Frequent mutated B2M, EZH2, IRF8, and TNFRSF14 in primary bone diffuse large B-cell lymphoma reflect a GCB phenotype

Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is a rare extranodal lymphoma subtype. This retrospective study elucidates the currently unknown genetic background of a large clinically well-annotated cohort of DLBCL with osseous localizations (O-DLBCL), including PB-DLBCL. A total of 103 patients with O-DLBCL were included and compared with 63 (extra)nodal non-osseous (NO)-DLBCLs with germinal center B-cell phenotype (NO-DLBCL-GCB). Cell-of-origin was determined by immunohistochemistry and gene-expression profiling (GEP) using (extended)-NanoString/Lymph2Cx analysis. Mutational profiles were identified with targeted next-generation deep sequencing, including 52 B-cell lymphoma-relevant genes. O-DLBCLs, including 34 PB-DLBCLs, were predominantly classified as GCB phenotype based on immunohistochemistry (74%) and NanoString analysis (88%). Unsupervised hierarchical clustering of an extended-NanoString/Lymph2Cx revealed significantly different GEP clusters for PB-DLBCL as opposed to NO-DLBCL-GCB (P < .001). Expression levels of 23 genes of 2 different targeted GEP panels indicated a centrocyte-like phenotype for PB-DLBCL, whereas NO-DLBCL-GCB exhibited a centroblast-like constitution. PB-DLBCL had significantly more frequent mutations in four GCB-associated genes (ie, B2M, EZH2, IRF8, TNFRSF14) compared with NO-DLBCL-GCB (P = .031, P = .010, P = .047, and P = .003, respectively). PB-DLBCL, with its corresponding specific mutational profile, was significantly associated with a superior survival compared with equivalent Ann Arbor limited-stage I/II NO-DLBCL-GCB (P = .016). This study is the first to show that PB-DLBCL is characterized by a GCB phenotype, with a centrocyte-like GEP pattern and a GCB-associated mutational profile (both involved in immune surveillance) and a favorable prognosis. These novel biology-associated features provide evidence that PB-DLBCL represents a distinct extranodal DLBCL entity, and its specific mutational landscape offers potential for targeted therapies (eg, EZH2 inhibitors)

Genome Haploidisation with Chromosome 7 Retention in Oncocytic Follicular Thyroid Carcinoma

Contains fulltext : 108012.pdf (publisher's version ) (Open Access)BACKGROUND: Recurrent non-medullary thyroid carcinoma (NMTC) is a rare disease. We initially characterized 27 recurrent NMTC: 13 papillary thyroid cancers (PTC), 10 oncocytic follicular carcinomas (FTC-OV), and 4 non-oncocytic follicular carcinomas (FTC). A validation cohort composed of benign and malignant (both recurrent and non-recurrent) thyroid tumours was subsequently analysed (n = 20). METHODS: Data from genome-wide SNP arrays and flow cytometry were combined to determine the chromosomal dosage (allelic state) in these tumours, including mutation analysis of components of PIK3CA/AKT and MAPK pathways. RESULTS: All FTC-OVs showed a very distinct pattern of genomic alterations. Ten out of 10 FTC-OV cases showed near-haploidisation with or without subsequent genome endoreduplication. Near-haploidisation was seen in 5/10 as extensive chromosome-wide monosomy (allelic state [A]) with near-haploid DNA indices and retention of especially chromosome 7 (seen as a heterozygous allelic state [AB]). In the remaining 5/10 chromosomal allelic states AA with near diploid DNA indices were seen with allelic state AABB of chromosome 7, suggesting endoreduplication after preceding haploidisation. The latter was supported by the presence of both near-haploid and endoreduplicated tumour fractions in some of the cases. Results were confirmed using FISH analysis. Relatively to FTC-OV limited numbers of genomic alterations were identified in other types of recurrent NMTC studied, except for chromosome 22q which showed alterations in 6 of 13 PTCs. Only two HRAS, but no mutations of EGFR or BRAF were found in FTC-OV. The validation cohort showed two additional tumours with the distinct pattern of genomic alterations (both with oncocytic features and recurrent). CONCLUSIONS: We demonstrate that recurrent FTC-OV is frequently characterised by genome-wide DNA haploidisation, heterozygous retention of chromosome 7, and endoreduplication of a near-haploid genome. Whether normal gene dosage on especially chromosome 7 (containing EGFR, BRAF, cMET) is crucial for FTC-OV tumour survival is an important topic for future research. MICROARRAYS: Data are made available at GEO (GSE31828)

