TruSeq-Based Gene Expression Analysis of Formalin-Fixed Paraffin-Embedded (FFPE) Cutaneous T-Cell Lymphoma Samples: Subgroup Analysis Results and Elucidation of Biases from FFPE Sample Processing on the TruSeq Platform

Abstract

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of malignancies with courses ranging from indolent to potentially lethal. We recently studied in a 157 patient cohort gene expression profiles generated by the TruSeq targeted RNA gene expression sequencing. We observed that the sequencing library quality and depth from formalin-fixed paraffin-embedded (FFPE) skin samples were significantly lower when biopsies were obtained prior to 2009. We also observed that the fresh CTCL samples clustered together, even though they included stage I–IV disease. In this study, we compared TruSeq gene expression patterns in older (≤2008) vs. more recent (≥2009) FFPE samples to determine whether these clustering analyses and earlier described differentially expressed gene findings are robust when analyzed based on the year of biopsy. We also explored biases found in FFPE samples when subjected to the TruSeq analysis of gene expression. Our results showed that ≤2008 and ≥2009 samples clustered equally well to the full data set and, importantly, both analyses produced nearly identical trends and findings. Specifically, both analyses enriched nearly identical DEGs when comparing benign vs. (1) stage I–IV and (2) stage IV (alone) CTCL samples. Results obtained using either ≤2008 or ≥2009 samples were strongly correlated. Furthermore, by using subgroup analyses, we were able to identify additional novel differentially expressed genes (DEGs), which did not reach statistical significance in the prior full data set analysis. Those included CTCL-upregulated BCL11A, SELL, IRF1, SMAD1, CASP1, BIRC5, and MAX and CTCL-downregulated MDM4, SERPINB3, and THBS4 genes. With respect to sample biases, no matter if we performed subgroup analyses or full data set analysis, fresh samples tightly clustered together. While principal component analysis revealed that fresh samples were spatially closer together, indicating some preprocessing batch effect, they remained in the proximity to other normal/benign and FFPE CTCL samples and were not clustering as outliers by themselves. Notably, this did not affect the determination of DEGs when analyzing ≥2009 samples (fresh and FFPE biopsies) vs. ≥2009 FFPE samples alone.

Introduction

Cutaneous T-cell lymphomas (CTCLs) represent ~4–8% of all non-Hodgkin’s lymphomas and are characterized by infiltration of malignant T lymphocytes into the skin (1). Most patients first present with stage I disease, limited to the skin, which can either follow an indolent course (in 70–80% of cases) or progress to a potentially devastating, deadly malignancy with a median survival of <3 years (2). The diagnosis of CTCL is rather challenging for several reasons. First, mycosis fungoides (MF) and Sézary syndrome (SS), the most recognized variants of CTCL, can have variable presentation (3). Second, other common and rare benign inflammatory dermatoses can mimic CTCL and vice versa. Classically, MF may present with centrally distributed erythematous patches and plaques that are not specific to CTCL and are commonly misdiagnosed as chronic eczema, psoriasis, pityriasis rubra pilaris, drug eruptions, and dermatophyte infections. Finally, histopathological analysis of skin biopsies and PCR evaluation of T-cell receptor clonality lacks sensitivity in early MF patients and in erythrodermic disease. Unfortunately, current time to CTCL diagnosis from its initial presentation averages ~6 years (4).

Factors involved in the pathogenesis and prognostication of CTCL have emerged from recent epidemiological (5–8), karyotype/chromosomal (9–23), exome sequencings (24–28), gene and microRNA expression profiling studies (3, 29–41), but remain incomplete and poorly elucidated. The lymphocyte precursor population was proposed to be different between MF (skin resident memory T lymphocytes) vs. SS (skin tropic central memory T lymphocytes with wide tropism) (42–45). Importantly, significant disease heterogeneity was noted on a molecular level, and genetic alterations in MF/SS were often not replicated between different studies. Pathways that are believed to be involved in CTCL pathogenesis include T-cell function/signaling/differentiation, JAK/STAT/NF-κB signaling, cytokine production, chromatin remodeling, cell cycle checkpoint regulation, DNA repair, as well as cancer testis and embryonic stem cell signaling and function (24, 25, 28, 46). The goal of discovery and validation of prognostic biomarkers for disease progression and patient survival remains critical to help identify the minority of stage I MF patients, who will eventually progress to advanced disease (~20–30% of patients). Poor disease outcome may be heralded by high expression of TOX, GTSF1, NOTCH1, CCR4, ITK, FYB, SYC1, LCK or miR155, miR21, and let-7i microRNAs (26, 31, 39, 47).

Recently, we analyzed using Illumina’s TruSeq targeted RNA gene expression platform a new cohort of 157 patients, with biopsy-confirmed CTCL and compared it to a cohort of patients with normal skin and benign skin conditions (41). A number of patients in this study provided longitudinal biopsy samples (41). Analyzed samples included (A) 29 formalin-fixed paraffin-embedded (FFPE) tissues from benign inflammatory dermatoses and skin tag biopsies (1 sample per patient; 7 skin tag samples and 22 benign inflammatory dermatoses samples); (B) 134 FFPE samples of lesional CTCL skin from 110 patients; and (C) an additional 18 samples of freshly obtained and liquid nitrogen snap-frozen skin samples from a different group of CTCL patients. We processed 181 skin biopsy samples either freshly obtained or FFPE using TruSeq platform, capturing 284 genes that were previously identified as important for CTCL diagnosis and/or prognosis (32, 48). We identified 75 statistically significant differentially expressed genes (DEGs) between benign skin samples and either all CTCL or stage IV CTCL samples (41) and validated a number of our previous diagnostic and prognostic expression markers (3, 41).

