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Keywords:

  • Affymetrix;
  • comparative genomic hybridization;
  • diffuse large B-cell lymphoma;
  • human immunodeficiency virus;
  • immunodeficiency;
  • solid organ transplant

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

Post-transplant lymphoproliferative disorders (PTLD) are complications of solid organ transplantation associated with severe morbidity and mortality. Diffuse large B-cell lymphoma (DLBCL) represents the most common form of monomorphic PTLD. We studied 44 cases of post-transplant DLBCL (PT-DLBCL) with high-density genome wide single nucleotide polymorphism-based arrays, and compared them with 105 cases of immunocompetent DLBCL (IC-DLBCL) and 28 cases of Human Immunodeficiency Virus-associated DLBCL (HIV-DLBCL). PT-DLBCL showed a genomic profile with specific features, although their genomic complexity was overall similar to that observed in IC- and HIV-DLBCL. Among the loci more frequently deleted in PT-DLBCL there were small interstitial deletions targeting known fragile sites, such as FRA1B, FRA2E and FRA3B. Deletions at 2p16.1 (FRA2E) were the most common lesions in PT-DLBCL, occurring at a frequency that was significantly higher than in IC-DLBCL. Genetic lesions that characterized post-germinal center IC-DLBCL were under-represented in our series of PT-DLBCL. Two other differences between IC-DLBCL and PT-DLBCL were the lack of del(13q14.3) (MIR15/MIR16) and of copy neutral LOH affecting 6p [major histocompatibility complex (MHC) locus] in the latter group. In conclusion, PT-DLBCL presented unique features when compared with IC-DLBCL. Changes in PT-DLBCL were partially different to those in HIV-DLBCL, suggesting different pathogenetic mechanisms in the two conditions linked to immunodeficiency.

Post-transplant lymphoproliferative disorders (PTLD) are an important complication of solid organ transplantation associated with severe morbidity and mortality (Knowles, 1999; Friedberg & Swinnen, 2006; Swerdlow et al, 2008; Vajdic & van Leeuwen, 2009). The incidence of PTLD can be as high as 20% of allo-transplant recipients, depending on the type of transplanted organ, immunosuppressive protocol, and pre-transplant Epstein-Barr virus (EBV) immune status. On the basis of the last World Health Organization (WHO) classification of haematopoietic tumors (Swerdlow et al, 2008), PTLD comprise a spectrum of lymphoproliferative disorders: early lesions, polymorphic PTLD, monomorphic PTLD, and classical Hodgkin lymphoma-type PTLD. While early lesions are mostly EBV-driven polyclonal lymphoproliferations, the remaining three categories are monoclonal disorders. The classical Hodgkin lymphoma-type PTLD is the least common entity and it resembles classical Hodgkin lymphoma. The polymorphic PTLD are morphologically polymorphic lesions showing different stages of the B cell maturation. The monomorphic PTLD fulfill the criteria for one of the lymphomas observed in immunocompetent host, and among them, diffuse large B-cell lymphoma (DLBCL) represents the most common form. The majority of PTLD arising after solid organ transplantation and from recipient lymphocytes are derived from post-germinal center (GC) B-cells (Brauninger et al, 2003; Capello et al, 2003, 2005; Vakiani et al, 2008). Apart from EBV infection, presenting as a latency-type III, knowledge regarding the pathogenesis of post-transplant DLBCL (PT-DLBCL) is limited. Molecular alterations of BCL6, as well as aberrant somatic hypermutation of several proto-oncogenes and epigenetic alterations have been reported (Cesarman et al, 1998; Rossi et al, 2003, 2004; Cerri et al, 2004; Capello et al, 2005; Vakiani et al, 2007). A conventional comparative genomic hybridization (CGH) study reported that PTLD bear several genomic aberrations common to lymphomas, such as 8q24, 3q27, 18q21 gains and 17p13 loss, but also aberrations that are less frequent in other lymphomas, such as 5p gain and 4q, 17q, Xp losses. In a previous analysis of 20 PTLD, including 13 PT-DLBCL, by first generation single nucleotide polymorphism (SNP)-based array-CGH, we noted the presence in PT-DLBCL of recurrent lesions similar to that observed in DLBCL occurring in immunocompetent host (IC-DLBCL), although with a lower frequency (Rinaldi et al, 2006). The application of techniques at a higher genomic resolution in Human Immunodeficiency Virus (HIV)-related DLBCL (HIV-DLBCL) has recently allowed us to detect genomic lesions distinct from that observed in IC-DLBCL, including deletions involving genes at overlapping fragile sites, such as FHIT or WWOX (Capello et al, 2010). As important pathogenetic lesions may not have been detected with the previous generations of microarrays, we analyzed a large series of PT-DLBCL with the same technique used for HIV-DLBCL.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

