Double Philadelphia Chromosomes- A Rare, Yet an Important Cytogenetic Phenomenon of Prognostic Significance in De Novo Acute Lymphoblastic Leukemia

Abstract

Presence of additional copies of Philadelphia chromosome (Ph) is characteristic of chronic myeloid leukemia in blast crisis, very rarely observed in de novo acute lymphoblastic leukemia (ALL). Ph positive (Ph+ve) ALL and CML in lymphoid blast crisis (CML-LBC) are biologically different with divergent clinical course. Double Ph+ve ALL has little data available as to its incidence and prognostic significance. We studied five cases of Ph+ve precursor B-cell ALL having an extra copy of Ph chromosome with regard to their clinical and laboratory features. An extensive review of literature was done on prognostic significance and molecular aspects of double Ph in ALL. The study confirms that double Ph was a rare phenomenon in precursor B-cell ALL. It is observed that molecular basis of double Ph positive ALL is less understood compared to CML in blast crisis. The study highlights fundamental role of cytogenetic and molecular studies in diagnosis and management of these patients. Long-term follow-up studies on a larger group of patients are required to understand the prognostic impact of extra Ph in Ph+ve ALL, which is usually resistant to standard chemotherapeutic regimen and often requiring bone marrow transplantation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12288-022-01525-1.

Background

Philadelphia chromosome was the first chromosomal abnormality to be found in leukemia in 1960s and is known to be present in 90–95% of CML cases [1]. The formation of a Ph chromosome generates an active chimeric BCR-ABL1 tyrosine kinase. It also occurs in ALL, the incidence of which increases with age, about 25% of adult ALL and 2–3% of childhood cases [2]. In acute leukemia the presence of the Ph is associated with a poor outcome.

Double Ph chromosomes have been reported in about 38% of CML during blast crisis [3]. Although BCR-ABL1 translocation is an initial disease-transforming event in CML, acquisition of additional chromosomal and molecular aberrations are responsible for progression of disease [4]. Double Ph+ve ALL has little data available as to its incidence and prognostic significance. Patients with double Ph chromosomes have less chances of attaining complete hematologic response, progress faster and have overall inferior survival outcome [5], requiring intensive chemotherapy along with Imatinib followed by allogeneic stem cell transplantation (SCT) after first remission [5]. With this background, we aimed to study a series of double Ph+ve ALL cases with review of literature to understand its prognostic significance.

Methods

This is a retrospective observational study from January 2016 to December 2019. First cytogenetic findings of patients diagnosed with precursor B-cell ALL and having Philadelphia chromosome were recorded. Later patients having double Ph chromosomes were noted and their clinical and laboratory findings were obtained from departmental records and case files.

The Cytogenetic procedure, FISH and PCR techniques were done according to our institution protocol (Details in ANNEXURE 1).

Results

Out of 1005 cases of ALL in four years, 74 cases of Ph+ve ALL were detected (0.5%). Five patients had double Ph (7%). Clinical and hematological findings are summarised in Table 1. Peripheral smear revealed no evidence of left shift and basophilia. The cells were large, with increased nuclear cytoplasmic ratio and inconspicuous nucleoli consistent with L1/ L2 morphology of FAB (French-American-British) classification. Immunophenotypically overall the blasts showed Positive expression for B-cell markers, with variable expression for CD20 (Table 1). Based on the morphology and flow cytometry results cases were diagnosed as precursor B-cell ALL.

Table 1.

Clinical and haematological features of patients with double Ph ALL

	Case 1	Case 2	Case 3	Case 4	Case 5
Age/gender	6 yr/Female	7 yr/ Male	12 yr/ Female	25 yr/ Female	28 yr/ Female
Lympadenopathy	Generalized	Submental & b/l inguinal	B/l cervical	Cervical and axillary	B/l cervical
Liver on ultrasound	18 cm	10 cm	14 cm	17.5 cm	14.8 cm
Spleen on ultrasound	14 cm	8.5 cm	14.5 cm	15 cm	14.5 cm
LDH	180 U/L	Normal	541 U/L	Normal	214 U/L
CSF infiltration	N/A	N/A	Nil	N/A	Nil
Hemoglobin	8.7 g/dL	6.8 g/dL	9.9 g/dL	9.5 g/dL	8.0 g/dL
TLC	54 × 109/L	4.4 × 109/L	2.2 × 109/L	3.6 × 109/L	5.84 × 109/L
Platelets	47 × 109/L	57 × 109/L	43 × 109/L	45 × 109/L	38 × 109/L
Peripheral Smear blasts	Blasts 56%	No blasts	Blasts 62%	No blasts	No blasts
BMA findings	Blasts 80%	Blasts 80%	Blasts 60%	Blasts 50%	Blasts 40%
Immunophenotyping	Positive for Tdt, HLA-DR, CD34, CD10, CD19, cCD79a, aberrant CD13. Negative for CD20	Positive for HLA-DR. Tdt, CD13, CD34, CD10, CD19, CD20 & cCD79a	Positive for Tdt, CD34, CD10, CD19, cCD79a & CD20	Positive for Tdt, CD19, CD10, cCD79a, Dim CD34. Negative for CD20	Positive for Tdt, CD34, CD10, CD19, CD20, cCD79a &, aberrant dimCD5