Pulmonary vein and atrial wall pathology in human total anomalous pulmonary venous connection

Background: Normally, the inside of the left atrial (LA) body and pulmonary veins (PVs) is lined by vessel wall tissue covered by myocardium. In total anomalous pulmonary venous connection (TAPVC), no connection of the PVs with the LA body exists. These veins have an increased incidence of PV stenosis. We describe the consequences of the absent connection for the histopathology of the wall of the LA body and the PVs, and hypothesize on a mechanism predisposing to PV stenosis. Methods and results: In 10 human neonates with TAPVC, the wall of the LA body and PVs were studied using histological and immunohistochemical techniques. As controls, 2 normal neonatal and adult hearts and 5 neonatal hearts with partial anomalous venous connection (PAPVC) or situs inversus were studied. In hearts with TAPVC no vessel wall tissue was found in the LA body and its myocardial layer was hypoplastic. No myocardial sleeve was found around the abnormally draining PVs. In hearts with PAPVC, only the non-LA draining PV lacked myocardial covering, whereas in situs inversus PVs connecting to the right-sided LA, were normally myocardialized. Conclusion: An open connection of the PVs with the morphological LA is necessary for the presence of vessel wall tissue in the LA and myocardialization of the PVs. Absence of myocardium covering the PVs is hypothesized to enhance susceptibility to PV stenosis and prevent onset of PV originating arrhythmias. The embryonic posterior heart field may be responsible for the abnormal myocardialization and smooth muscle cell formation in TAPVC. (C) 2008 Elsevier Ireland Ltd. All rights reserved

Examples of DNA content analysis of recurrent NMTC. Multiparameter DNA content analysis was performed on FFPE NMTC, as described.

<p><b>A</b>. Multiparameter DNA content analysis of a bimodal PTC with a DI of 1.02 and 2.05 (case No. 19), <b>B</b>. a PTC-OV with a DI of 0.97 (case No. 25) and <b>C</b>. a bi-modal FTC-OV with a DI of 0.53 and 1.04, respectively (case No. 13). <b>a</b>. Haematoxylin – eosin staining 200×. <b>b</b>. keratin vs. vimentin density plot (note the vimentin co-expression of these tumours and the clear separation between the stromal and the epithelial cell fraction. The expression of keratin and vimentin are high, relative to the controls showing background fluorescence [<b>d</b>]). Twenty-five samples, 93% (25/27), showed high vimentin co-expression in more than 50% of the cancer cells (data not shown). <b>c</b>. DNA histogram generated after gating on the epithelial cell fraction. <b>e</b>. DNA histogram generated after gating on the normal DNA diploid stromal cell fraction. This fraction was used as a DNA content reference. <b>f</b>. DNA histogram of the epithelial cell fraction after modelling by ModFit (note that the presence of a second cell cycling population in the bimodal PTC and the FTC-OV DNA histograms is significant and demonstrates endoreduplication. In addition, the FTC-OV shows a dominant DNA near-haploid population [<b>c</b>, <b>f</b>]).</p

Interphase FISH analysis in relation to allelic state analysis.

<p>In FTC-OV, chromosome 6 was always observed in allelic state [A] or [AA], whereas chromosome 7 was always retained in a heterozygous state or amplified heterozygous state. To confirm these results, interphase FISH was performed for chromosomes 6 and 7. Examples are shown, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038287#pone-0038287-t001" target="_blank">Table 1</a>. <b>A</b>. FISH on normal thyroid epithelium. <b>B</b> and <b>C</b> show FTC-OV case No. 10. <b>B</b>: Allelic state analysis illustrating allelic state [A] for chromosome 6 and allelic state [AABB] for chromosome 7. <b>C</b>. Left panel: green signal, centromere 6 shows 1 copy, confirming the allelic state [A] in three of the four nuclei. Right panel: four green and four red signals representing EGFR and centromere 7, respectively and confirming the [AABB] allelic state. <b>D–E</b> show FTC-OV, case no 11. <b>D</b>. The allelic state [AAB] of chromosome 7 could not be confirmed definitively and showed a mixture of nuclei containing three green (EGFR) and three red (centromere) signals or four green and four red signals, respectively (<b>E</b>). This may be due to intra-tumour heterogeneity.</p

Summary of the genomic alterations found after LAIR analysis (see Materials and Methods) in 27 recurrent NMTCs.