Tumor panel

Frozen samples of 44 PT-DLBCL were obtained in the course of routine diagnostic work-up prior to initiating therapy. Cases were selected for the study based upon the availability of frozen material and for having a fraction of malignant cells in the pathological specimen representing >70% of overall cellularity as determined by morphological and/or immunophenotypic studies. Median time from transplant to PT-DLBCL was 84 months (range 3–343). In a subset of 24 cases, information on the cell of origin based on the algorithm reported by Hans et al (2004) was available, showing a majority (71%, 17/24) of post-GC DLBCL. For comparative purposes, 105 IC-DLBCL and 28 HIV-related clinical specimens were also investigated, as previously described (Capello et al, 2010). The procedures and protocols for sample collection were approved by the ethics committees and institutional review boards of the participating Institutions.

DNA extraction, array-CGH analysis and data mining

DNA was extracted and its integrity was verified as previously described (Capello et al, 2010). DNA samples were analyzed using the GeneChip Human Mapping 250K NspI (Affymetrix, Santa Clara, CA, USA), as previously described (Capello et al, 2010). Data mining was performed as previously reported (Rancoita et al, 2009; Capello et al, 2010). The modified Bayesian Piecewise Regression (mBPCR) method (Rancoita et al, 2009) was used to estimate the copy number (CN) starting from values obtained with Affymetrix CNAT 4.01. After normalization of each profile to a median log2-ratio of zero, thresholds for loss and gain were defined as six times the median absolute deviation symmetrically around zero with an associated P-value lower than <0·001 after Bonferroni multiple test correction. Loss of heterozygosity (LOH) profiles were obtained applying the method with haplotype correction for tumor-only LOH inference available in the dChip software (Beroukhim et al, 2006). The recurrent minimal common regions (MCR) were defined using the algorithm by Lenz et al (2008a). For MCR occurring in at least 15% of cases, differences in MCR frequencies between subgroups were evaluated using a Fisher’s exact test followed by Benjamini and Hochberg multiple test correction (q-value). The commonly affected regions were compared with the Database of Genomic Variants (http://projects.tcag.ca/variation/): regions showing an overlap above 80% between probes and known copy number variations (CNV) were considered bona fide CNV. The University of California, Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu) (Karolchik et al, 2008) was used to retrieve additional information.

Results

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

Recurrent lesions in PT-DLBCL

We analyzed a total of 44 PT-DLBCL by high-resolution SNP-based a-CGH. Fig 1 shows the frequency of CN changes and LOH in PT-DLBCL. To obtain a more precise representation of the lesions characterizing these disorders, we determined the MCR, which are the minimal recurrent alterations that are supposed to contain loci relevant for the tumor. Four types of MCR were defined: abnormal chromosome arm (ACA), abnormal whole chromosome (AWC), short recurrent abnormality (SRA) and long recurrent abnormality (LRA) (Table SI).

image

Figure 1.  Frequency of DNA gains (up) and losses (down) (upper panel) and LOH (lower panel) observed in 44 PT-DLBCL primary samples. X-axis, chromosome localization and physical mapping; Y-axis, percentage of cases showing the aberrations.

Download figure to PowerPoint

The most common SRA and LRA are shown on Table I. Recurrent AWC and ACA gains affected chromosome 11 (11q, 18%, 8/44; 11p, 7%, 3/44), chromosome 7 (7q, 7p, 16%, 7/44; whole 7, 9%, 4/44), 1q (11%, 5/44), 21p (9%, 4/44), 9p and 20q (7%, 3/44). Regarding DNA losses, the most common AWC and ACA were del(17p) (11%, 5/44), followed by del(4p), and del(4q) (11%, each 4/44).