B/l: bilateral; TLC: Total leukocyte count; BMA: Bone marrow aspiration; FAB: French-American-British; N/A- Information not available

Cytogenetic and molecular findings are summarised in Table 2. Karyotypes of Case 2 and Case 5 with double Ph are shown in Figs. 1,  2 respectively. Fluorescent in-situ hybridization (FISH) showed a single red-blue-green fusion signal and two red-green fusion signals in 95% of the cells indicative of BCR-ABL1 translocation (Fig. 3). Treatment and clinical outcome of patients are summarised in Table 3. Post-induction phase, bone marrow examination showed both morphologic and cytogenetic remission in all the cases.

Table 3.

Treatment, overall survival of patients and their remission status

	Chemotherapeutic regimen	Follow-up period	Remission status
Case 1	MCP 841	36 months	In remission
Case 2	MCP 841	22 months	In remission
Case 3	MCP 841	53 months	In remission
Case 4	Refused treatment	3 months	died of disease
Case 5	BFM 95	18 months	In remission

Table 2.

Cytogenetic and molecular findings of patients with double Ph ALL

Case	KARYOTYPE	PCR	BCR-ABL1/ABL1 ratio at diagnosis
1	45,XX, + X,-7,t(9;22)(q34;q11.2),-15,-20, + der(22)t(9:22)(q34;q11.2)[5]	p190*	12.5%
2	49,XY,t(9;22)(q34;q11.2), + 19, + der(22)t(9;22)(q34;q11.2), + mar[3]/46,XY,t(9;22)(q34;q11.2)[2]/48,XY, + 21, + mar[2]/46,XY[10]	p190	5.67%
3	47,XX,t(9;22)(q34;q11.2), + der(22)t(9;22)(q34;q11.2)[8]	p190	8.838%
4	47,XX,t(9;22)(q34;q11.2), + der(22)t(9;22)(q34;q11.2)[2]/46,XX[4]	Not done	––––-
5	47,XX,t(9;22)(q34;q11.2),add(19)(p13), + der(22)t(9;22)(q34;q11.2)[3]/46,XX[3]	p190	1.13%

PCR: Polymerase chain reaction; BCR: Breakpoint cluster region; ABL1: ABL proto-oncogene1; *Minor BCR-ABL mRNA transcript

Fig. 1.

Karyotype: 47,XX,t(9;22)(q34;q11.2),add(19)(p13), + der(22)t(9;22)(q34;q11.2)[3]

Fig. 2.

Karyotype: 48,XY,t(9;22)(q34;q11.2), + der(22)t(9;22)(q34;q11.2), + mar[3]

Fig. 3.

Tri-colour FISH using probes for ASS1 gene (blue), ABL1 gene (red) and BCR gene (green) produced three fusion signals- one red/green/blue (positive for BCR/ASS1/ABL1 fusion) and two yellow (red/green- positive for BCR/ABL1 fusion). One red/blue and one green signals indicate normal homologue chromosomes 9 and 22

One adult patient (Case 4) refused chemotherapy and succumbed to the disease in 3 months after discharge from the hospital against medical advice.

Discussion

Ph positive ALL is found in 25% of adults and less than 5% of paediatric patients [2]. Although clinical presentation of Ph + ve precursor B-cell ALL (pre-B-ALL) mimics ALL without Ph chromosome, the former is more resistant to chemotherapy and associated with a shorter survival [7].

Double Ph chromosome is among the most common chromosomal changes observed in CML with blast crisis (38%) [3]. Double Ph + ve pre-B-ALL is also reported in several case reports worldwide [8–11]. The possible mechanism of double Ph chromosome is considered to be the result of non-dis-junction occurring during mitosis and endoduplication [8]. The role of double Ph chromosomes in disease progression is unclear. Perhaps the presence of additional copies of Ph leads to over-expression of BCR/ABL1 transcripts, which has also been reported in advanced disease stages.

Hyperdiploid karyotypes with double Ph along with other numerical abnormalities are more commonly encountered in CML than ALL. Over 500 cases of CML with double Ph and hyperdiploidy have been reported. In contrast only 66 cases of double Ph are reported [12]. Except one patient (Case 2) all other patients in our study had only an extra copy of Ph chromosome as an additional numerical abnormality.

