Dec 1, 2019
This JCO Podcast provides observations and commentary on the JCO article “Progression of Disease Within 24 Months (POD24) in Follicular Lymphoma Is Associated With Reduced Intratumoral Immune-Infiltration” by Dr. Tobin and colleagues. My name is Dr. Carla Casulo, and I am Associate Professor of Medicine, Hematology and Oncology at the Wilmot Cancer Institute of the University of Rochester in Rochester, NY, USA. My oncologic specialty is Lymphoma.
Follicular lymphoma is the most frequently occurring indolent non-Hodgkin lymphoma and has a long natural history, with median overall survival nearing two decades. Patients with follicular lymphoma may experience a variable clinical course, with periods of long remission punctuated by episodes of recurrent lymphoma requiring re-treatment. Among all patients, up to one third will have early disease recurrence, defined as occurring within 24 months of diagnosis. Please note that progression of disease within 24 months will be referred to as POD24 for the remainder of this podcast. These patients have inferior survival, ranging from 25-50% at 5 years. Consequently, POD24 has become a robust and well accepted indicator of identifying high-risk patients.
The implications of POD24 were first identified through our analysis of the National Lymphocare Study, which sought to test the hypothesis that time to disease progression had an impact on subsequent patient outcomes. 588 patients treated with RCHOP were included. Patients with POD24 were defined as early progressors, and those without relapse or death within 24 months were defined as the reference group. Patients with POD24 had OS of 50% at 5 years compared to 90% in the reference group. These findings have subsequently been independently validated by numerous investigators worldwide, corroborating the adverse prognostic impact of an early disease related event in follicular lymphoma. The largest of these validation studies pooled individual patient data from 5,453 patients on 13 Clinical Trials using the Follicular Lymphoma Analysis of Surrogacy Hypothesis (FLASH) Investigation.
In the FLASH analysis, we identified that male gender, poor performance status, high follicular lymphoma international Prognostic index (FLIPI) score, and elevated baseline beta 2 microglobulin B2M as predictors of early death and progression. Moreover, it confirmed POD24 as an early clinical endpoint of poor survival in follicular lymphoma that should be utilized to identify patients for prospective clinical trials.
The current status of biomarkers in follicular lymphoma has emerged from a wealth of clinical and laboratory-based factors to classify risk, towards a biologic based, molecular approach merging clinical factors with our current understanding of the follicular lymphoma genomic landscape.
There are numerous well-established and emerging clinical prognostic indices used at the time of diagnosis in follicular lymphoma that can help discriminate general outcome. These include the FLIPI and FLIPI -2. To an extent, these prognostic markers can identify subsets of patients with an expected POD24 with a sensitivity between 60-78%, and a specificity between 56-58%. However, in an attempt to use a precision approach, investigators from the German Low-Grade Lymphoma Study Group harmonized clinical and pathologic data to create a clinico-genetic risk model aimed at more accurate risk prognostication in patients receiving front line chemoimmunotherapy. They performed deep DNA sequencing from formalin fixed pre-treatment biopsies to analyze the mutational status of genes in 151 patients with follicular lymphoma tumor samples. The resulting prognostic tool, called the m7-FLIPI, distilled down 74 genes into 7 genes with non-silent mutations occurring at a variant allele frequency of 10% or greater, and combined these with high risk FLIPI status and ECOG performance status. These included genes that increased risk of progression, including EP300, FOX01, CREBBP, CARD11, and those that decreased risk of progression, including EZH2, ARID1A, and MEF2B. The cumulative risk score was calculated by combining relative weights of these genes in a multivariate analysis predicting failure-free survival. This m7-FLIPI score was tested to identify POD24 but only captured about 50% of patients as high risk. A later model included only 3 genes, including EP300, FOX01 and EZH2, performance status and FLIPI score. Defined as the POD24-PI, this was more sensitive at identifying POD24 patients but did not outperform other metrics due to lower specificity.