However, we noticed non-trivial heterogeneity when performing clustering based on the TruSeq gene expression data, where early-stage CTCL samples and benign samples were admixed in the same clusters with the stage IV advanced CTCL disease. We hypothesized that this could be due to differences in TruSeq library sequencing depth and/or variation in the quality of the FFPE samples obtained during 2007–2008 (older) vs. 2009–2012 (more recent) years. Indeed, recent samples that were freshly obtained and snap frozen had comparable total number of sequencing reads (400–1,000 K reads), while older FFPE samples had often <300 K sequencing reads (41). In addition, we observed that freshly obtained snap-frozen CTCL samples were often tightly grouped in the same cluster, independent of their disease stage (41). This may indicate that TruSeq gene expression analysis may be affected by intrinsic biases based on the very natures of the samples analyzed (e.g., FFPE vs. fresh-frozen biopsies).

Notably, these variables (i.e., old vs. new; FFPE vs. freshly obtained snap frozen) were not formally evaluated in the prior publication but may contribute to the observed heterogeneity. These variations contribute toward a larger problem, known as the batch effect, in the field of gene expression-based analyses that utilize TruSeq, RNA-Seq, gene expression microarrays, and other approaches to identify DEGs. Differences in preprocessing, sequencing runs, technicians/centers, date of experiments, populations, and experimental design can account for heterogeneity that will remain despite normalization and use of control samples. Potential consequences of batch effect include reduction of statistical accuracy, introduction of spurious DEGs, and discrepancies between observed and true correlations (49). Several techniques can be used to minimize batch effects without removing true signals including surrogate variable analysis (50), ComBat (51), and principal component-based approaches (i.e., EIGENSTRAT among others) (52).

In this study, we aimed to characterize TruSeq gene expression patterns separately in older (≤2008) vs. more recent (≥2009) FFPE samples to determine whether clustering analyses results display robustness when compared to the full data set. We also explored sample processing biases (old vs. new and FFPE vs. freshly obtained snap frozen).

Materials and Methods

Patients and Samples

As described before (41), all patients were enrolled in the study in accordance with the IRB-approved protocols: PA12-0267, PA12-0497, and Lab97-256 at the MD Anderson Cancer Center (MDACC) and A09-M106-13A and 13-201-GEN at McGill University/McGill University Health Centre (MUHC). This study was carried out in accordance with the recommendations of the Research Ethics Board of the McGill University/MUHC with written informed consent from all subjects in accordance with the Declaration of Helsinki. This study was carried out in accordance with the recommendations of the MDACC Research Ethics Board, which exempted us from obtaining written informed consent from patients, who earlier signed a hospital consent allowing their stored biopsy samples to be used for research.

Data Acquisition

Processed TruSeq data from Litvinov et al (41) were re-analyzed in this study based on transcripts per million (TPM) and RNA integrity number (RIN) parameters. Raw data were deposited in the NCBI SRA, accession number SRP114956. We separated CTCL FFPE samples obtained from the MDACC into two subgroups: older (≤2008) vs. more recent (≥2009).

Clustering

Unsupervised hierarchal clustering was performed in R, using packages stats, cluster, and gplots. Pairwise dissimilarity (distance) matrix was calculated using Gower’s method, which performs well in the case of incomplete/missing data when compared to other methods (53). Clusters were obtained using Ward’s clustering method and criteria (54). Silhouette plots followed by visual inspection (to ensure appropriately sized clusters) were used to assess clusters and subclusters divisions. We repeated similar comparisons for all samples, benign samples vs. stage IV CTCL disease, and early (stage ≤IIA) vs. intermediate (stages IIB and III) vs. advanced (stage IV) CTCL.

Principal Component Analysis (PCA)

Principal component analysis was performed on scaled, centered TPM data using package pcaMethods (55). Probabilistic PCA was used to account for missing data. Score plots of principal components 1 and 2 were generated.

Statistical Analyses

Differences in mean TPMs were determined using two-tailed Ward’s t-test. Power analysis showed an 86% power to detect a twofold expression change at a significance level of 0.05 for the comparison between the smallest subgroups, with complete data points. Correlations were computed using Spearman’s rho, on log-2 ratios. Mean RINs were compared using a Bayesian analysis with Markov Chain Monte Carlo (MCMC) simulations, using R package rjags; at least 100,000 iterations were performed to estimate p values.

Results

Subgroup Clustering Analysis of All Samples

We previously noted that the ≤2008 FFPE samples had significantly decreased number of sequence reads per sample when compared to the ≥2009 samples (mean 103,406 ± 96,620 vs. 437,218 ± 550,840 reads, respectively). Therefore, we repeated unsupervised hierarchical clustering for benign samples (skin tags and benign inflammatory dermatoses), fresh liquid nitrogen snap-frozen CTCL samples, and either ≤2008 or ≥2009 FFPE CTCL samples. For ≤2008 FFPE sample analysis (Figure 1), we observed three major clusters. Cluster 1 comprised exclusively the FFPE CTCL samples, mostly early-stage (≤IIA) (12/33), along with two mid-stage (IIB and III) (2/8) and one late-stage (IV) (1/14) disease. In Cluster 2, 21 of 22 samples were from CTCL patients representing advanced stages (mid = 4/8 and late = 9/14), along with one eczema sample and a number of early-stage CTCL samples (8/33). Cluster 3 formed multiple subgroups (~4) that comprised mostly benign samples (28/29) and fresh CTCL samples, along with many early-stage and mid-late stage FFPE CTCL samples (early = 13/33, mid = 2/8, and late = 4/14). As previously discussed (41), one of the subgroups encompassed all fresh CTCL samples, which tightly clustered together (18/18). Two of the subgroups contained mostly benign samples, while the last one had early-stage FFPE CTCL samples. For ≥2009 FFPE samples (Figure 2), we noted two small clusters and two larger clusters. The first small cluster on the left panel (Cluster 1) contained six FFPE CTCL samples (two early, one mid, and three late). The second small cluster on the right (Cluster 4) included 18/18 fresh CTCL samples similarly to our previous analyses along with 2 benign biopsies. The first large cluster on the center left panel (Cluster 2) exhibited significant molecular disease heterogeneity. The first subgroup (A) had primarily mid-stage (n = 9) CTCL skin biopsies, one early and two late-stage samples, while the other two subgroups (B and C) were very heterogeneous with respect to their composition. For the second large cluster on the center right panel (Cluster 3), a similar admixture was observed with three subgroups, one subgroup being comprised primarily benign skin samples (C) and the other two containing predominantly early (A) and advanced (B) stage CTCL disease samples.

