages of maturation. One of these, t(8;21)(q22;q22), is found mainly in patients with acute myeloblastic leukemia with maturation (AML-M2). The other, t(15;17)(q22?;q21?), is seen only in patients with acute promyelocytic leukemia (APL-M3). Various translocations have been observed in B-cell acute lymphoblastic leukemia or in Burkitt lymphoma. The most common is t(8;14)(q24;q32), but variants of this, namely t(2;8)(p13?;q24) and t(8;22)(q24;q11), have also been observed; in all of these, the consistent change involves 8q24. The various immunoglobulin loci are located on chromosomes 2, 14, and 22 in the same chromosome band affected by the translocations in B-cell leukemia. These translocations may occur randomly. If a specific translocation provides a particular cell type with a growth advantage, then selection could act to cause the proliferation of this aneuploid cell line vis-a-vis cells with a normal karyotype. In this view, the chromosome change could be the fundamental event leading to the leukemic transformation of an otherwise normal cell. The challenge for the future is to define the genes located at the sites of consistent translocations in myeloid leukemias and to determine the alterations in gene function that are associated with the translocation.     

Variant Ph translocations in chronic myeloid leukemia

Variant translocations were found in eight of 142 consecutive patients with Ph-positive, chronic myeloid leukemia encountered in our laboratory during the last decade. Two patients had simple, two-way variant translocations: t(17;22)(p13;q11) and t(16;22)(q24;q11). Both of these patients had an additional translocation involving chromosomes #9: t(7;9)(q22;q34) and t(9;17)(q34;q21), respectively. Complex variant translocations were found in four cases: t(2;9;22)(p23q12;q34;q11), t(3;9;22)(p21;q34;q11), t(9;12;22)(q34;q13;q11q13), and t(13;17;22)(p11;p11q21;q11). In two cases, the only discernable cytogenetic aberration was del(22)(q11). A review of the chromosomal breakpoints involved in this series and in 185 cases of variant Ph translocations previously reported in the literature reveals that a disproportionately large number of breakpoints are located in light-staining regions of G-banded chromosomes. Furthermore, the breakpoints in simple variant translocations are more often located in terminal chromosomal regions, whereas, the breakpoints in complex translocations typically affect nonterminal bands. No obvious correlation was detected between variant Ph translocation breakpoints and either fragile sites, oncogene locations, or consistent chromosome breakpoints in other malignancies.     

Chronic myeloid leukemia with variation of translocation at (Ph) [ins (22;9) (q11;q21q34)]: a case report

Chronic myeloid leukemia (CML) is most frequently observed in middle-aged individuals. In most patients, normal marrow cells are replaced by cells with an abnormal G-group chromosome, the Philadelphia (Ph) chromosome. The Ph chromosome that is characterized by the translocation (9;22) (q34;q11) is noted in 90-95% of patients diagnosed with CML. Studies have also shown that CML can be associated with various other cytogenetic abnormalities, with 5-10% of these cases showing complex translocation involving another chromosome in addition to the Ph chromosome. Here, we report the case of a Ph(+) CML patient with an inserted karyotype who presented clinically in the chronic phase but with atypical features. This case highlights the significance of cytogenetic abnormalities on the prognosis in CML.     

A new translocation involving chromosomes 8 and 9 in a Philadelphia-negative chronic myelogenous leukemia

A new case is presented displaying typical features of the stable phase of chronic myelogenous leukemia (CML), with a complex translocation involving chromosomes 8q and 9q. Cytogenetic evaluation revealed an abnormal karyotype, 46,XY,t(8;9)(q22;q34). Both chromosomes 22 were found to be cytogenetically normal. After molecular evaluation the cytogenetic diagnosis was revised to 46,XY,t(8;9;22)(q22;q34;q11). The importance of the chimeric abl/bcr gene fusion product in the pathogenesis of CML is suggested as a characteristic feature, even in some patients with a so-called Philadelphia (Ph) negative CML. Utilization of molecular probes in the evaluation of such cases must become a routine diagnostic procedure. Our patient received the potential benefit of Ph-positive directed therapy because of the present approach.     

Variant Ph1 translocations in CML and their incidence,including two cases with sequential lymphoid and myeloid crises

A serial cytogenetic study of 110 cases of chronic myelogenous leukemia (CML) has been performed with G- and/or Q-banding techniques with the following results. (1) Seven out of the 110 cases were karyotypically normal. (2) A variant Ph¹ translocation was observed in three cases. In one case, the leukemic cells contained two reciprocal translocations, i.e., a t(3;9) (q21;q34) and a t(17;22)(q21;q11); therefore, a Ph¹ chromosome was masked by a translocation of the deleted material from the 17q onto the band q11 of the long arm of a chromosome No. 22. In the second case, a variant Ph¹ translocation involved chromosomes No. 9, 20, and 22, resulting in a karyotype interpreted as 46,XX,t(9q+;20q+;22q?); in this rearrangement, one of the segments, i.e., 9q31 or 9q33, seemed to be interstitially deleted and inserted into the interstitial region (q11) of a chromosome No. 20 and the 22q11→qter was translocated onto the 9q. This is the first case in which chromosome No. 20 was involved in a variant Ph¹ translocation. In the third case, the karyotype of leukemic cells was interpreted as 46,XY,t(5;9;22)(q13;q34;q11). (3) The frequency of Ph¹-negative CML and that of Ph¹-positive CML with various types of Ph¹ translocation from 15 studies reported as series of 25 or more cases, including the present study, have been tabulated. The incidence of a variant Ph¹ translocation was 4.1% (42/1027 cases of Ph¹-positive CML); of the 42, 13 were of a simple type and 29 of a complex type. (4) In one case of the present study, a masked Ph¹ by a translocation of material onto the short arm of the 22q? was observed in the blastic crisis but not in the chronic phase. From the present study and a review of the published cases, it appears that the incidence of such a “masked” Ph¹, which cannot be detected by conventional Giemsa staining, is less than 0.6% in CML cases. (5) The first and the second cases with a variant Ph¹ translocation mentioned above developed a myeloid blastic crisis after the induction of remission of a lymphoid blastic crisis. For the present, it is unclear whether the occurrence of such blast cells in the two cases and the cytogenetic findings are coincidental. However, the evidence supports the notion of “lymphoid-myeloid” multipotentiality of certain leukemic cells.     

Two new chromosomal abnormalities in chronic myelogenous leukemia 46,XY,t(9;15;22)(q34;q22;q11) and 46,XY,t(6;9;12;22)(p21;q34;q24;q11)

Two new variant cases of chronic myelogenous leukemia (CML) are presented. The first case is a 19-year-old male with a 46,XY,t(9;15;22)(q34;q22;q11) karyotype. The second case is a 75-year-old man with a 46,XY,t(6;9;12;22)(p21;q34;q24;q11) karyotype. In both cases, the prognosis was no different from those cases of CML with the standard t(9;22) as the only abnormality. We recommend that all unusual translocations be reported.