Table I.   Most common short and long recurrent abnormalities in PT-DLBCL. Bona fide copy number variations are not included.
Type and percentageLocationMCR core start*Size
  1. *, start of the minimal core region, defined as the region covered by at least two-thirds of the aberrations in the overlapping group, according to the human reference sequence NCBI Build 36.1.

Gains
2511q219.43E + 071·98E + 06
11q23.21·15E + 082·60E + 05
231q31.21·90E + 084·42E + 05
7p15.13·15E + 071·78E + 04
11q24.11·22E + 081·55E + 05
204q21.217·96E + 075·12E + 05
7p12.34·72E + 076·16E + 05
11p133·49E + 073·33E + 05
11q14.2-q258·60E + 074·84E + 07
11q24.31·28E + 089·40E + 05
181q25.21·77E + 081·24E + 05
2q32.31·97E + 083·76E + 05
7p22.3-q31.21·41E + 051·17E + 08
7q341·39E + 086·72E + 04
1612q21.338·85E + 075·20E + 05
16q23.17·47E + 072·21E + 05
16q24.18·36E + 075·40E + 04
141q21.3-q441·53E + 089·45E + 07
2p24.21·77E + 072·03E + 05
2p22.3-p23.13·11E + 072·50E + 06
6p22.31·85E + 074·91E + 05
6p22.22·50E + 076·71E + 04
6p12.15·32E + 071·95E + 05
9p13.33·56E + 074·19E + 05
Losses
302p16.15·79E + 074·00E + 05
161p36.31-p36.337·76E + 055·36E + 06
4p11-p124·82E + 075·72E + 05
6q23.31·37E + 083·54E + 05
17p12-p13.31·89E + 041·12E + 07
144q25-q35.21·09E + 088·19E + 07

MCR of amplification were detected at 17q21.31 and 9p24.1. Amplification at 17q21.31 was probably due to copy number variation, whereas lesions targeting 9p24.1 (C9orf123 and PTPRD) occurred in two patients, and they were associated with interstitial deletions targeting the CDKN2A locus. In one case, the 9p alterations were confirmed by the available G-band karyotype analysis. No MCR were detected for homozygous deletions.

EBV-positive PT-DLBCL presented less recurrent lesions than EBV-negative PT-DLBCL, including gain of 7p (0/23, 0%, vs. 6/19, 32%; P-value = 0·005; q-value = 0·33), del(4q25-q35) (0/23, 0%, vs. 5/19, 26%; P-value = 0·014; q-value = 0·33), gains of 7q (1/23, 4%, vs. 7/19, 37%; P-value = 0·015; q-value = 0·33), 11q24-q25 (2/23, 9%, vs. 8/19, 42%; P-value = 0·026; q-value = 0·33) (Table SII).

PT-DLBCL following heart transplant showed a trend for a higher prevalence of gains affecting 6p when compared with other cases: 5/14, 35% vs. 1/30 (P = 0·009; q-value = 0·23). No specific CN changes could be observed for the 16 cases after kidney transplant. There were only seven cases of PT-DLBCL after liver transplant, but they comprised the two cases bearing CDKN2A (9p21.3) deletions with concomitant amplification of KDM4A (JMJD2), C9orf123 and PTPRD (9p23-p24.1).

PT- and IC-DLBCL have differences in their genomic profiles

With the aim of evaluating genomic differences between PT- and IC-DLBCL, we compared the genomic profiles of 44 PT-DLBCL and 105 IC-DLBCL analyzed with the same technique (Capello et al, 2010) (Table II, Fig S1 and Table SIII). Del(2p16.1), which contains the fragile site FRA2E and the FANCL and VRK2 genes, and gain of 4q21.21 were more frequently observed among PT-DLBCL. In contrast, del(13q14.3) (MIR15/MIR16), gains of 18q (BCL2 and NFATC1), and LOH at 6q21-q22 (approximately 7 Mb telomeric from PRDM1[BLIMP1]) and at 6p21.32-p21.33 (HLA-DR locus) were more frequent among IC-DLBCL. A trend to a higher frequency of loss at 6q, including PRDM1 and TNFAIP3 (A20), was observed in IC-PTLD, compared to PT-DLBCL (24/105, 23%, vs. 4/44, 9%; P-value = 0·065, q-value = 0·35). Recurrent lesions including gains of 1q, 11q and of chromosome 7, as well as losses at 17p were detected with similar frequencies in PT- and IC-DLBCL.