Figure 1.

Unsupervised hierarchical clustering analysis based on TruSeq targeted RNA gene expression analysis of 284 select genes in benign inflammatory dermatoses (green), freshly obtained and snap-frozen cutaneous T-cell lymphoma (CTCL) samples (gray), and ≤2008 formalin-fixed paraffin-embedded (FFPE) CTCL samples (early stage, yellow; mid stage, orange; and late stage, dark red). A color key refers to gene expression in log(transcripts per million).

Figure 2.

Unsupervised hierarchical clustering analysis based on TruSeq targeted RNA gene expression analysis of 284 select genes in benign inflammatory dermatoses (green), freshly obtained and snap-frozen cutaneous T-cell lymphoma (CTCL) samples (gray), and ≥2009 formalin-fixed paraffin-embedded (FFPE) CTCL samples (early stage, yellow; mid stage, orange; and late stage, dark red). A color key refers to gene expression in log(transcripts per million).

Subgroup Clustering Analysis of Healthy Skin/Benign Inflammatory Dermatoses Samples vs. Stage IV CTCL Samples

We then performed unsupervised hierarchical clustering for benign samples (which included skin tags and benign dermatoses that often clinically mimic CTCL) vs. stage IV CTCL disease. Similarly, two analyses were performed for ≤2008 and ≥2009 FFPE biopsies. In the case of ≤2008 samples (Figure 3), there were two major clusters that separated quite well these biopsies based on gene expression changes. Cluster 1 had 13 samples, 12 of which were stage IV CTCL disease (including 12/14 of total stage IV CTCL samples) and 1 sample form a patient with chronic eczema. Cluster 2 contained 30 samples in total and comprised mostly benign dermatoses and skin tags (n = 28) and 2 stage IV CTCL samples.

Figure 3.

Unsupervised hierarchical clustering analysis based on TruSeq targeted RNA gene expression analysis of 284 select genes in benign inflammatory dermatoses (green) vs. ≤2008 stage IV formalin-fixed paraffin-embedded cutaneous T-cell lymphoma (CTCL) samples (red). A color key refers to gene expression in log(transcripts per million).

Surprisingly, for ≥2009 samples (Figure 4), greater overall heterogeneity was observed. However, we noted one small cluster in the right panel and one large cluster with three subgroups in the center. Cluster 1 (right panel) had nine samples, eight of which were stage IV samples (8/20 total stage IV CTCL samples). Cluster 2 was subdivided into three subgroups, where 2A samples (n = 7) with advanced CTCL disease tightly clustered together, while 2B (n = 20) and 2C (n = 11) samples included primarily benign dermatoses and skin tags (85 and 82%, respectively, for each subcluster).

Figure 4.

Unsupervised hierarchical clustering analysis based on TruSeq targeted RNA gene expression analysis of 284 select genes in benign inflammatory dermatoses (green), vs. ≥2009 stage IV formalin-fixed paraffin-embedded cutaneous T-cell lymphoma (CTCL) samples (red). A color key refers to gene expression in log(transcripts per million).

Identification of Differentially Expressed Genes (DEGs) in All Samples Using Subgroup Analyses Based on the Year of Biopsy and Benign vs. Malignant Nature of Samples

We then analyzed our full data set, by performing a Wald’s t-test to compare either benign dermatoses vs. (1) all CTCL samples or (2) stage IV CTCL. In our initial report (41), we identified important differentially expressed genes (DEGs) including TOX, FYB, LEF1, CCR4, ITK, EED, POU2AF, IL-26, STAT5, BLK, GTSF1, PSORS1C2, CD70, and STAT signaling genes; LTA, NFKB1, NFKB2, and IL-15; and other inflammatory cytokines. In this study, we repeated the analysis of the FFPE samples obtained ≤2008 vs. ≥2009.

As presented in Table 1, our analysis revealed 54 DEGs (p < 0.05), when ≤2008 stage I–IV CTCL or ≤2008 stage IV CTCL samples were compared to benign skin samples. This list included 47/75 DEGs that were enriched in the initially reported full data set (41). New highlighted CTCL-upregulated targets in this analysis included BCL11A, SELL, IRF1, SMAD1, CASP1, and BIRC5, while THBS4 was upregulated in benign skin samples. For ≥2009 samples, 41 significant DEGs (p < 0.05) were found when freshly obtained and for ≥2009 samples, FFPE CTCL biopsies were analyzed together in a similar way (Table 2). Importantly, the same 41 DEGs were identified using only the ≥2009 FFPE samples alone (i.e., excluding the freshly obtained biopsies from this analysis). In the latter analysis, four additional CTCL-upregulated DEGs (EP400, NFKB1, TRRAP, and MAX) were revealed as being statistically significant (Table 2).

Table 1.

Genes with statistically significant differences in expression both between benign skin dermatoses vs. all ≤2008 CTCL samples (left panel) and between benign skin lesions vs. ≤2008 stage IV CTCL samples (right panel).

