GDP and MDB equally contributed as first authors
Clinical significance of c.7544-7545 delCT NOTCH1 mutation in chronic lymphocytic leukaemia
Version of Record online: 20 NOV 2012
© 2012 Blackwell Publishing Ltd
British Journal of Haematology
Volume 160, Issue 3, pages 415–418, February 2013
How to Cite
Del Poeta, G., Dal Bo, M., Del Principe, M. I., Pozzo, F., Rossi, F. M., Zucchetto, A., Bomben, R., Degan, M., Rasi, S., Rossi, D., Bulian, P., Gaidano, G., Amadori, S. and Gattei, V. (2013), Clinical significance of c.7544-7545 delCT NOTCH1 mutation in chronic lymphocytic leukaemia. British Journal of Haematology, 160: 415–418. doi: 10.1111/bjh.12128
- Issue online: 17 JAN 2013
- Version of Record online: 20 NOV 2012
- CLL ;
- prognostic factors;
Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with highly variable clinical courses and survivals ranging from months to decades. In particular, a subset of CLL patients is known to experience rapidly progressive symptomatic disease requiring treatment. Given that only a fraction of these high risk CLL can be identified by screening for TP53 disruption by mutation/deletion, other genetic lesions have been thought allegedly implicated in the development of this aggressive CLL phenotype (Hallek et al, 2008).
Recently, it has been reported that activating mutations of NOTCH1 are recurrently associated with CLL, being identified in about 10% of CLL at diagnosis and with higher frequencies in chemorefractory CLL, CLL in advanced disease phases, and in CLL transformation to Richter Syndrome (Sportoletti et al, 2010; Fabbri et al, 2011; Puente et al, 2011; Wang et al, 2011; Rossi et al, 2012). Notably, in CLL, these mutations have tended to be mutually exclusive with genetic lesions involving TP53 (Fabbri et al, 2011). All NOTCH1 mutations disrupt the C-terminal PEST domain, this lesion is known to cause an accumulation of a stabilized NOTCH1 isoform in cellular models other than CLL (Paganin & Ferrando, 2011).
Clinically, in consecutive CLL series, NOTCH1 mutations have been identified as an independent predictor of overall survival, identifying a subset of high risk patients with an unfavourable prognosis similar to that associated with TP53 disruption (Rossi et al, 2012). As about 80% of NOTCH1 mutations in CLL are represented by a c.7544-7545delCT frameshift deletion (hereafter delCT NOTCH1 mutation) (Sportoletti et al, 2010; Fabbri et al, 2011; Puente et al, 2011), an ad-hoc amplification refractory mutation system polymerase chain reaction (ARMS PCR)-based strategy for the screening of this NOTCH1 alteration has been proposed to avoid the need for DNA sequencing (Rossi et al, 2012), although its validation as independent predictor is still to be fully elucidated.
The present study includes peripheral blood samples from 463 patients affected by CLL and recruited in a single centre (Department of Haematology, S. Eugenio Hospital, University of Tor Vergata, Rome, Italy). CLL diagnosis was made according to the current International Workshop on CLL-National Cancer Institute criteria (Hallek et al, 2008). Approval of the study was obtained from the Institutional Review Board, and informed consent was obtained in accordance with institutional requirements and the Declaration of Helsinki. The main clinical and biological features of CLL patients and methods employed in this study are reported in Appendix S1.
The presence of delCT NOTCH1 mutation was investigated by ARMS PCR (Fabbri et al, 2011; Rossi et al, 2012). This method showed that the delCT NOTCH1 mutation occurred in 41 out of 463 (8·9%) CLL at diagnosis (Fig 1A) and was significantly associated with all the investigated prognostic markers, utilized as dichotomous variables as reported (Hallek et al, 2008), including the markers of tumour burden modified Rai (mod-Rai) staging (P = 0·0031), β2-microglobulin (B2M, P = 0·0270), lymphocyte doubling time (LDT, P < 0·0001), and soluble CD23 (sCD23, P < 0·0001), and the biological markers IGHV status (P < 0·0001), ZAP70 (P < 0·0001), CD38 (P < 0·0001), and CD49d (P = 0·0053; Table SI). Regarding specific chromosomal abnormalities, a significant association was found between delCT NOTCH1 mutation and trisomy 12 (P = 0·0003), as reported previously (Balatti et al, 2012; Del Giudice et al, 2012; López et al, 2012), whereas delCT NOTCH1 mutation and del11q22-23 or del17p13 were not associated (P > 0·05 in both cases).
