NGS Analysis Confirms Common TP53 and RB1 Mutations, and Suggests MYC Amplification in Ocular Adnexal Sebaceous Carcinomas

Ocular adnexal (OA) sebaceous carcinomas generally demonstrate more aggressive clinical and histopathological phenotypes than extraocular cases, but the molecular drivers implicated in their oncogenesis remain poorly defined. A retrospective review of surgical and ocular pathology archives identified eleven primary resection specimens of OA sebaceous carcinomas with adequate tissue for molecular analysis; two extraocular cases were also examined. Next-generation sequencing was used to evaluate mutations and copy number changes in a large panel of cancer-associated genes. Fluorescence in situ hybridization (FISH) confirmed MYC copy number gain in select cases, and immunohistochemistry to evaluate MYC protein expression. The commonest mutations occurred in TP53 (10/13) and RB1 (7/13). Additional mutations in clinically actionable genes, or mutations with a frequency of at least 25%, included the NF1 (3/12), PMS2 (4/12), ROS1 (3/12), KMT2C (4/12), MNX1 (6/12), NOTCH1 (4/12), PCLO (3/12), and PTPRT (3/12) loci. Low level copy number gain suggestive of amplification of the MYC locus was seen in two cases, and confirmed using FISH. MYC protein expression, as assessed by immunohistochemistry, was present in almost all sebaceous carcinoma cases. Our findings support the concept that alterations in TP53 and RB1 are the commonest alterations in sebaceous carcinoma, and suggest that MYC may contribute to the oncogenesis of these tumors

Ring Chromosomes in Hematological Malignancies Are Associated with <i>TP53</i> Gene Mutations and Characteristic Copy Number Variants

Ring chromosomes (RC) are present in 13,000 patients diagnosed with hematological malignancies at the Johns Hopkins University Hospital and identified 98 patients with RCs—90 with myeloid malignancies and 8 with lymphoid malignancies. We also performed a targeted Next-Generation Sequencing (NGS) assay, using a panel of 642 cancer genes, to identify whether these patients harbor relevant pathogenic variants. Cytogenetic analyses revealed that RCs and marker chromosomes of unknown origin are concurrently present in most patients by karyotyping, and 93% of patients with NGS data have complex karyotypes. A total of 72% of these individuals have pathogenic mutations in TP53, most of whom also possess cytogenetic abnormalities resulting in the loss of 17p, including the loss of TP53. All patients with a detected RC and without complex karyotypes also lack TP53 mutations but have pathogenic mutations in TET2. Further, 70% of RCs that map to a known chromosome are detected in individuals without TP53 mutations. Our data suggest that RCs in hematological malignancies may arise through different mechanisms, but ultimately promote widespread chromosomal instability

An Integrated Approach Including CRISPR/Cas9-Mediated Nanopore Sequencing, Mate Pair Sequencing, and Cytogenomic Methods to Characterize Complex Structural Rearrangements in Acute Myeloid Leukemia

Complex structural chromosome abnormalities such as chromoanagenesis have been reported in acute myeloid leukemia (AML). They are usually not well characterized by conventional genetic methods, and the characterization of chromoanagenesis structural abnormalities from short-read sequencing still presents challenges. Here, we characterized complex structural abnormalities involving chromosomes 2, 3, and 7 in an AML patient using an integrated approach including CRISPR/Cas9-mediated nanopore sequencing, mate pair sequencing (MPseq), and SNP microarray analysis along with cytogenetic methods. SNP microarray analysis revealed chromoanagenesis involving chromosomes 3 and 7, and a pseudotricentric chromosome 7 was revealed by cytogenetic methods. MPseq revealed 138 structural variants (SVs) as putative junctions of complex rearrangements involving chromosomes 2, 3, and 7, which led to 16 novel gene fusions and 33 truncated genes. Thirty CRISPR RNA (crRNA) sequences were designed to map 29 SVs, of which 27 (93.1%) were on-target based on CRISPR/Cas9 crRNA nanopore sequencing. In addition to simple SVs, complex SVs involving over two breakpoints were also revealed. Twenty-one SVs (77.8% of the on-target SVs) were also revealed by MPseq with shared SV breakpoints. Approximately three-quarters of breakpoints were located within genes, especially intronic regions, and one-quarter of breakpoints were intergenic. Alu and LINE repeat elements were frequent among breakpoints. Amplification of the chromosome 7 centromere was also detected by nanopore sequencing. Given the high amplification of the chromosome 7 centromere, extra chromosome 7 centromere sequences (tricentric), and more gains than losses of genomic material, chromoanasynthesis and chromothripsis may be responsible for forming this highly complex structural abnormality. We showed this combination approach’s value in characterizing complex structural abnormalities for clinical and research applications. Characterization of these complex structural chromosome abnormalities not only will help understand the molecular mechanisms responsible for the process of chromoanagenesis, but also may identify specific molecular targets and their impact on therapy and overall survival

Tumor-Infiltrating Macrophages in Post-Transplant, Relapsed Classical Hodgkin Lymphoma Are Donor-Derived