Genes	Average of benign [transcripts per million (TPM)]	Average of all CTCL (TPM)	log2 ratio (all CTCL vs. benign)	p Value (all CTCL vs. benign)	Average of stage IV CTCL (TPM)	log2 ratio (stage IV CTCL vs. benign)	p Value (stage IV CTCL vs. benign)
CCR7	91.6375	1,571.482	4.100044	0.049964	748.8	3.03057	0.001363
LTA	189.1833	2,300.565	3.604131	0.006095	1,473.638	2.961525	0.015005
CD70	813.88	8,678.515	3.41456	0.001653	7,529.575	3.20968	0.003482
ITK	140.1125	1,447.92	3.369324	4.33E−05	1,136.309	3.019698	0.002318
TOX	410.12	3,459.284	3.076355	2.65E−08	4,502.264	3.456533	0.000852
LEF1	161.0417	1,283.983	2.995121	0.000612	1,056.588	2.713906	0.00929
CCR4	1,375.88	7,700.756	2.484645	6.15E−05	8,003.146	2.540213	0.004464
IL21R	256.3111	1,344.811	2.391435	0.000208	1,600.418	2.642481	0.004746
FYB	1,337.875	6,587.762	2.299845	5.97E−08	8,462.607	2.661159	0.002439
TRAF1	221.0818	1,055.033	2.254636	2.77E−05	1,357.833	2.618654	0.010125
ZAP70	915.675	4,096.65	2.161537	2.87E−05	4,007.87	2.129928	0.040324
IL7R	523.8217	2,339.003	2.158746	0.001063	2,376.782	2.181862	0.04109
SELL	1,160.508	5,064.55	2.125677	0.003904	5,525.386	2.251318	0.041623
ZBTB16	1,100.665	4,684.436	2.0895	0.000107	3,581.977	1.70238	0.012754
CDKN2B	476.1875	2,021.157	2.08558	0.000255	1,735.944	1.866119	0.019539
NFKB2	320.1294	1,356.579	2.083246	0.000131	1,336.467	2.061697	0.045613
MMP9	149.2333	609.0611	2.029017	0.00312	708.9625	2.24814	0.013971
PILRB	2562.259	10,430.87	2.025371	1.22E−05	8,270.423	1.690545	0.039467
IL32	2,781.636	11,217.26	2.011714	2.44E−05	10,043.51	1.852258	0.00489
STAT1	1,714.134	6,868.114	2.002434	2.52E−08	6,162.807	1.846107	0.000347
STAG3	1,118.487	4,312.054	1.946827	5.32E−07	4,394.586	1.974179	0.001446
EED	1,110.654	4,229.108	1.928944	6.99E−06	3,032.208	1.44896	0.039
LCP2	535.9733	2,010.365	1.907224	2.45E−06	2,396.473	2.160679	1.96E−05
EZH2	466.14	1,715.855	1.880092	6E−07	1,339.258	1.522599	0.004833
NME4	3075.109	11,318.73	1.880003	0.000737	9,229.838	1.585668	0.022129
PTPN6	746.3091	2,663.14	1.835283	8.96E−08	2,509.582	1.749602	0.005967
IRF7	841.7043	2,956.561	1.812534	0.008642	2,596.145	1.624986	0.01711
IRF4	192.3	652.3552	1.762299	0.000497	493.4556	1.359561	0.041528
KLHL42	136.4864	462.55	1.760853	0.004419	388.9889	1.510972	0.015901
RASA1	338.0591	1,118.067	1.725659	2.71E−06	951.1375	1.492378	0.014139
CXCL9	2,507.891	8,268.812	1.721205	0.00098	9,713.692	1.953545	0.010393
MTF2	217.3882	704.213	1.695738	0.000696	728.3556	1.744369	0.009782
BIRC5	338.0077	1,056.554	1.644238	0.007351	1,149.533	1.76592	0.006444
RAC2	4,323.963	13,309.44	1.622024	0.000209	10,852.03	1.327539	0.027541
MAP2K1	412.5632	1,209.649	1.551901	0.000416	1,143.84	1.471198	0.021317
EP400	1,879.341	5,316.209	1.500171	3.7E−05	4,616.664	1.296624	0.009013
TRAF2	2,425.25	6,748.69	1.476474	0.000693	5,677.97	1.227242	0.017582
SUZ12	340.216	943.5389	1.471631	1.3E−06	963.2273	1.501425	0.00096
JARID2	698.2037	1,913.181	1.454253	1.7E−05	1,853.242	1.408331	0.00949
TGFB1	394.75	1,065.353	1.43232	3.62E−05	1,000.375	1.34153	0.008951
SOCS3	1,008.825	2,703.836	1.422332	0.008306	3,391.209	1.749124	0.019409
MCL1	1,349.318	3,542.977	1.392732	7.2E−07	2,977.636	1.141937	0.005681
MYC	380.7762	996.8	1.388361	0.00144	1,117.25	1.552937	0.005836
STAT5B	210.0545	538.47	1.358102	0.002673	559.9091	1.414429	0.008889
WWOX	953.3938	2401.292	1.332667	0.002271	2,353.325	1.303557	0.048617
BCL11A	6,489.772	1,5869.53	1.29002	0.004204	15,268.44	1.234313	0.026839
ANPEP	820.7069	1,975.305	1.267136	0.000199	1,934.646	1.237131	0.019324
SMAD1	829.5667	1,947.6	1.231268	0.001198	2,076.155	1.323484	0.045054
CCL5	18,304.19	42,227.59	1.206012	0.006711	43,568.9	1.251125	0.006718
IRF1	690.6826	1,493.441	1.112546	0.005323	1,314.473	0.928389	0.032976
NOTCH1	393.3417	847.8263	1.107986	0.003446	834.7364	1.085538	0.046229
CD52	39,313.92	79,582.7	1.017415	0.001914	95,658.19	1.282848	0.016828
CASP1	18,518.75	33,218.6	0.843004	0.002609	39,462.42	1.091493	0.027117
THBS4	221.55	3.4	−6.02595	0.031067	<1	−7.79149	0.030685

Average expression is presented as TPM. Positive log-2 ratios indicate upregulation in CTCL samples and negative log-2 ratios indicate downregulation in CTCL samples. p Values from Wald’s t-test are presented.

Table 2.

Genes with statistically significant differences in expression both between benign skin dermatoses vs. all ≥2009 CTCL samples (left panel) and between benign skin lesions vs. ≥2009 stage IV CTCL samples (right panel).

