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B-cell receptor signature in mantle cell lymphoma
ate a reduced signature (Figure 2B). In this way, 18 probes corresponding to 15 genes, all over-expressed in BCRhigh cases were identified (Figure 2B). Among these genes, a subgroup of six genes (AKT3, BCL2, BTK, CD79B, PIK3CD, and SYK) was selected for further validations due to their direct involvement in the BCR pathway and/or the existence of drugs targeting the related pro- teins. A hierarchical cluster using only these six genes was able to discriminate patients belonging to the BCRlow or BCRhigh groups (Figure 2C).
Development of a qRT-PCR-based predictor for BCRlow and BCRhigh in MCL samples
By analyzing the expression levels of the selected six genes in the same 27 MCL PB samples by qRT-PCR approach, a strict correlation with GEP data was found (Online Supplementary Figure S3B). Moreover, the 27 MCL cases were randomly divided into a training set (17 cases; 8 BCRlow and 9 BCRhigh samples) and a validation set (10 cases; 6 BCRlow and 4 BCRhigh samples) to develop and test a decision tree (DT) model based on qRT-PCR data capa- ble of categorizing patients into one of the two categories. The DT model based on qRT-PCR data correctly classified 16 of 17 cases belonging to the training set and 10 of 10 cases of the validation cohort, and allowed the classifica- tion of 19 additional PB samples screened with qRT-PCR (9 BCRlow and 10 BCRhigh (Figure 2D and E and Online Supplementary Table S2).
Association between BCR categories and biological and clinical parameters
Collectively, the 6-gene signature was re-evaluated by setting up a validated qRT-PCR approach (see Online Supplementary Appendix and Online Supplementary Table S6) in PB samples from 46 MCL cases, 23 were identified as BCRlow and 23 as BCRhigh. By correlating the BCR groups with the available biological parameters, no association was found between the 6-gene signature and IGHV gene status (P=0.93) (Online Supplementary Table S2 and Online Supplementary Figure S4A), Ki-67 expression, white blood cells, hemoglobin, lymphocytes, platelets, and neutrophil count (data not shown). The only significant difference was between the BCR classification and lactate dehydrogenase (LDH) levels; BCRhigh cases showed a higher level of LDH
low
with respect to BCR MCL (416.6±191.6 vs. 292.2±127.4;
P=0.023) (Online Supplementary Figure S4B).
Clinically, MCL patients classified as BCRhigh experi-
enced shorter PFS with respect to BCRlow MCL cases
Table 2. Cox regression analysis on mantle cell lymphoma cases. Univariable
(median PFS: 21.6 months vs. not reached; P=0.0375) (Figure 3).
Application of the 6-gene signature to LN samples from MCL patients
To evaluate the capability of the 6-gene signature to identify different subgroups also in the context of MCL LN cases, we tested our qRT-PCR approach in a series of 43 LN samples preserved as FFPE LN specimens. Thirty- five (81%) out of 43 samples were amplifiable for all six genes, and using a DT model based on qRT-PCR values from FFPE, 23 cases were classified as BCRlow and 20 clas- sified as BCRhigh (Online Supplementary Table S2). Notably, for 6 out of 43 LN samples, a PB matched sample was available, and by comparing qRT-PCR results performed on PB samples and LN FFPE samples from these cases, a good concordance was overall observed, although FFPE samples generally amplified at higher Ct values (Online Supplementary Figure S5). Of note, 5 out of 6 these MCL cases were consistently classified. The misclassified case
Figure 3. BCR
clinical outcome. Kaplan-Meier curves obtained by comparing progression-free survival intervals of 23 BCRlow MCL cases with 23 BCRhigh MCL cases. The number of patients in each group is reported under relative categories; P refers to log- rank test.
mantle cell lymphoma (MCL) group is associated with a worse
Variable
BCR signature
BCR high MIPI-c
Low/intermediate
High/intermediate
High
HR (95%CI) P 2.81 (1.28-6.19) 0.01
1.5 (0.6-3.73) 0.384
1.41 (0.45-4.43) 0.557
3.46 (1.1-10.91) 0.034
high
Multivariable HR (95%CI)
3.48 (1.47-8.25)
4.17 (1.3-13.34)
P
0.005
0.016
Multivariable Cox regression analysis of progression-free survival was performed by including the 6-gene BCR categorization and the mantle cell lymphoma International Prognostic Index-combined (MIPI-c) score as defined by Hoster et al.13 HR: Hazard Ratio; CI: Confidence Interval; BCR: B-cell receptor.
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