<div><p>Tumor-associated inflammatory cells in classical Hodgkin lymphoma (CHL) typically outnumber the neoplastic Hodgkin/Reed-Sternberg (H/RS) cells. The composition of the inflammatory infiltrate, particularly the fraction of macrophages, has been associated with clinical behavior. Emerging work from animal models demonstrates that most tissue macrophages are maintained by a process of self-renewal under physiologic circumstances and certain inflammatory states, but the contribution from circulating monocytes may be increased in some disease states. This raises the question of the source of macrophages involved in human disease, particularly that of CHL. Patients with relapsed CHL following allogeneic bone marrow transplant (BMT) provide a unique opportunity to begin to address this issue. We identified 4 such patients in our archives. Through molecular chimerism and/or XY FISH studies, we demonstrated the DNA content in the post-BMT recurrent CHL was predominantly donor-derived, while the H/RS cells were derived from the patient. Where possible to evaluate, the cellular composition of the inflammatory infiltrate, including the percentage of macrophages, was similar to that of the original tumor. Our findings suggest that the H/RS cells themselves define the inflammatory environment. In addition, our results demonstrate that tumor-associated macrophages in CHL are predominantly derived from circulating monocytes rather than resident tissue macrophages. Given the association between tumor microenvironment and disease progression, a better understanding of macrophage recruitment to CHL may open new strategies for therapeutic intervention.</p></div

Complex/cryptic <i>EWSR1::FLI1/ERG</i> Gene Fusions and 1q Jumping Translocation in Pediatric Ewing Sarcomas

Ewing sarcomas (ES) are rare small round cell sarcomas often affecting children and characterized by gene fusions involving one member of the FET family of genes (usually EWSR1) and a member of the ETS family of transcription factors (usually FLI1 or ERG). The detection of EWSR1 rearrangements has important diagnostic value. Here, we conducted a retrospective review of 218 consecutive pediatric ES at diagnosis and found eight patients having data from chromosome analysis, FISH/microarray, and gene-fusion assay. Three of these eight ES had novel complex/cryptic EWSR1 rearrangements/fusions by chromosome analysis. One case had a t(9;11;22)(q22;q24;q12) three-way translocation involving EWSR1::FLI1 fusion and 1q jumping translocation. Two cases had cryptic EWSR1 rearrangements/fusions, including one case with a cryptic t(4;11;22)(q35;q24;q12) three-way translocation involving EWSR1::FLI1 fusion, and the other had a cryptic EWSR1::ERG rearrangement/fusion on an abnormal chromosome 22. All patients in this study had various aneuploidies with a gain of chromosome 8 (75%), the most common, followed by a gain of chromosomes 20 (50%) and 4 (37.5%), respectively. Recognition of complex and/or cryptic EWSR1 gene rearrangements/fusions and other chromosome abnormalities (such as jumping translocation and aneuploidies) using a combination of various genetic methods is important for accurate diagnosis, prognosis, and treatment outcomes of pediatric ES

H/RS cells are patient-derived while the majority of the inflammatory infiltrate is donor-derived.

<p>Sections of lung from a female patient (Patient D) with recurrent Hodgkin lymphoma following an allogeneic BMT (brother) are shown. An overview of one of the lesional areas is shown (A, DAPI nuclear staining). XY FISH was performed on this section, and a high power view of the boxed area in red is shown in B (X = red, Y = green). An adjacent tissue section was stained for CD30 (C), highlighting numerous H/RS cells (arrow, higher magnification, D). While not possible to align perfectly, the H/RS cells in this patient were positive for EBV (in situ hybridization for EBER, panels E, F). The majority of the smaller nuclei are donor-derived (XY, red and green, 78% including only DAPI-positive nuclei with distinct FISH signals). By comparison with the H&E, these cells predominantly represent an inflammatory infiltrate. A separate area from this specimen demonstrating similar findings is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163559#pone.0163559.s001" target="_blank">S1 Fig</a>.</p

Tumor-infiltrating macrophages in recurrent Hodgkin lymphoma are predominantly donor- and, therefore, bone marrow-derived.

<p>A portion of the same field highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163559#pone.0163559.g001" target="_blank">Fig 1A</a> (white box) is shown at higher magnification. Prior to analysis by XY FISH, the same slide was stained for CD68 using standard immunohistochemical techniques in order to identify macrophages (A). The images of the DAPI nuclear stain and the CD68 cytoplasmic stain are overlaid (B) to better identify individual cells in the corresponding FISH images (C). Where possible to discern, the tumor-infiltrating macrophages are all derived from the male donor (arrows, XY, red and green). However, by this method we found that in 21 +/- 4% of DAPI-positive nuclei, it was not possible to score X, Y status due to sectioning and/or other technical limitations. Of note, this patient had 100% donor chimerism in her bone marrow when tested near the time of this biopsy. H/RS cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163559#pone.0163559.g001" target="_blank">Fig 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163559#pone.0163559.s001" target="_blank">S1 Fig</a>) and areas of residual, uninvolved lung tissue were female (not shown). An overview of the density of macrophage infiltrate is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163559#pone.0163559.s002" target="_blank">S2 Fig</a>.</p

Demographic information.

<p>Demographic information.</p

Composition of the Inflammatory Infiltrate.

<p>Composition of the Inflammatory Infiltrate.</p