AbstractThis report deals with a case of Sézary syndrome, a rare peripheral T-cell lymphoproliferative disorder, in which cytogenetic analysis performed during the disease transformation revealed the presence of a t(9;22) (q34;q11.2) translocation. Molecular analyses identified a new transcript, an e8a4 BCR-ABL fusion mRNA which could be responsible for the disease transformation.
Sézary syndrome (SS), characterized by a pruritic exfoliative erythroderma, lymphadenopathies and atypical peripheral blood T lymphocytes,1 is considered an advanced stage of mycosis fungoides (MF). Transformation to a large cell lymphoma has been reported to occur in 8–55% of cases2 usually with complex karyotypes.3 Though a single case has been reported,4 a Philadelphia chromosome (Ph) is unlikely to be found in cutaneous peripheral T-lineage lymphoproliferative disorders. A 74 year-old female with an MF diagnosed in June 1998 developed a SS in December 1998, with the appearance of lymph nodes and a lymphocyte count of 2 G/L. She presented 15% atypical small cells with no myelo-proliferative features. PCR analysis, according to the report of BIOMED-2,5 demonstrated a clonal rearrangement of T-cell receptor genes (TCRG) without clonal rearrangement of B-cell receptor genes. In 1999, the patient developed generalized lymphadenopathies, splenomegaly, had elevated LDH level (4508UI) and a leukocyte count of 263G/L with massive infiltration (93%) of tumoral cells, indicating a transformation to a large T-cell lymphoma.
Flow cytometry analysis of peripheral blood confirmed the T surface phenotype (CD3+, CD4+) of the tumoral population. No residual CD19 or CD20 positive cells could be identified.
The disease rapidly progressed and the patient died in October 1999. Chromosome studies, supplemented by fluorescence in situ hybridization (FISH) using the LSI BCR/ABL ES dual color translocation probe (Vysis Downer’s Grove, IL, USA) and by M-FISH experiments, were performed on tumoral cells when the patient was referred to our department for the treatment of T-cell lymphoma in August 1999. Complex abnormalities were identified with the following karyotype: 45,X,der(X) t(X;1)(p22;p?35),del(1)(p34),der(1)t(1;10)(q2?4;q2?5),der( 2)t(2;15)(q37;q22),der(4)ins(4;8)(q11;q12~22),der(6)t(6;9) (q11;p21),+8,− 9,t(9;22)(q34;q11.2).isht(9;22)(ABL+, BCR; ABL+,BCR+),der(10)t(1;10)(q25;q21)t(10;10)(p14;p1?2),d er(11)del(11)(p11)del(11)(q11), − 13,der(15)t(8;15) (q2?3;q21),der(19)t(6;9;19)(q13~21; q34; p13).ish der(19) (ABL+) . Interestingly, the patient presented structural changes involving chromosomes 1p, 2q, 6q and 19p, recurrently reported in SS.3 FISH analysis demonstrated the typical M-BCR pattern in 52% of interphase nuclei (Figure 1A). Reverse transcription (RT) and quantitative real-time polymerase chain reaction (RQ-PCR) were performed on the same sample according to Standardised EAC (European Against Cancer) protocols previously reported6 and expressed as BCR-ABL/ABL ratios according to the international scale now required.7 The transcripts detected in the patient using the M-primers were quantified at the BCR-ABL/ABL rate of 0.8% which was not in keeping with the cytogenetic results positive in 52% of interphase nuclei. The direct sequencing of these PCR products showed an e15a2 fusion transcript with no preservation of the open reading frame (ORF). A larger PCR analysis using a forward primer in the BCR gene (ENF4026) and a reverse primer (5’GCCCTCGTACAC-CTCCCCGTA3’) in the ABL exon a4 revealed an atypical e8a4 after direct sequencing (Figure 1B). However, unlike the e8a2 transcripts previously described,8–10 the direct junction between BCR exon e8 and ABL exon a4 led to the preservation of the ORF encompassing the whole oncogenic domains of the ABL tyrosine kinase. The quantification of the e8a4 was carried out using RQ-PCR with an e8 forward primer from BCR gene,10 the same reverse primer and a new probe (FAM 5’TCTACGTCTC-CTCCGAGAGC3’TAMRA) in ABL exon a4. With a serial 10-fold dilution series for e8a4 cDNA from the patient’s sample, the assay was found to be linear over at least 4 orders of magnitude (slope, −3.33; intercept, 45,3). The number of e8a4 copies in the non diluted sample was estimated to be 13,600 for 10,000 copies of ABL which agreed with data obtained by cytogenetic analysis and confirmed that the e8a4 must be the oncogenic fusion transcript. The detection of minority e15a2 spliceoforms suggests that the breakpoint could be located downstream of the Major breakpoint. This genomic rearrangement could then produce multiple spliceoforms without ORF and then undergo nonsense-mediated mRNA decay as previously described. Unfortunately, no retrospective studies of the rearrangement could be performed at diagnosis because of lack of available frozen material.
Since this observation occurred in 1999, before the imatinib era, the clinical outcome for patients bearing this unusual e8a4 transcript could not be predicted. It has to be underlined that the RQ-PCR method designed here is able to detect all known BCR-ABL variants and could represent as an interesting molecular strategy for patients with Ph cytogenetic but negative molecular analyses. Indeed, the previous case of Ph lymphoma did not show BCR-ABL rearrangement at molecular level.4 Therefore, it is possible that this case may correspond to this rare e8a4 transcript. Further studies of unusual BCR-ABL junctions and their associated diseases are required to gain further insight into their pathologic consequences and their sensitivity to new tyrosine kinase inhibitors.
we thank A. Bertholin, C. Ollagnier and B. Santa-Lucia for their technical assistance
- Funding: this work was supported by the Hospices Civils de Lyon.
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