Biologic classification of POD24 patients remains an ongoing international research priority to seek actionable targets that might change the natural history of follicular lymphoma and improve survival of patients more likely to have morbidity and death from their disease. Several years ago, in the pre-rituximab era, the Leukemia and Lymphoma Molecular profiling project identified the importance of the follicular lymphoma tumor microenvironment in predicting favorable or poor outcome. The follicular lymphoma tumor microenvironment is composed of tumor infiltrating T cells, regulatory T cells, and lymphoma-associated macrophages. The immune survival score or ISS established that differential expression of gene expression signatures from intratumoral immune cells in the tumor microenvironment affected survival, and particular increased expression of macrophages was associated with poor prognosis.
The impact of the follicular lymphoma immune microenvironment on survival is changing in the current era of chemotherapy combined with antiCD20 immunotherapy, and particularly by available drugs that reverse tumoral immunosuppression by blocking programmed cell death. Studies from solid tumor literature demonstrate that low levels of tumor infiltration immune cells are associated with inferior survival. In follicular lymphoma, then, precise characterization of high or low immune infiltrating cells in the tumor microenvironment may have a critical effect on understanding the mechanisms of POD24. This particular relationship is what Tobin and colleagues investigated in the current study.
In this analysis, targeted gene sequencing using NanoString technology from paraffin-embedded tissue and multi-spectral immunofluorescence on a tissue microarray was applied to two groups: a discovery cohort of 132 patients from Princess Alexandria Hospital with early- and advanced-stage follicular lymphoma who received either chemotherapy or observation and two independent validation cohorts of 198 patients with advanced-stage disease treated with RCHOP and RCVP from the German Low grade lymphoma Study Group and the British Columbia Cancer Agency. They also performed T cell repertoire analysis, flow cytometry, immunofluorescence, and next-generation sequencing.
Here, they defined POD24 as primary refractory disease following treatment, transformation to a more aggressive histology, and relapse within 24 months of diagnosis, which is a more liberal definition from what was initially described in the national lymphocare study but does encompass patients at highest risk of lymphoma-related death.
Gene expression profiling revealed distinct clustering of follicular lymphoma samples based on high or low expression of immune infiltrating cells. Low expression of four immune markers, including PDL-2, TNFalpha, CD4, and CD68 were all associated with poor outcome, but the most specific marker with the highest specificity and sensitivity was PD-L2. They then dichotomized PD-L2 expression into “immune infiltration high” and “immune infiltration low” in subsequent analyses. PD-L2 is an immune checkpoint present broadly on both tumor cells and the tumor microenvironment. To localize its distribution, they performed flow cytometry in fresh FL samples and quantified PD-L2 expression by PCR. They identified that PD-L2 gene expression was distributed in both CD20+ tumor cells as well as non CD20+ cells in the tumor microenvironment. However, the proportion of PD-L2 was lower in the CD20+ cells. Overall, there was lower expression of all immune cells in the immune infiltration low phenotype compare to the immune infiltration high phenotype.
They tested the relevance of immune infiltration and POD24. Consistent with numerous other studies, POD24 events occurred in about 24% of patients. Patients with POD24 events in the Princess Alexandria discovery set were more enriched for the immune infiltration low phenotype. These findings were also validated in the British Columbia cancer agency and German lymphoma study group populations. Nearly 50% of patients with low PD-L2 had POD24 events, compared to 16% in those with high PD-L2, concluding that low PD-L2 identifies a subset of patients enriched for POD24. When evaluating the mutational profiles of those with immune infiltration high versus immune infiltration low based on the m7-FLIPI genes, mutations were detected equally among both populations, suggesting that this mutational profiling is not influenced by the immune infiltration phenotype.
The data presented by Tobin and colleagues makes an important contribution to our understanding of the biological and immune-based mechanism influencing early disease-related events in follicular lymphoma. They demonstrate that reduced immune infiltration is associated with greater chance of POD24. While the sensitivity of this was high, similar to other prognostic markers, it still did not capture the entire subset of future POD24 cases, underscoring the significant heterogeneity within this high-risk patient population. However, there remains opportunity to include PD-L2 as a surrogate for poor risk disease. If further applied to immunohistochemistry or other widespread diagnostic methods, it has the potential for more widespread application. Further validation is still required, particularly to patients treated with novel agents or other immunotherapies. However, the assessment of immune infiltration as described by these findings appears to be an encouraging step in determining risk of POD24.
This concludes this JCO Podcast. Thank you for listening.