Double Ph chromosomes are very rare in ALL. Our study showed double Ph chromosomes at an overall frequency of 7% in Ph+ve ALL, about 10% in paediatric and 5% in adult Ph + ve ALL. Earlier studies have reported 13–17% incidence of double Ph in Ph + ve ALL [9, 13–15]. These discordant results could be attributed to diagnostic dilemma that may arise between CML-LBC and double Ph+ve ALL, since the two entities have often overlapping features.

A constellation of clinical, hematological, cytogenetic and molecular features might help in differentiating between the two entities. Clinically an acute presentation without long standing antecedent blood disorder favors ALL. While splenomegaly may be seen in one third of ALL patients, massive splenomegaly is more characteristic of CML. Lab investigations suggestive of CML includes peripheral smear picture of ‘Myelocyte buldge’ with left shift, maturing granulocyte precursors and basophilia. The Ph+ ve clone is restricted o lymphoid series in patients with ALL and thus shows normal karyotype in all metaphases post successful induction. Most common additional cytogenetic abnormalities suggestive of ALL includes hyperdiploidy, t(12;21), t(1;19), t(4;11), deletions of 12p, 9p, 6q, whereas CML with blast crisis is usually associated with trisomies of 8, 19, 21, isochromosome 17 and inversion 3q [16]. RT-PCR showing a minor transcript (p190) favors ALL but the major transcript may be seen in 40% adults and 10% paediatric cases [17].

Several studies have highlighted the role of additional chromosomal abnormalities and genomic instability in clonal evolution of CML to an aggressive clinical phenotype, owing to high proliferation and survival of progenitors, independent of BCR-ABL1. [4, 18–20].

Vinhas R et al.hypothesized that as atypical shorter BCR-ABL1 transcripts induce an aggressive clinical phenotype due to the lack of important regulatory BCR sequences [18] Hence the importance of identifying the type of BCR-ABL1 transcripts and their quantification.

Wassman et al.in their study of 23 double Ph + ve ALL cases out of 64 relapsed or refractory Ph + ve ALL treated with imatinib, double Ph + ve ALL patients were shown to have lower probability of complete haematological response (13% vs 39%; p = 0.04). Median TTP and OS were inferior to those in patients without double Ph (1.6 months versus 3.2 months [P 0.006], and 5.2 months versus 9.6 months [P 0.01], respectively) [21]. They found that pretreatment variables including BCR-ABL1 amplification (extra copies of Ph), may contribute to primary refractoriness to imatinib whereas point mutations of BCR-ABL1 are a cause of secondary resistance in Ph + ve ALL.

In another study which Included 1322 children, 30 patients had Ph including 5 children with double Ph [15]. Nearly all Ph + ve (96.7%) and Ph-ve (98.3%) patients achieved remission after induction therapy, yet event free survival outcome was significantly worse for Ph + ve patients compared with Ph-ve patients, with 4-year estimates of 20.1% and 75.8% (p, 0.0001). But five patients with double Ph did not appear to be different from that of the other Ph + ve patients.

The administration of imatinib in addition to induction and consolidation chemotherapy for Ph + ve B-ALL is the current standard therapy. Owing to the dismal outcome associated with chemotherapy, allogeneic SCT is considered to be the treatment of choice in adult Ph chromosome-positive B-ALL[5].

In conclusion, double Ph chromosomes is very rare in ALL. The theory of clonal evolution and genomic instability that has been widely studied in accelerated or blastic phase of CML may be extrapolated to double Ph + ve ALL, given the aggressive clinical phenotype and reduced overall survival of the latter. We suggest larger and long-term studies to provide further insight into this rare cytogenetic subgroup which apparently carries poor prognosis despite aggressive treatment with chemotherapy.

Supplementary Information

Below is the link to the electronic supplementary material.

Author’s contribution

Concepts: SS, P, LKR, PM, Design: SS, SAH, P, PM, LD, Definition of Intellectual content: SS, LD, Literature search: SS, SAH, P, Clinical studies: SAH, Experimental studies: SAH, Data acquisition: SS, RLK, LD, Data analysis: SS, SAH, P, PM, Statistical analysis: SAH, Manuscript preparation: SS, SAH, RLK, Manuscript editing: SS, SAH, P, RLK, PM, LD, Manuscript review: SS, P, RLK, PM, Guarantor: RLK.

Funding

The authors did not receive any funding for conducting this study.

Declarations

Conflict of interest

The authors have no conflicts of interest to declare that are relevant to the content of this article.

Ethical approval

Ethical approval was waived by the local Ethics Committee of Institution in view of retrospective nature of the study and all the procedures being performed were part of the routine care.

Consent for publication

Consent was felt not necessary as the study involves reported results of routine cytogenetic work. Also the study does not reveal identifiable personal data/ photographs/ sensitive images of study participants.