Low Bax/Bcl-2 Ratio and NOTCH1 Mutations Represent Powerful and Synergistic Adverse Prognostic Factors within Trisomy 12 Chronic Lymphocytic Leukemia (CLL)

Trisomy 12 is the third most common cytogenetic abnormality in CLL with several distinguishing features including abormal morphology, high prevalence of NOTCH1 mutations (NOTCH1 M) and increased expression of the alpha-integrins CD11a and CD49d (Balatti, 2012; Zucchetto, 2013). Trisomy 12 marks a disease subset characterized by high rates of cell proliferation, disease progression and Richter syndrome transformation (Rossi, 2013). Noteworthy, NOTCH1 M implicate constitutive activation of NOTCH1 signaling which triggers apoptosis resistance and increased survival of CLL cells (Rosati, 2009). Moreover, bax/bcl-2 ratio (bax/bcl-2), marker of apoptosis, predicts chemoresistance and progressive disease in CLL (Del Principe, 2016). The today availability in clinical use of venetoclax (ABT-199), a potent oral anti-bcl-2 peptidomimetic (Seymour, 2014), prompted us to analyze the real impact of the apoptosis (bax/bcl-2) on trisomy 12 CLL prognosis. The primary aims of our research were: 1) to demonstrate a significant association between NOTCH1 M and bax/bcl-2; 2) to correlate NOTCH1 M and bax/bcl-2 with biological and clinical prognosticators; 3) to determine progression-free survival (PFS) and overall survival (OS) upon NOTCH1 M and bax/bcl-2; 4) to evaluate NOTCH1 M and bax/bcl-2 as independent prognostic factors. Therefore we investigated 653 CLL patients, median age 66 years, 363 males and 290 females. Using the 5% cutoff for the FISH cytogenetic abnormalities, 140 (21.4%) cases were classified as trisomy 12 CLL. Bax/bcl-2 was calculated by flow cytometry, dividing mean florescence intensity (MFI) of bax by MFI of bcl-2 on CLL cells. The threshold was set at the median value >1.5 (range 0.32-5.30). The presence of NOTCH1 M was investigated with ARMS PCR for c.75447545delCT and by Sanger sequencing of NOTCH1 exon 34. CD49d was >20% in almost all trisomy 12 pts (116/140; 83%). Forty-five patients were NOTCH1 M (32.1%) and 45 were bax/bcl-2 positive (32.1%). There was a very strong correlation between bax/bcl-2 <1.5 and NOTCH1 M (42/45; p<0.0001), corroborating the close dependence of the lacking apoptosis on NOTCH1 M. Both lower bax/bcl-2 and NOTCH1 M were significantly associated with lymphocyte doubling time <12 months (p=0.0077 and p=0.0006), with beta-₂ microglobulin >2.2 mg/dl (p=0.0001 and p=0.0007) and LDH >300 U/L (p=0.004 and p=0.00001), thus confirming their significant association both with a high proliferative rate and a high tumor burden. Strong correlations were found between NOTCH1 M or bax/bcl-2 <1.5 and IGHV unmutated status (p<0.0001 and p=0.0005) or ZAP-70 >30% (p<0.0001 and p=0.00002). With regard to clinical outcome, significant shorter PFS and OS were observed in patients with NOTCH1 M vs NOTCH1 wild type [WT] (0% vs 27% at 10 years, p=0.001 and 26% vs 78% at 14 years, p=0.005) or lower bax/bcl-2 (9% vs 38% at 10 years, p=0.0007 and 45% vs 89% at 14 years, p=0.002). Interestingly, NOTCH1 M and bax/bcl-2 showed synergistic prognostic properties, since NOTCH1 M and bax/bcl-2 <1.5 identified a trisomy 12 CLL subset at worst prognosis with regard to PFS (0% vs 40% at 10 years, p=0.00004, Figure) and OS (20% vs 100% at 14 years, p=0.0001, Figure). Therefore, bax/bcl-2 and NOTCH1 M are powerful prognosticators, showing synergistic clinical effects. In multivariate analysis of OS, bax/bcl-2 (p=0.004) together with age (p=0.0006), CD49d (p=0.01) and IGHV status (p=0.03) was confirmed as an independent prognostic factor. In conclusion, the modern strategies to trigger apoptosis and block proliferation, such as venetoclax, may be very effective in this CLL subset, also taking into account that trisomy 12 CD49d+ CLL has abbreviated lymphocytosis with retention of CLL cells within tissues (Thompson, 2014) and consequent poor reduction of lymphadenopathy during treatment with ibrutinib.

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