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, 9 (28), 19961-19979

MicroRNA Signatures and Foxp3 + Cell Count Correlate With Relapse Occurrence in Follicular Lymphoma

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MicroRNA Signatures and Foxp3 + Cell Count Correlate With Relapse Occurrence in Follicular Lymphoma

Giorgio Malpeli et al. Oncotarget.

Abstract

First line drug treatment of follicular lymphoma (FL) patients is followed by a highly variable disease-free time before relapse in about one third of patients. No molecular marker is able to predict efficiently the risk of relapse. We investigated the expression profile of microRNAs (miRNAs) by microarrays and of the tumor microenvironment by immunohistochemistry in 26 FLs and 12 reactive lymph nodes (rLN) as reference. Twenty-nine miRNAs were differentially expressed in FLs compared to rLNs and some of them discriminated grade 1 from 3a FLs. Both FLs and rLNs displayed molecular heterogeneity. FLs grouped into two clusters mostly driven by the tumor T-cell content. Among 21 drug-treated FL patients with an average follow-up of 13.5 years, eight cases relapsed. Twenty-six miRNAs discriminated between relapsed and non-relapsed FLs. Ten miRNAs also correlated with Foxp3+ cells number. Notably, Foxp3+ cells were significantly less in relapsed patients and lower Foxp3+ cell number associated with shorter time-to-relapse. Foxp3+ cells did not co-expressed follicular helper T-cell markers and were therefore classified as regulatory T cells rather than follicular regulatory T-cells. These findings introduce new knowledge about the relationship between miRNA alterations and infiltrating immune cells and show that Foxp3+ cells might be predictive of disease relapse.