Genes	Average of benign [transcripts per million (TPM)]	Average of all CTCL (TPM)	log2 ratio of all CTCL vs. benign	p Value of all CTCL vs. benign	Average of stage IV CTCL (TPM)	log2 ratio of stage IV CTCL vs. benign	p Value of stage IV CTCL vs. benign
GTSF1	<1	1,755.165	10.77739	7.05E−05	2,350.786	11.19893	0.021712
TOX	410.12	2,315.593	2.497264	9.6E−10	3,506.295	3.095829	0.000662
LTA	189.1833	1,059.592	2.485652	1.6E−05	1,438.912	2.927121	0.016594
CCR4	1375.88	6,972.679	2.341358	4.11E−06	8,994.879	2.708749	0.002939
FYB	1337.875	6,662.626	2.316148	3.26E−13	10,651.85	2.993088	3.22E−05
ITK	140.1125	683.6844	2.286745	1.5E−05	765.1688	2.449192	0.005206
SKAP1	373.9	1,741.68	2.219756	1.47E−06	2,487.344	2.733882	0.000207
LEF1	161.0417	665.3026	2.046577	1.8E−05	769.1615	2.255853	0.000553
MMP9	149.2333	509.9797	1.77287	0.016193	1,277.873	3.098102	0.042027
IL21R	256.3111	805.0809	1.651238	0.000823	867.8737	1.759589	0.023029
SH2D1A	162.8714	504.3029	1.630557	0.000264	422.3357	1.374657	0.02604
MDM4	69.4	206.8441	1.575536	0.034407	269.8571	1.959188	0.037649
ZAP70	915.675	2616.744	1.514865	0.000397	2,726.044	1.573901	0.032018
TRAF1	221.0818	621.6043	1.491416	0.000464	862.61	1.964128	0.024488
EED	1110.654	3,032.809	1.449245	1.92E−07	3,058.529	1.461429	0.005258
IL7R	523.8217	1,253.655	1.258992	0.000674	1,282.056	1.291311	0.027339
IRF4	192.3	457.9822	1.251933	1.51E−05	468.6125	1.285037	0.00781
PTPN6	746.3091	1,754.427	1.233155	7.66E−06	1,674.979	1.166298	0.012107
PILRB	2562.259	5,979.205	1.222537	5.22E−07	5,874.39	1.197023	0.001808
STAG3	1118.487	2,561.473	1.195425	7.14E−05	3,466.835	1.632071	0.001586
LCP2	535.9733	1,203.391	1.166873	0.000358	1,290.937	1.268185	0.007005
STAT1	1714.134	3,738.915	1.125139	9.17E−05	4,591.761	1.421567	0.024235
IL32	2781.636	5,963.653	1.100262	0.000424	6,458.989	1.215375	0.005436
CD52	39313.92	82,527.71	1.069838	0.000555	87,585.14	1.155646	0.004942
NFKB2	320.1294	661.9056	1.04797	0.001318	719.4333	1.168206	0.011523
STAT2	324.7545	668.2443	1.041026	0.00217	1,074.281	1.72595	0.043189
RAC2	4323.963	8,859.232	1.034828	0.012875	12,229.12	1.499894	0.003941
CCL5	18304.19	37,008.56	1.015685	0.006721	43,941.66	1.263415	0.003185
CNOT3	268.192	538.7935	1.006466	0.000799	718.7158	1.422155	0.01338
HDAC1	1224.993	2441.145	0.994785	5.61E−05	3,158.984	1.366688	0.004638
ZFX	116.128	228.9473	0.979299	0.000605	259.8667	1.162056	0.048005
MTF2	217.3882	382.8481	0.816498	0.007382	369.1313	0.76386	0.032394
ANKRD11	987.6292	1,734.318	0.812327	0.000253	2,046.583	1.051176	0.033996
SUZ12	340.216	573.6457	0.753709	0.002437	703.375	1.047843	0.030192
NUB1	5078.115	8,463.066	0.736887	0.017489	9,530.422	0.908247	0.035693
ZBTB16	1100.665	1,818.246	0.724172	0.035544	2,833.213	1.364063	0.020919
WWOX	953.3938	1,551.22	0.70226	0.039474	1,921.327	1.010959	0.039726
SERPINB13	4,197.017	2,456.495	−0.77276	0.025735	2,005.013	−1.06575	0.010905
SERPINB3	12,386.85	5,653.013	−1.13172	0.012455	4,564.794	−1.44019	0.008324
PSORS1C2	56,092.96	15,415.36	−1.86345	0.001104	19,264.48	−1.54188	0.003512
SERPINB4	37,557.08	9,788.951	−1.93986	0.010726	8,392.617	−2.16189	0.01035
EP400	1,879.341	2,833.862	0.592543	4.28E−03	3,732.626	0.989964	0.021786
TRRAP	113.8296	154.2161	0.438078	1.14E−03	204.5421	0.845522	0.039173
NFKB1	498.076	620.1367	0.31622	2.43E−02	915.6556	0.878439	0.034389
MAX	8,108.728	8,575.909	0.080814	1.76E−03	11,353.27	0.48556	0.039695

In light gray are presented four additional genes achieving statistical significance when using ≥2009 FFPE samples only (i.e., excluding freshly obtained samples from the analysis). Average expression is presented as TPM. Positive log-2 ratios indicate upregulation in CTCL samples and negative log-2 ratios indicate downregulation in CTCL samples. p Values from Wald’s t-test are presented.

Based on these combined results, 42/75 DEGs were confirmed in both analyses, which highlights significant robustness of these tests. Of course, many of the initially identified DEGs did not achieve statistical significance since the number of samples analyzed in each of these subanalyses (i.e., ≤2008 and ≥2009) was significantly smaller than when all the data were analyzed as one set. Moreover, based on the original TruSeq data, subgroup analysis showed consistency in log-2 ratios between ≤2008 and ≥2009 CTCL samples. Indeed, rank–rank correlation when comparing benign dermatoses vs. all FFPE CTCL samples was ρ = 0.71 (strong; p < 10⁻¹⁶), while this indicator was ρ = 0.55 (medium; p < 10⁻¹⁶) when comparison was made between benign dermatoses and stage IV FFPE biopsies.

Clustering Analysis of All FFPE CTCL Samples Using Subgroup Analyses Based on the Year of Biopsy and CTCL Clinical Cancer Stage

We then performed unsupervised hierarchical clustering analysis for early (≤IIA) vs. mid (IIB and III) vs. late (IV) stage CTCL for ≤2008 vs. ≥2009 samples. Similarly, we noted a significant molecular heterogeneity that was seen in our original report (41). However, for the ≤2008 CTCL FFPE samples (Figure 5), there were two major clusters. Cluster 1 had 12 samples, 10 of which were early-stage CTCL biopsies (10/31 of the total early-stage CTCL samples). Cluster 2 was rather heterogeneous with respect to its composition and could be subdivided into two subclusters: 2A, larger, with samples from all different stages and 2B with the well-defined subgroup of early-stage CTCL biopsies (10/11) on the right side of this subcluster. For ≥2009 samples (Figure 6), the distribution of samples was very heterogeneous as was seen in our earlier report (41).