Overall survival (OS) data were available for all the 463 patients entering the study. Median follow-up was 76 months (range 3–271 months), with 56 deaths and 407 censored patients. All the investigated prognostic markers were also proven to maintain their prognostic impact as OS predictors in our CLL series (Table SII). When testing the prognostic relevance of delCT NOTCH1 mutation, a significantly shorter survival was found in patients carrying delCT NOTCH1 mutation compared to patients lacking delCT NOTCH1 mutation (P < 0·0001, Fig 1B and Table SII). Of note, the presence of delCT NOTCH1 mutation behaved as negative prognosticator also in the context of patients characterized by an unfavourable configuration of all the investigated prognosticators, either markers of tumour burden (Fig 1C), or biological markers (Fig 1D) and, in some instances, in the context of patient subgroups with a favourable configuration (Fig S1). Consistently, delCT NOTCH1 mutation retained its independent prognostic value in bivariate Cox regression analyses (Table SIII), without interaction effect with all the investigated prognosticators (Table SIV). The impact of delCT NOTCH1 mutation as an independent prognostic factor for OS was also demonstrated by multivariate Cox proportional hazards analysis in a 4-variable model that included IGHV status (unmutated IGHV vs. mutated IGHV), fluorescence in situ hybridization (FISH) subgroups (del11q22-23/del17p13 vs. tris12/del13q14/normal) and mod-Rai staging (intermediate/high vs. low mod-Rai stages, Table 1).
|Sample size||HR (95% CI)||P value|
|Intermediate and high Mod-Rai stage||413||5·48 (1·32–22·73)||0·0197|
|Unmutated IGHV status||4·13 (2·13–8·01)||<0·0001|
|Del11q22-23 and del17p13 FISH subgroups||2·26 (1·25–4·11)||0·0075|
|DelCT NOTCH1 mutation||2·47 (1·29–4·75)||0·0067|
Although an ARMS PCR-based qualitative assay was suitable for revealing only about 80% of total NOTCH1 mutations in CLL, the results here reported demonstrate the capability of delCT NOTCH1 mutation, assessed by this rapid and simple approach, to select a patient subset with worse prognosis in the context of bad prognosis subgroups, as indentified by the main tumour burden and biological markers for CLL. Moreover, delCT NOTCH1 mutation consistently behaved as independent prognosticator in a multivariate model that included the main molecular/cytogenetic prognostic markers in CLL (Hallek et al, 2008). The demonstration of the prognostic relevance of a simple qualitative ARMS PCR detectable delCT NOTCH1 mutation may be relevant for at least two reasons: (i) it strengthens the importance of NOTCH1 assessment for determining the clinical and biological features of CLL, (ii) operationally, it proposes the ARMS PCR strategy, presented here and previously (Fabbri et al, 2011; Rossi et al, 2012), as a simple and straightforward test for delCT NOTCH1 mutation screening also in the setting of routine diagnostic laboratories.
Altogether, delCT NOTCH1 mutation has been confirmed as a novel important prognostic parameter to be considered in determining prognostic scores for CLL, and in the perspective of identifying NOTCH1-mutated CLL patients for specific target therapies (Paganin & Ferrando, 2011). Larger cohorts of patients are needed to definitely validate delCT NOTCH1 mutation as independent prognosticator and to compare the clinical behaviour of CLL patients bearing delCT NOTCH1 mutation vs. other NOTCH1 mutations.
Supported in part by: Ministero della Salute (Ricerca Finalizzata I.R.C.C.S., ‘Alleanza Contro il Cancro’; Rete Nazionale Bio-Informatica Oncologica/RN-BIO; Progetto Giovani Ricercatori no. GR-2010-2317594, no GR-2009-1475467, and no GR-2008-1138053, Ministero della salute, Rome, Italy; Fondazione Internazionale di Ricerca in Medicina Sperimentale (FIRMS); Associazione Italiana contro le Leucemie, linfomi e mielomi (AIL), Venezia Section, Pramaggiore Group, Italy; Ricerca Scientifica Applicata, Regione Friuli Venezia Giulia (‘Linfonet’ Project), Trieste, Italy; the Associazione Italiana Ricerca Cancro (AIRC), Special Program Molecular Clinical Oncology, 5 × 1000, no 10007; Investigator Grant IG-8701, and MFAG-10327, Milan, Italy; ‘5 × 1000 Intramural Program’, Centro di Riferimento Oncologico, Aviano, Italy.
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Fig S1. Clinical significance of c.7544-7545delCT NOTCH1 mutation in the context of a favourable configuration of the main clinical and biological prognostic markers used in chronic lymphocytic leukaemia.
Table SI. Association between delCT NOTCH1 mutation and the main tumor burden and biological prognosticators for CLL.
Table SII. Univariate Cox regression analyses of OS.
Table SIII. Bivariate Cox regression analyses of OS.
Table SIV. Bivariate Cox regression model of OS with interaction term.
Appendix S1. Supplemental materials and methods
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