Keywords: Foxp3; follicular lymphoma; heterogeneity; microRNAs; relapse.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Profile of miRNAs differentially expressed between FLs and rLNs
The heat map describes the expression of 36 spots, corresponding to 29 single miRNAs, differentially expressed among 38 samples: 26 follicular lymphoma (FLs) and 12 reactive lymph nodes (rLNs) (FDR 10%). FL-1, FL-2, FL-3a, FL-3b are FL of grade 1, 2, 3a and 3b, respectively. Red, higher expression (log2, +4); green, lower expression (log2, -4). The tables report the list of 17 miRNAs upregulated in FLs and of 12 miRNAs downregulated in FL in comparison to rLNs and the corresponding Q values. White circles: upregulated in Rohele et al. [23]. Black circles: downregulated in Rohele et al. [23]. White triangles: upregulated in isolated FL cells in Wang et al. [24]. R, relapsed; NR, not relapsed; n.a. not available.
Figure 2
Figure 2. Profile of miRNAs differentially expressed between FL grade 1 or 3a and rLNs
FL-1 and FL-3a are follicular lymphoma (FL) of grade 1 and 3a, respectively. Red, higher expression (log2, +4); green, lower expression (log2, –4). (A) The heat maps describe the expression of 14 spots, corresponding to 11 single miRNAs, differentially expressed in 20 samples: eight FLs grade 1 and 12 reactive lymph nodes (rLNs) (FDR 10%). (B) The heat maps describe the expression of 5 spots, corresponding to 4 single miRNAs, differentially expressed among 20 samples: eight FLs grade 3a and 12 rLNs (FDR 10%). (C) The table reports the list of 11 miRNAs upregulated (up) or downregulated (down) in FL grade 1 and 3a in comparison to rLNs.
Figure 3
Figure 3. Combined expression levels and trend of 5p and 3p variants of miR-9-2 and miR-21 in FLs and rLNs
(A) Graph reports the expression level of miR-9-2 and miR-9* (from microarray data) in 21 follicular lymphomas (FLs) cases having clinical information (black circles) and in 12 reactive lymph nodes (rLNs) (empty circles). (B) Graph reports the expression level of miR-21 and miR-21* (from microarray data) in 21 FLs cases having clinical information (black circles) and in 12 rLNs (empty circles). Bottom graphs report slope and 95% confidence interval of the trend lines passing through the symbols of the two series represented in top graphs.
Figure 4
Figure 4. Profile of miRNAs differentially expressed among FLs, CD4+ T-cells and CD8+ T-cells
FL-HT, follicular lymphoma (FL) with high T-cell level. FL-LT, with low T-cell level. (A) The heat map describes the expression levels of miRNAs differentially expressed among 30 samples: 26 FLs, two CD4+ and two CD8+ T-cell samples (FDR 1%). FL-1, FL-2, FL-3a, FL-3b are FLs of grade 1, 2, 3a and 3b, respectively. Red, higher expression (log2, +4); green, lower expression (log2, -4). On the right of the heat map a partial list of miRNA differentially expressed among the three sets of samples, which are expressed at higher level in T-cells than in FLs (miRNA cluster 1). On the top of the heat map is the average mRNA expression level of three CD3 chains gamma, delta and epsilon for each of the 26 FL samples. Expression data were obtained by qRT-PCR. (B) Array tree of 30 samples based on the expression levels of miRNAs differentially expressed. (C) Correlation between the expression levels of miR-342 or miR-370 (from microrray analysis) and the expression level (log2) of CD3 and CD28 mRNAs by qRT-PCR in 26 FLs samples. P was calculated by Spearman's correlation.
Figure 5
Figure 5. Intrafollicular, interfollicular and total Foxp3+ cell count in FLs and rLNs
White circles, follicular lymphomas (FLs); black circles, reactive lymph nodes (rLNs); black line, median. Intra, inter and total are intrafollicular, interfollicular and intrafollicular plus interfollicular Foxp3+ cells, respectively. Number of Foxp3+ cells in each FL and rLN case was the average number of cells in two consecutive slides. The average number of positive cells was normalized on the whole number of nuclei present in a slide. P values were calculated by t-test. (A) Foxp3+ cell counts in intrafollicular, interfollicular, total and interfollicular/intrafollicular ratio in FLs and rLN. (B) Intrafollicular Foxp3+ cell counts in FL grades and LNs. (C) Interfollicular Foxp3+ cell counts in FL grades and LNs. (D) Interfollicular/intrafollicular ratio Foxp3+ cell counts in FL grades and LNs.
Figure 6
Figure 6. Intrafollicular, interfollicular and total PD1+ cell counts in FLs and rLNs
White circles, follicular lymphomas (FLs); black circles, reactive lymph nodes (rLNs); black line, median. Intra, inter and total are intrafollicular, interfollicular and intrafollicular plus interfollicular PD1+ cells, respectively. Number of PD1+ cells in each FL and rLN case was the average number of cells in two consecutive slides. The average number of positive cells was normalized on the whole number of nuclei present in a slide. P values were calculated by t-test. (A) PD1+ cell counts in intrafollicular, interfollicular, PD1+ total and interfollicular/intrafollicular ratio in FLs and rLNs. (B) Intrafollicular PD1+cell counts in FL grades and rLNs. (C) Interfollicular PD1+cell counts in FL grades and rLNs. (D) Interfollicular/intrafollicular ratio PD1+ cell counts in FL grades and rLNs.
Figure 7
Figure 7. Foxp3+ cell counts in relapsed and not relapsed FLs and Venn diagram of miRNAs upregulated in relapsed or not relapsed FLs and of miRNAs correlated directly or inversely with Foxp3+ cell counts
Number of Foxp3+ cells in each follicular lymphoma (FL) was the average of cells in two consecutive slides. The average number of positive cells was normalized on the whole number of nuclei present in a slide. (A) Comparison of intrafollicular, interfollicular and total Foxp3+ cells in relapsed and not relapsed FLs. P values were calculated by t-test. (B) Venn diagram of miRNAs upregulated in relapsed or not relapsed FLs (dark grey) and the intersection (intermediate grey) with miRNAs correlated directly or inversely with Foxp3+ cell counts (light grey).
Figure 8
Figure 8. Time to relapse probability curves based on total Foxp3+ cell counts in FL patients
Top curve represents FL cases with total Foxp3+ cells higher than median. Bottom curve represents FL cases with total Foxp3+ cells lower than median.

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