<p>In this heatmap, rows represent tumours and columns represent chromosomes. The first column shows the tumour number, type and DNA index (DI). The tumours have been grouped according to their subtype, with ten FTC-OV tumours in the upper group and 17 NON FTC-OV in the lower group. The combined frequency of genomic alterations for each group is indicated in a separate row. Black indicates allelic states with >2 copies and at least one B allele retained, e.g. [AABB] and [AAB]. Grey indicates allelic states of [A] and [AA]. The colours in the heatmap indicate: white, allelic state [AB] = normal heterozygous state. Dark red, allelic states [AABB], [AAABBB], etc. = amplified heterozygous states. Light red, allelic states [AAB], [AAABB], etc. = imbalanced gain. Dark blue, allelic state [A] = LOH or physical loss in the context of a diploid genome but monosomy in the context of a haploid genome. Light blue, allelic states [AA], [AAA], etc. = copy neutral LOH and amplified LOH, respectively. Notice the retention of chromosome 7 for five out of ten FTC-OV tumours (allelic states [AB]) with the remaining five showing endoreduplication with allelic states [AABB] (n = 4) or [AAB] (n = 1). The Integrative Genomics Viewer (IGV) was used to produce this image.</p

Hypothetic model of oncocytic follicular thyroid carcinoma development and progression.

<p>Mutations in mtDNA underlie low levels of ATP productions. In order to compensate for these low energy levels mitochondria carrying these mutations proliferate and accumulate in the cytoplasm of affected cells. Oncocytic FTC is also characterised by a mitochondria-rich cytoplasm, are known to harbour mtDNA mutations (mainly complex I) and do show a disturbed energy production. DNA replication and progression through the cell cycle are energy demanding processes. Low energy levels might also disturb normal formation and function of the mitotic spindle, resulting in an unbalanced mitosis. Cells that have lost chromosomes during several rounds of cell division become DNA near-haploid (haploidisation process). Sustaining a near-haploid genome may require less energy than of normal 2n cells. Consequently near-haploid cells show a growth advantage and are selected for during tumour development. Maintaining chromosome 7 in heterozygous state ([AB]) seems to be essential for tumour survival. This might be indicative for the presence of genes playing an important role in oncocytic FTC. Endoreduplication of these genes into an [AABB] allelic state increases the gene dosage which might be beneficial for further progression.</p

Summary of results of the analysis of the 20 tumours in the validation cohort: diagnosis, near-homozygous phenotype and recurrence.

<p>The presence of homozygous chromosomes was determined by high-density SNP-arrays.</p><p>min. inv. = minimal invasive.</p>*<p> = incomplete resection and death of disease <5 months.</p>**<p> = sorafenib study, FFPE sample No. 9.</p>&<p> = other tumour fraction dedifferentiated to ATC.</p>$<p> = possible intratumour heterogeneity.</p

Examples of genome-wide allelic state analysis of an FTC-OV and a PTC-OV.

<p><b>A</b>. FTC-OV (case No. 13, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038287#pone-0038287-g001" target="_blank">Figure 1</a>) with DIs of 0.53 and 1.04 shows allelic state [A] for most chromosomes, except for chromosomes 7 and 12 and a segment of chromosome 18 showing retention (allelic state [AB]). Chromosome X also shows an allelic state [A]. <b>B</b>. The PTC-OV sample with a DI of 0.94 (case No. 24) shows a relatively limited number of genomic alterations. Chromosomes 1q and 7p showed an [AAA] allelic state after LAIR analysis. Another segment of 1q showed 1 copy but was heterozygous, which can be explained by a balanced mixture of two populations, one with an allelic state [A] and one with an allelic state [B], representing intra-tumour heterogeneity. In comparison with normal cells, one copy of chromosome 9, 13 and 22 was lost, as shown by the allelic state [A]. Both X chromosomes were detected in this female patient [AB].</p

Summary of results of the analysis of 27 recurrent NMTC tumours: DNA index, allelic state and chromosome copy number determined by FISH for chromosomes 6 (centromeric probe) and 7 (centromeric and EGFR locus specific probes) and mutation analysis of <i>BRAF</i>, <i>EGFR</i>, RAS (<i>HRAS, KRAS</i> and <i>NRAS</i>) genes and <i>PIK3CA</i>.

<p>Underlined: discrepancy between FISH copy number and allelic state.</p>#<p> = bold indicates the dominant population as determined by flow cytometry, in cases where multiple populations are present.</p>*<p> = an intermingled pattern of three and four copies was found both for the centromeric and the EGFR locus specific probes.</p><p>+ = presence of mutation.</p><p>− = mutation not found.</p><p>nd = not determined.</p