Table II.   The most significantly different regions between PT-DLBCL and IC-DLBCL (A), PT-DLBCL and IC-GCB-DLBCL (B), and PT-DLBCL and IC-ABC-DLBCL (C) as evaluated by applying Fisher’s exact test (P-value) followed by multiple test correction (q-value). Higher frequencies are in bold face.
A
RegionPT-DLBCL%IC-DLBCL%P valueq value
del(2p16.1) (FRA2E)13/44304/1054<0·00010·0006
4q21.21 gain9/44200/1050<0·00010·0004
del(13q14.3) (MIR15/MIR16)0/44016/105150·0030·04
6q21-q22 LOH0/44016/105150·0030·03
6p21.32-p21.33 LOH (HLA-locus, TNF)2/44425/105240·0050·04
18q gain (BCL2, NFATC1)2/44520/105190·0230·21
B
RegionPT-DLBCL%IC-GCB-DLBCL%P valueq value
LOH at 1p36.32-p36.232/44511/3037<0·00010·025
del(2p16.1) (FRA2E)13/44301/3030·0050·07
6p21.32-p21.33 LOH (HLA-locus, TNF)2/4449/30300·0060·07
17p11.2-p13.3 LOH (TP53)1/4427/30230·0060·07
6q21-q22 LOH0/4405/30170·0090·07
2p14-p16.1 gain (REL, BCL11A)5/441111/30370·0190·13
7q gain7/441612/30400·0290·18
del(18q22.1-q23)3/4478/30270·0410·18
C
RegionPT-DLBCL%IC-ABC-DLBCL%P valueq value
del(2p16.1) (FRA2E)13/44300/1900·00630·06
3q gain (BCL6, NFKBIZ)0/4404/19210·00650·063
18q gain (BCL2, NFATC1)2/4456/19320·00720·072
6p21.32-p21.33 LOH (HLA-locus, TNF)2/4446/19320·00790·063

Given that 49/105 (47%) IC-DLBCL had been previously characterized for their cell of origin using gene expression profile (Lenz et al, 2008b), we also compared PTLD-DLBCL versus 19 activated B-cell like (ABC)-DLBC and 30 GCB-DLBCL. When compared with the latter, PT-DLBCL presented more commonly del(2p16.1) (FANCL and VRK2) but less gains of 7q and at 2p14-p16.1 (REL/BCL11A), and del(18q22.1-q23), suggestive of translocations involving BCL2), LOH at 1p36.32-p36.23 (containing also TP73), at 6p21.32-p21.33 (HLA and TNF), 17p11.2-p13.3 (TP53), and at 6q21-q22 (Table SIII). ABC-DLBCL had more gains of 18q (BCL2, NFATC1) and 3q (BCL6, NFKBIZ), and LOH at 6p21.32-p21.33 (HLA and TNF locus) while PTLD-DLBCL presented more del(2p16.1) (FANCL and VRK2) (Table SIII).

PT- and HIV-DLBCL have differences in their genomic profiles

Comparison of the genomic profiles of the 44 PT-DLBCL with that of 28 HIV-DLBCL that we reported recently (Capello et al, 2010) led to the identification of significant differences between the two groups of DLBCL (Fig S2 and Table SIV). PT-DLBCL presented more frequently del(2p16.1) (FANCL and VRK2; 13/44, 30%, vs. 1/28, 4%; P-value = 0·0063, q-value = 0·11) and gains at 4q21.21 (9/44, 20%, vs. 0/28, 0%; P-value = 0·01, q-value = 0·15) and at 9p13 (10/44, 23%, vs. 1/28, 4%; P-value = 0·042, q-value = 0·17) and displayed fewer gains at 2p16.1-p25.3 (1/44, 2%, vs. 7/28, 25%; P-value = 0·0046, q-value = 0·11) and at 12q21.31 (2/44, 9%, vs. 6/28, 43%; P-value = 0·012, q-value = 0·08). PT- and HIV-DLBCL showed similar occurrence of gains at 1q, chromosome 7, 11q, and deletion at 17p. Deletions of FHIT were seen in 25% of HIV-DLBCL and in 9% of PT-DLBCL (P = 0·095), in which, however, deletions on WWOX alterations, present in 11% of HIV-DLBCL, were not found in any case (P-value = 0·055).

Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

This report presents the results of comparative high density genome-wide analysis of a large series of PT-DLBCL, IC-DLBCL and HIV-DLBCL.

The genomic profile of PT-DLBCL was overall similar to that observed in IC- and HIV-DLBCL. Nevertheless, PT-DLBCL showed specific features. Among the loci more frequently deleted in PT-DLBCL there were a series of small interstitial deletions at 1p32.2, 2p16.1, 3p14.2, 4p14, 14q13.2, 20p12.3, 20q13.32. At least some of these aberrations targeted known fragile sites, such as FRA1B, FRA2E and FRA3B. In particular, del(2p16.1) (FRA2E) was the most common lesion in PT-DLBCL, occurring at a frequency that was highly statistically different from IC-DLBCL. Fragile sites are chromosomal regions that tend to break when cells are exposed to specific culture conditions or chemical agents (Freudenreich, 2007). Interestingly, we have recently shown that HIV-DLBCL also differs from IC-DLBCL due to a higher frequency of fragile sites deletions (Capello et al, 2010). Deletion of Fragile Histidine Triad (FHIT), a gene overlapping the FRA3B fragile site, was the most common event in HIV-DLBCL, and it was often associated with deletion of another fragile site-associated tumor suppressor gene, WW domain-containing Oxidoreductase (WWOX), at 16q23.1 (Capello et al, 2010). Among PT-DLBCL, although FHIT was deleted in a fraction of cases, alterations of WWOX were not observed.

Deletions occurring at FRA2E targeted two genes, Vaccinia Related Kinase 2 (VRK2) and Fanconi anemia, complementation group L (FANCL). The two genes were not always concomitantly deleted, nor there was correlation with FHIT deletion. VRK2 acts as a negative regulator of the mitogen-associated protein kinases (MAPK) pathway (Blanco et al, 2008; Blonska & Lin, 2009), while FANCL is an E3 ubiquitin ligase component of the FA nuclear protein complex mainly involved in the DNA repair, but also in telomere stability, chromatin remodeling, cell cycle regulation and apoptosis (Bogliolo et al, 2002; Taniguchi & D’Andrea, 2006). FANCL is responsible for mono-ubiquitination of FANCD2, a necessary step for the accumulation of this molecule at the sites of DNA damage (Taniguchi & D’Andrea, 2006). A deficit of FANCD2 has been associated with increased instability at fragile sites (Howlett et al, 2005). Patients with FA have an increased risk of cancer and inactivation of different members of the FA complex has been reported in human tumors (Taniguchi & D’Andrea, 2006), but data on FANCL deregulation in tumors are scarce (Zhang et al, 2006; Hess et al, 2008). No other genes of the FA core complex were recurrently altered in our series of PT-DLBCL (data not shown).

As suggested for HIV-DLBCL, an increased breakage at fragile sites could indicate a possible contribution of viruses in the pathogenesis of immunodeficiency-related DLBCL. In PT-DLBCL, the iatrogenic immunodeficiency status would expose the individuals to a multitude of viruses, which could infect B cells and integrate in the genome, preferentially at fragile sites (Rassool et al, 1992; Thorland et al, 2003; Luo et al, 2004; Feitelson & Lee, 2007). Indeed, the VRK2/FANCL locus is closely related to the EBV insertion site described in a Burkitt lymphoma cell line (Luo et al, 2004). The apparent different patterns of fragile site breakage that we have observed in PT- and in HIV-DLBCL could be due to different viruses that have preferential sites of integration (Ciuffi & Bushman, 2006) or, alternately, to distinct mechanisms of selection pressure exerted on lymphomatous cells by the immunological environment.