Figure 5.

Unsupervised hierarchical clustering analysis based on TruSeq targeted RNA gene expression analysis of 284 select genes in ≤2008 early-stage (stage ≤IIA, yellow), mid-stage (stages IIB and III, orange), and late-stage (stage IV, dark red) formalin-fixed paraffin-embedded cutaneous T-cell lymphoma (CTCL) samples. A color key refers to gene expression in log(transcripts per million).

Figure 6.

Unsupervised hierarchical clustering analysis based on TruSeq targeted RNA gene expression analysis of 284 select genes in ≥2009 early-stage (stage ≤IIA, yellow), mid-stage (stages IIB and III, orange), and late-stage (stage IV, dark red) formalin-fixed paraffin-embedded cutaneous T-cell lymphoma (CTCL) samples. A color key refers to gene expression in log(transcripts per million).

Identification of Differentially Expressed Genes (DEGs) in All FFPE CTCL Samples Using Subgroup Analyses Based on the Year of Biopsy and CTCL Clinical Cancer Stage

We next searched for the DEGs that were highlighted when we compared (1) early-stage (≤IIA) to mid and late CTCL stages (≥IIB) samples and (2) stage I vs. stage IV CTCL samples. Similarly, in this case, we analyzed ≤2008 and ≥2009 CTCL samples separately to test the robustness of the TruSeq results based on the year of skin biopsy. For ≤2008 samples, 12 genes were highlighted as being statistically significant: TOX, EED, and LCP2 were upregulated in late-stage CTCL, while ATXN7, CHD1, HUNK, TP63, KIT, JUNB, LTBP4, HDAC2, and OTUB2 were expressed preferentially in early-stage CTCL samples (Table 3). For ≥2009 samples, three different genes were identified: SKAP1 and GTSF1 were upregulated in late-stage CTCL, while BCL11A was upregulated in early-stage CTCL (Table 4). Overall, merging both subgroups, we validated three of the four genes observed in the full data set when performing the same analysis: TOX and GTSF1 were upregulated in late-stage CTCL, and LTBP4 was upregulated in early-stage CTCL.

Table 3.

Genes with statistically significant differences in expression both between ≤2008 early-stage (≤IIA) vs. mid- and late-stage (≥IIB) formalin-fixed paraffin-embedded (FFPE) cutaneous T-cell lymphoma (CTCL) samples (left panel) and between ≤2008 stage I vs. stage IV FFPE CTCL samples (right panel).

Genes	Average of early CTCL [transcripts per million (TPM)]	Average of mid and late stages of CTCL (TPM)	log2 ratio early vs. mid and late stages of CTCL	p Value early vs. mid and late stages of CTCL	Average of stage I CTCL (TPM)	Average of stage IV CTCL (TPM)	log2 ratio of stage I vs. IV CTCL	p Value of stage I vs. IV CTCL
LCP2	1,279.782	2,740.947	1.098776	0.001332	1,351.527	2,396.473	0.826323	0.009062
TOX	2,286.086	4,579.155	1.002202	0.008709	2,343.758	4,502.264	0.941827	0.049764
JUNB	29,594.68	18,186.07	−0.7025	0.004493	28,825.75	19,214.87	−0.58513	0.040127
EED	5,408.253	2,911.241	−0.89353	0.025434	5,518.367	3,032.208	−0.86387	0.048117
ATXN7	936.3	494.4571	−0.92113	0.028724	932.8667	436.17	−1.09678	0.042606
KIT	1,493.838	629.5737	−1.24658	0.00016	1,529.729	695.6857	−1.13677	0.001263
TP63	13,320.81	5,028.95	−1.40535	0.005549	13,088.99	6,156.644	−1.08814	0.04105
CHD1	1,214.245	349.3444	−1.79734	0.017354	1,291.15	339.2167	−1.92838	0.017985
HUNK	512.48	112.225	−2.1911	0.046422	512.48	112.225	−2.1911	0.046422
LTBP4	732.3111	119.0667	−2.62069	0.014127	604.2125	142.45	−2.0846	0.029265
HDAC2	554.4625	83.8	−2.72607	0.025829	554.4625	111.4667	−2.31448	0.033172
OTUB2	8,321.478	1,190	−2.80588	0.012208	9,104.825	1,190	−2.93567	0.010448

Average expression is presented as TPM. Positive log-2 ratios indicate upregulation in CTCL samples and negative log-2 ratios indicate downregulation in CTCL samples. p Values from Wald’s t-test are presented.

Table 4.

Genes with statistically significant differences in expression both between ≥2009 early stage (≤IIA) vs. mid and late stage (≥IIB) formalin-fixed paraffin-embedded (FFPE) cutaneous T-cell lymphoma (CTCL) samples (left panel) and between ≥2009 stage I vs. stage IV FFPE CTCL samples (right panel).

Genes	Average of early CTCL [transcripts per million (TPM)]	Average of mid and late stages of CTCL (TPM)	log2 ratio of early vs. mid and late stages of CTCL	p Value early vs. mid and late stages of CTCL	Average of stage I CTCL (TPM)	Average of stage IV CTCL (TPM)	log2 ratio of stage I vs. IV CTCL	p Value of stage I vs. IV CTCL
GTSF1	739.5667	3,103.076	2.068947	0.006142	434.14	2,350.786	2.436911	0.046242
SKAP1	1,361.83	2,508.23	0.881123	0.011364	1,350.189	2,487.344	0.881445	0.03311
BCL11A	12,154.45	7,382.605	−0.71928	0.011902	12,530.53	7660.058	−0.71002	0.037322

Average expression is presented as TPM. Positive log-2 ratios indicate upregulation in CTCL samples and negative log-2 ratios indicate downregulation in CTCL samples. p values from Wald’s t-test are presented.

