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B-cell receptor signature in mantle cell lymphoma
Validations of BCR signature
To verify whether the BCR signature maintained its prognostic impact in an independent set of patients, we used the gene expression data of MCL LN biopsies report- ed by Saba et al.30 Also in this different setting, a high expression of the 6-gene signature, as in the context of BCRhigh cases, identified an MCL patient subset with infe- rior PFS (P=0.049) (Online Supplementary Figure S6).
In another set of analyses, by taking advantage of our 27 MCL cases with GEP data available, we correlated our BCR signature with other MCL signatures with proven clinical impact.30,38 As reported in Online Supplementary Figure S7A, the BCR signature reported in Saba et al.30 divided MCL cases in two groups that corresponded exactly to our BCR definition (Online Supplementary Figure S7B).30 Similarly, the 17 genes of the proliferation signa- ture reported by Scott et al.38 split our MCL cases in 3 dif- ferent groups resembling the 3 different groups originally defined (Online Supplementary Figure S8A). In this context, the shortest PFS and OS intervals were observed in the third group characterized by a higher expression of genes related to proliferation and a BCRhigh phenotype in keeping with our findings (Online Supplementary Figure S8A-C).
Discussion
In this study, we demonstrated that a BCR-derived sig- nature based on the differential expression of six genes correlated with shorter PFS intervals in the context of a Phase III prospective clinical trial (FIL-MCL-0208) for younger MCL patients receiving R-CHOP induction, fol- lowed by high-dose cytarabine and autologous stem cell transplantation (clinicaltrials.gov identifier: 023541313).32
Notably, the BCR-related 6-gene signature reported here was able to identify an MCL subset with shorter PFS inter- vals also in the context of an external independent MCL cohort homogenously treated with different schedules.30 On the other hand, when the signature described by Saba et al.30 and Scott et al.38 was applied to our MCL cases, the patient subsets with the worse prognosis turned out to be particularly enriched in BCRhigh cases, even though these signatures did not include any gene from our signature. Therefore, although composed of genes located upstream of the BCR machinery, our signature was able to identify cases with an active BCR pathway as defined by other sig- natures.30 In this regard, however, experiments with pri- mary MCL cases and/or MCL cell lines combining BCR stimulation with the use of specific BCR inhibitors should be performed to investigate the contribution of the 6-gene signatures described here to the actual activation of the BCR pathway.
Again in agreement with this line of reasoning, BCRhigh samples presented a significant upregulation of PAX5 (see GEP data in Online Supplementary Table S3), a gene whose product is known to prevent plasma cell differentiation thus preserving the capacity to respond to antigen- induced activation and proliferation.39 Taken together these data corroborate recent findings of ongoing active BCR signaling in MCL cell in vivo,29,30 and further underline the role of antigen stimulation in the ontogeny of MCL, as suggested by the skewed IGVH gene repertoire found in MCL cells.40
In order to discriminate between BCRlow and BCRhigh MCL samples, we developed a DT model based on the
expression of the selected six genes.28 This DT model was applied in an independent cohort of PB samples and then to a further series of FFPE LN samples, thus demonstrating that two MCL subsets with different expression levels of BCR-related genes could also be recognized in the LN compartment, mirroring PB. Taken together, by combin- ing data from the PB and LN compartments, MCL cases classified as BCRhigh showed higher LDH levels and shorter PFS with respect to BCRlow patients, suggesting that activa- tion of BCR signaling drives tumor proliferation and deter- mines clinical outcome of MCL patients, which is in keep- ing with recent findings.30
By combining the predictive capacity of the 6-gene BCR signature with the Ki-67 index, we identified a particularly unfavorable category (BCRhigh and high Ki-67) with a sub- stantially shorter PFS and OS than the other groups. Consistently, the BCRhigh signature turned out to be an independent prognosticator along with the high-risk MIPI-c category for short PFS by multivariate analysis. There is no indication that the validity of the model may be affected by the different recruitment site (PB vs. LN), or by different sample storage (frozen vs. FFPE) because the main clinical parameters were equally distributed between the different series (PB/frozen vs. LN/FFPE) (R Bomben et al., 2018, unpublished observation). In this regard, an important feature of this model/assay is its applicabili- ty to both PB and LN FFPE samples, having, therefore, the chance to combine results of qRT-PCR with Ki-67 staining in all the cases.
Our data underscore the increasing importance of BCR- related genes in the pathogenesis and development of MCL, further underlined by the clinical significance of drugs specifically targeting genes belonging to this path- way. In particular, therapeutic targeting of BTK41 can be rationally exploited in lymphoid malignancies that have been proved to be dependent on an antigen-dependent BCR-mediated active signaling. However, despite the rel- atively high response rate to single agent ibrutinib in relapsed/refractory MCL, it remained unclear as to why some patients showed clear responses, while others received little therapeutic benefit.31,42 The BCR-related sig- nature described here may provide insights into molecular factors that explain the divergent responses of MCL patients to ibrutinib, although other causes of primary resistance might be related to gene mutations in the other pathways, e.g. NF-κB pathway and epigenetic modifiers, as recently reported.43,44
In conclusion, in the present study we developed a sur- vival model for patients with MCL composed of six genes (AKT3, BTK, CD79B, PIK3CD, SYK, BCL2) whose expres- sion can easily be investigated by qRT-PCR and also in FFPE specimens. The signature was associated with a poor clinical response in the context of a high-dose chemo- immunotherapy regimen, and might, therefore, be con- sidered for validation and application in future clinical tri- als.
Acknowledgments
The authors would like to thank Progetto Giovani Ricercatori GR-2011-02347441, GR-2009-1475467, and GR-2011- 02351370, Ministero della Salute, Rome, Italy; Progetto Ricerca Finalizzata RF-2009-1469205, and RF-2010-2307262, Ministero della Salute, Rome, Italy; Associazione Italiana contro le Leucemie, linfomi e mielomi (AIL), Venezia Section, Pramaggiore Group, Italy; Associazione Italiana Ricerca Cancro
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