Although immunoglobulin variable gene mutational status and gene expression profile of PT-DLBCL are reminiscent of post-GC B-cells (Capello et al, 2005; Swerdlow et al, 2008; Vakiani et al, 2008), genetic lesions that characterize post-GC IC-DLBCL, such as gains of chromosome 3 (FOXP1, BCL6, NFKBIZ) and 18q (BCL2, NFATC1) together with losses of 6q (PRDM1 and TNFAIP3) (Lenz et al, 2008a; Compagno et al, 2009), were under-represented in our series of PT-DLBCL, reinforcing the notion that PT-DLBCL and other types of DLBCL that have similar immunophenotypic profiles are genetically heterogeneous (Capello et al, 2010), the most striking finding being the lack of chromosome 3q gains.

The most significant difference between PT-DLBCL and IC-DLBCL was the lack of del(13q14.3) in PT-DLBCL. A similar phenomenon was observed in the HIV-setting (Capello et al, 2010). The del(13q14.3) targets the locus coding for different non-coding RNAs, in particular MIR15/MIR16, and it is a common event also in other B cell neoplasms (Ferreira et al, 2008). Our data suggest that del(13q14.3) might contribute to immune surveillance escape, because it is not present in immunodeficiency-related lymphomas.

Another observation could also be linked to immune surveillance. Differently from other lymphomas (Ross et al, 2007; O’Shea et al, 2009) and from both IC- and HIV-DLBCL (Capello et al, 2010), PT-DLBCL did not present copy neutral LOH affecting 6p. An absence or reduced expression of major histocompatibility class II (MHC-II) proteins is a relatively common feature of DLBCL (Riemersma et al, 2000; Rimsza et al, 2004; Roberts et al, 2006; Booman et al, 2008). While in cases arising in immuno-privileged sites, such as testis or central nervous system, low MHC-II expression seems to be due to genomic events, in the remaining DLBCL epigenetic changes or transcriptional mechanisms seem to be involved. Copy neutral LOH of 6p, due to mitotic recombinations, could contribute to the silencing of the MHC complex (Riemersma et al, 2000). DLBCL cases with low MHC-II expression have a lower number of infiltrating T cells and an impaired activation of cytotoxic CD8+ T cells (Booman et al, 2006). Interestingly, HIV-DLBCL present 6p copy neutral LOH at a frequency similar to IC-DLBCL (Capello et al, 2010). It can be speculated that PT-DLBCL cells, which arise in the context of an iatrogenic immunosuppression lowering both CD4+ and CD8+ T cells do not need to down-regulate the MHC complexes to escape. However, HIV-DLBCL do, as they show a more pronounced loss of CD4+ than of cytotoxic CD8+ T-cells.

Recurrent gains of 1q, 11q and of chromosome 7, as well as losses at 17p (TP53) could be observed at a similar frequency in PT-, HIV- and IC-DLBCL.

PT-DLBCL frequently presented losses affecting chromosome 4. Interestingly, losses of chromosome 4 together with 9p gains are among the recurrent lesions more commonly observed in primary mediastinal B-cell lymphomas (PMBCL) compared to DLBCL from other sites (Palanisamy et al, 2002; Kimm et al, 2007). As mentioned above, in our series recurrent gains and amplifications of 9p were detected, apparently targeting KDM4C, C9orf123 and PTPRD. Two regions have been reported as selectively gained in PMBCL, 9p13.1-p13.3 and 9p24.1, which are the same found in PT-DLBCL in the current series, and in another study (amplification at 9p22-p24) (Poirel et al, 2005). Similar to PMBCL, JAK2 is not part of the minimal common region in PT-DLBCL. JAK2 is, however, over-expressed in PBMCL, (Savage et al, 2003; Meier et al, 2009), but no data are available for PT-DLBCL. KDM4C encodes a nuclear protein that functions as a trimethylation-specific demethylase, converting specific trimethylated histone residues to the dimethylated form. KDM4C was recently reported to be translocated in extranodal marginal zone lymphomas (Vinatzer et al, 2008).