This subgroup analysis showed moderate consistency in log-2 ratios ≤2008 and ≥2009 samples when comparing early vs. mid and late FFPE CTCL samples (ρ = 0.28; low; p < 10⁻⁴). However, there was no correlation when comparing stage I vs. stage IV FFPE tissues (ρ = 0.06; no correlation; p = 0.36).

Comparison of the TruSeq Data Quality in FFPE vs. Freshly Obtained Snap-Frozen Samples

With respect to the RINs, a measure of sample quality prior to conducting the TruSeq analysis, we observed lower RINs for FFPE samples than freshly obtained snap-frozen samples. However, these RINs were within the expected range for FFPE samples (56, 57). RINs were much higher in fresh samples than in the FFPE samples, as expected (fresh: mean 6.1, 95% CI 5.5–6.8; FFPE: mean 2.4, 95% CI 2.3–2.5, p < 10⁻⁶ with MCMC). RNA libraries were also more concentrated in the freshly obtained samples (fresh: mean 227 ng/µL, 95% CI 110–239; FFPE: mean 64 ng/µL, 95% CI 53–75; p = 0.0012 with MCMC). There was no difference in RINs between ≤2008 and ≥2009 FFPE samples [≤2008: mean 2.3, 95% confidence interval (95% CI) 2.2–2.3; ≥2009: mean 2.4, 95% CI 2.3–2.6; p = 0.92 with MCMC]. RNA libraries were less concentrated in ≤2008 FFPE samples vs. ≥2009 samples, possibly explaining in part the lower TruSeq sequencing depth in ≤2008 samples (≤2008: mean 44 ng/µL, 95% CI 33–55; ≥2009: mean 76 ng/µL, 95% CI 60–92; p = 0.0007 with MCMC).

To detect possible batch effects, we performed PCA on TPM data. We aimed to determine whether (1) freshly obtained flash snap-frozen samples cluster together and (2) ≤2008 and ≥2009 FFPE samples cluster in different areas. We observed a tight cluster of fresh CTCL samples (gray), whether using ≤2008 (Figures 7A,B) or ≥2009 (Figures 7C,D) FFPE CTCL samples, indicating that differences in preprocessing protocols might explain these findings (tight associations in clustering analyses). However, these freshly obtained samples were also in close spatial proximity to normal/benign samples and a number of FFPE samples. When comparing ≤2008 and ≥2009 FFPE samples, we observed no clear clusters (Figures 7E,F), but rather two loose associations. First, many newer samples (≥2009) were clustering around the center of the distribution, toward normal/benign and freshly obtained samples, indicating less preprocessing batch effect. Second, samples showing greater variability were mostly older samples (≤2008), indicating that there might be some processing batch effect among these. Taken together, these findings may explain why performing individual subgroup analyses enabled us to uncover additional DEGs.

Figure 7.

Principal component score plots. (A,B) First and second principal component scores of normal/benign (green), freshly obtained and liquid nitrogen snapped-frozen cutaneous T-cell lymphoma (CTCL) (gray), ≤2008 early-stage formalin-fixed paraffin-embedded (FFPE) CTCL (yellow), ≤2008 mid-stage FFPE CTCL (orange), and ≤2008 advanced stage FFPE CTCL (red) samples are plotted. (C,D) First and second principal component scores of normal/benign (green), freshly obtained and liquid nitrogen snapped-frozen CTCL (gray), ≥2009 early-stage FFPE CTCL (yellow), ≥2009 mid-stage FFPE CTCL (orange), and ≥2009 advanced stage FFPE CTCL (red) samples are plotted. (E,F) First and second principal component scores of normal/benign (green), freshly obtained, and liquid nitrogen snapped-frozen CTCL (gray), ≤2008 FFPE CTCL (red), and ≥2009 FFPE CTCL (yellow) samples are plotted.

Discussion

In this study, we used subgroup analysis to determine whether older ≤2008 FFPE samples, which were sequenced at a lower depth on the TruSeq platform, were comparable to those obtained ≥2009. In this study, we also systematically analyzed sample processing biases based on the year of biopsy and the nature (i.e., FFPE vs. freshly obtained snap frozen) of the samples. Clustering analysis showed that ≤2008 and ≥2009 samples clustered equally well to the full data set and, furthermore, in a number of instances they demonstrated even better defined clusters. In particular, for ≤2008 samples, clusters were more reminiscent of the three clusters found in the landmark Boston CTCL cohort (3, 32, 48) when looking at all samples. There was also a better discrimination between benign and stage IV CTCL samples in ≤2008 samples than in the ≥2009 samples. Both analyses produced nearly identical trends and findings. Specifically, both analyses enriched nearly identical DEGs when comparing benign vs. (1) stage I–IV and (2) stage IV (alone) CTCL samples. Importantly, in this subgroup analysis, we recapitulated most of the targets seen within the full data set. Results obtained using either ≤2008 or ≥2009 samples were strongly correlated. Known upregulated targets in CTCL vs. benign dermatoses were validated, including TOX, FYB, LEF, and STAT signaling genes, inflammatory interleukins, NF-κB pathway signaling members, cancer testis genes, etc. We had previously reviewed in detail how these genes relate to the biology of CTCL tumorigenesis (3, 31).

Furthermore, this subgroup analysis enabled us to discover additional genes, which did not reach statistical significance in the full data set analysis. One may find it to be counterintuitive. However, indeed, despite the inherently decreased power, potential reasons why additional DEGs can be identified through subgroup analysis may include reduced variability on a per-sample basis due to increased in-group similarity and removal of outliers in some groups.

