New Molecular Assay for the Proliferation Signature in Mantle Cell Lymphoma Applicable to Formalin-Fixed Paraffin-Embedded Biopsies

Abstract

Purpose Mantle cell lymphoma is an aggressive B-cell neoplasm that displays heterogeneous outcomes after treatment. In 2003, the Lymphoma/Leukemia Molecular Profiling Project described a powerful biomarker-the proliferation signature-using gene expression in fresh frozen material. Herein, we describe the training and validation of a new assay that measures the proliferation signature in RNA derived from routinely available formalin-fixed paraffin-embedded (FFPE) biopsies. Methods Forty-seven FFPE biopsies were used to train an assay on the NanoString platform, using microarray gene expression data of matched fresh frozen biopsies as a gold standard. The locked assay was applied to pretreatment FFPE lymph node biopsies from an independent cohort of 110 patients uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. Seventeen biopsies were tested across three laboratories to assess assay reproducibility. Results The MCL35 assay, which contained a 17-gene proliferation signature, yielded gene expression of sufficient quality to assign an assay score and risk group in 108 (98%) of 110 archival FFPE biopsies. The MCL35 assay assigned patients to high-risk (26%), standard-risk (29%), and low-risk (45%) groups, with different lengths of overall survival (OS): a median of 1.1, 2.6, and 8.6 years, respectively (log-rank for trend, P < .001). In multivariable analysis, these risk groups and the Mantle Cell Lymphoma International Prognostic Index were independently associated with OS ( P < .001 for both variables). Concordance of risk assignment across the three independent laboratories was 100%. Conclusion The newly developed and validated MCL35 assay for FFPE biopsies uses the proliferation signature to define groups of patients with significantly different OS independent of the Mantle Cell Lymphoma International Prognostic Index. Importantly, the analytic and clinical validity of this assay defines it as a reliable biomarker to support risk-adapted clinical trials.

Fig 1.

Patient flow for the validation cohort. B+R, bendamustine plus rituximab; CLB, chlorambucil; MCL: mantle cell lymphoma; R, rituximab; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone; R+CP, rituximab plus cyclophosphamide and prednisone; R+CVP, rituximab plus cyclophosphamide, vincristine, and prednisone.

Fig 2.

Gene expression data in the training cohort. The correlation of the expression of individual genes to the proliferation signature calculated in Rosenwald et al plotted against the Wald test Z-score for overall survival (OS) for that gene. The data are from gene expression profiling of 80 fresh frozen biopsies from Rosenwald et al using Affymetrix U133 plus 2.0 microarrays. Gold and gray dots represent genes that were included in the NanoString gene set, which was used to select genes to replicate the proliferation signature. Gray dots represent genes that were selected for the MCL35 assay.

Fig 3.

The gene expression–based model for the proliferation signature in the training cohort. (A) The MLC35 assay is shown in the form of a heat map, with the 17 informative genes shown as rows and the 47 patient biopsies shown as columns. The three patient groups identified by the assay are shown below the heat map. (B) Kaplan-Meier curves of the overall survival (OS) of the three patient groups identified by the MCL35 assay. Outcome data were available for 44 of the 47 patients.

Fig 4.

The gene expression–based model for the proliferation signature in the validation cohort. (A) The MLC35 assay is shown in the form of a heat map, with the 17 informative genes shown as rows and the 108 patient biopsies shown as columns. The three patient groups identified by the assay are shown below the heat map. Shown below are the Ki-67 proliferation index (PI), pathologic characteristics, and the mantle cell lymphoma International Prognostic Index (MIPI). (B) Kaplan-Meier curves of the overall survival (OS) of the three patient groups in the validation cohort identified by the MCL35 assay. Hazard ratios (HR) are reported with the standard-risk group used as the reference. (C) Kaplan-Meier curves of the overall survival of the three patient groups within the subgroup of patients for whom there was an intention to consolidate response with an autologous stem-cell transplantation (ASCT). HRs are reported with the standard-risk group used as the reference. IHC, immunohistochemistry; UTR, untranslated region.

Fig 5.

Studies of the analytic validity of the MCL35 assay. (A) MCL35 scores are shown in ascending order, left to right, in the validation cohort. Gold dots represent the scores of the 17 biopsies (equally spread across the spectrum of scores) selected for the analytic validation studies. Blue dots represent the scores of the biopsies not selected. (B) MCL35 scores of RNA from the 17 biopsies identified in (A) run in triplicate (y-axis) plotted against the average of the three scores (x-axis). The gold dot represents an outlier score. (C) MCL35 scores of RNA from the 17 biopsies that were extracted and run at two independent laboratories in Würzburg, Germany and Barcelona, Spain (y-axis) plotted against the original MCL35 score (x-axis) generated at the BC Cancer Agency, Vancouver, BC, Canada. Calculation of the MCL35 scores was performed centrally. (D) MCL35 scores from 100 ng of RNA from the 17 biopsies (y-axis) plotted against the score when 200 ng was loaded. The solid line represents the line-of-best-fit. (E) MCL35 scores from 50 ng of RNA run in duplicate from the 17 biopsies (y-axis) plotted against the score when 200 ng was loaded. The solid line represents the line-of-best-fit. (F) MCL35 scores from 25 ng of RNA from the 17 biopsies (y-axis) plotted against the score when 200 ng was loaded. The solid line represents the line-of-best-fit.