As EBV-positive PT-DLBCL had less recurrent lesions than EBV-negative PT-DLBCL, we also compared these two groups against the IC-DLBCL patients. All the described differences with IC-DLBCL were maintained irrespective of EBV status, although not reaching statistical significance due to the small samples sizes (data not shown). Del(2p16.1) was common in both EBV-negative and positive EBV-PT-DLBCL and almost absent in IC-DLBCL. The lack in PT-DLBCL of lesions common in IC-DLBCL (3q gain, 6p copy neutral LOH) was not affected by EBV status.

In conclusion, PT-DLBCL showed, in comparison with IC-DLBCL, distinctive features, such as the lack of del(13q.14.3), the lack of copy neutral LOH at 6p (HLA-DR locus) and the frequent breakage at fragile sites. The latter appeared partially different from what was observed in HIV-DLBCL, suggesting different pathogenetic mechanisms, albeit in the context of immunodeficiency. Importantly, the comparison with IC-, GC- and ABC-DLBCL subtypes indicated discrepancies between the PT-DLBCL phenotype and their genomic profile, as well suggesting possible similarities to PMBCL. At least a fraction of DLBCL showing similar morphological and phenotypic features but occurring within a distinct immunological setting could differ in their genomic profiles. Future studies will be needed to determine whether these divergent pathogenetic pathways are a consequence of a different histogenetic origin of these tumors or the result of the microenvironment stimulation and immunological selection or the effect of both conditions.

Acknowledgements

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

Work supported by: Oncosuisse grant OCS-1939-8-2006; Swiss National Science Foundation (grants 205321-112430, 205320-121886/1); Cantone Ticino (“Computational life science/Ticino in rete” program); Fondazione per la Ricerca e la Cura sui Linfomi (Lugano, Switzerland); Ricerca Sanitaria Finalizzata 2008 and 2009, Regione Piemonte, Torino, Italy; VI Programma Nazionale di Ricerca sull’AIDS, ISS, Rome, Italy; Novara-AIL Onlus. M.S. is enrolled in the PhD program in Pharmaceutical Sciences, University of Geneva, Switzerland. M.M. is recipient of fellowship from Alto Adige Bolzano-AIL Onlus. E.C. is recipient of an European Society for Medical Oncology (ESMO) Fellowship Grant.

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  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interest
  8. References
  9. Supporting Information

Figure S1. Comparison of frequency of DNA gains and losses between 44 PT-DLBCL (upper part) and 105 IC-DLBCL (lower part).

Figure S2. Comparison of frequency of DNA gains and losses between 44 PT-DLBCL (upper part) and 28 HIV-DLBCL (lower part).

Table SI. Minimal Common Regions (MCR) occurring in PT-DLBCL. "mcr.core.start" and "mcr.core.end" indicate the start and the end, according to the human reference sequence NCBI Build 36.1, of the MCR core region, defined as the region covered by at least two-thirds of the aberrations in the overlapping group. *, > 80% overlap with known CNV.

Table SII. Differences in terms of MCR occurrence between 23 EBV-positive and 19 EBV-negative PT-DLBCL as evaluated by applying Fisher's exact test (p-value) followed by multiple test correction (q-value). "mcr.core.start" and "mcr.core.end" indicate the start and the end of the MCR core region, defined as the region covered by at least two-thirds of the aberrations in the overlapping group. *, > 80% overlap with known CNV.

Table SIII. Differences in terms of MCR occurrence between 44 PT-DLBCL and 105 IC-DLBCL, 44 PT-DLBCL and 30 GCB-DLBCL, and 44 PT-DLBCL and 19 ABC-DLBCL, as evaluated by applying Fisher’s exact test (p-value) followed by multiple test correction (q-value). “mcr.core.start” and “mcr.core.end” indicate the start and the end of the MCR core region, defined as the region covered by at least two-thirds of the aberrations in the overlapping group. *, > 80% overlap with known CNV.

Table SIV. Differences in terms of MCR occurrence between 44 PT-DLBCL and 28 HIV-DLBCL, as evaluated by applying Fisher’s exact test (p-value) followed by multiple test correction (q-value). “mcr.core.start” and “mcr.core.end” indicate the start and the end of the MCR core region, defined as the region covered by at least two-thirds of the aberrations in the overlapping group. *, > 80% overlap with known CNV.

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