Those new DEGs included CTCL-upregulated BCL11A (regulation of RNA transcription), SELL (cell adhesion molecule in the selectin family), IRF1 (Interferon transcription factor), SMAD1 (BMP signaling and gene expression), CASP1 (caspase involved in proteolysis), BIRC5 (inhibitor of apoptosis, survivin), MAX (Myc-associated transcription factor), and CTCL-downregulated MDM4 (negative regulator of p53), SERPINB3 (serine protease involved in inflammatory response), and THBS4 (cell-cell and cell-matrix interactions) genes. Of note, THBS4 promoter was previously found to be frequently hypermethylated in 52% of CTCL samples, which leads to the downregulation in expression of this tumor suppressor gene (58). CASP1 single-nucleotide polymorphisms were associated with changes in NF-κB signaling and development of other non-Hodgkin lymphomas, including diffuse-large B cell lymphomas and small lymphocytic lymphoma/chronic lymphocytic leukemia (59).

By using the full data set analysis, we found significant heterogeneity in our clusters (41). When we performed clustering on ≤2008 or ≥2009 FFPE CTCL samples, we still did not obtain three clusters that were previously described in the historic Boston cohort of CTCL patients (3, 32, 48). However, in this study of subgroup analyses, we noted less heterogeneity than we observed in the full data set analysis (41). PCA results also supported this conclusion. Indeed, in this subgroup analysis, samples of similar clinical disease stages were most often grouped together.

Subsequently, when we studied the DEGs enriched in both (1) early vs. mid and late CTCL and (2) stage I vs. stage IV disease, four genes were differentially expressed: TOX (involved in chromatin processes and T-cell development), FYB (T-cell adaptor protein), and GTSF1 (germ cell maintenance) were upregulated, and LTBP4 (latent TGF-beta binding protein) was downregulated in later CTCL stages. By merging subgroup analysis of ≤2008 and ≥2009 FFPE samples, our targets included TOX, GTSF1, and LTBP4 as well. In particular, TOX overexpression is a hallmark of poor prognosis in CTCL, although low level of TOX expression has been previously reported in benign dermatoses (31, 60). TOX and GTSF1 are aberrantly expressed developmental and meiotic genes that can prognosticate CTCL progression toward advanced disease (29, 31, 34). We also found that EED (Polycomb complex member expressed in embryonic stem cells), SKAP1 (T-cell adhesion), and LCP2 (T-cell receptor-mediated signaling) were upregulated in advanced CTCL stages. Surprisingly, we also found multiple genes with higher expression in early-stage tumors. These included BCL11A (see above), ATXN7 (chromatin remodeling, AKT signaling), HUNK (AMPK-related kinase), CHD1 (chromatin remodeling), TP63 (transcription factor), KIT (receptor tyrosine kinase), JUNB (transcription factor), HDAC2 (histone deacetylase), and OTUB2 (deubiquitinase, inhibits proteolysis). Based on these combined results, transcription factors, chromatin remodelers, and global cell signaling processes are upregulated early in the disease, while in the advanced stages of CTCL, T-cell-specific genes, inflammatory mediators, and stem cell/germ cell maintenance genes appear to be driving cancer progression. These results further argue that subgroup analysis can often yield additional clues into the biology of cancers.

Formalin-fixed paraffin-embedded samples have RNA of lesser quality than the freshly obtained snap-frozen samples (61). However, FFPE samples are much easier to obtain in the clinical setting, have longer storage half-life, and are suitable for immunohistochemistry in a clinical pathology lab (62). Our FFPE RINs were comparable to those obtained in previous studies (56, 57). No matter if we performed subgroup analyses or full data set analysis, fresh samples tightly clustered together. While PCA revealed that fresh samples were spatially closer together, indicating some preprocessing batch effect, they remained in the proximity to other normal/benign and FFPE CTCL samples and were not clustering as outliers by themselves. However, this observed batch effect did not affect the determination of DEGs when analyzing all ≥2009 samples (fresh and FFPE biopsies) vs. ≥2009 FFPE samples alone. Other reports comparing freshly obtained frozen samples to FFPE samples showed a strong correlation (ρ > 0.70) in gene expression analysis (63). Formalin acts as a crosslinking agent for protein–protein, DNA–protein, and RNA–protein interactions (64). Crosslinking nucleic acid to proteins has its advantages in molecular medicine and is especially useful in characterizing transcription factor binding sites via chromatin immunoprecipitation (65) or RNA–protein interactions using RNA immunoprecipitation (66). In this study, we have successfully applied TruSeq targeted RNA sequencing to CTCL samples, both fresh and FFPE. A recent, direct comparison of TruSeq-analyzed RNA obtained from matched FFPE vs. fresh samples produced strongly correlated gene expression findings (R² > 0.70) (67). Interestingly, previous studies showed that the RINs can range from 2.2 to 2.8 (median 2.3) for FFPE samples and 3.8 to 8.0 (median 6.8) for freshly obtained samples (67), which is consistent with our findings detailed in this report. In the study by Graw et al., illumina sequence reads between FFPE and freshly obtained matched samples showed a 0.33% error rate (67), which is consistent to previous reports for identical samples processed on the Illumina platform, when a 0.30% error rate was reported (68). In summary, our results indicate that performing targeted gene expression studies on the TruSeq platform from FFPE samples is a viable option that can be used in the real-life, clinical medicine setting.

Ethics Statement

All patients were enrolled in the study in accordance with the IRB-approved protocols: PA12-0267, PA12-0497, and Lab97-256 at the MD Anderson Cancer Center (MDACC) and A09-M106-13A and 13-201-GEN at McGill University/McGill University Health Centre (MUHC). This study was carried out in accordance with the recommendations of the Research Ethics Board of the McGill University/McGill University Health Centre with written informed consent from all subjects in accordance with the Declaration of Helsinki. This study was carried out in accordance with the recommendations of the MD Anderson Cancer Center (MDACC) Research Ethics Board, which exempted us from obtaining written informed consent from patients, who earlier signed a hospital consent allowing their stored biopsy samples to be used for research.

Author Contributions

PL, EN, MT, LM, AW, DS, XN, NP, MG, MD, and IL procured and analyzed patient sample data presented in this paper. PL, EN, and IL performed bioinformatic and statistical analyses. MT and AW performed pathological analysis of the original skin samples. PL, EN, MT, LM, AW, DS, XN, NP, MG, MD, and IL wrote the paper. NP, LM, MG, DS, MD, and IL supervised the study.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YG and handling editor declared their shared affiliation.