Massive genomic rearrangement acquired in a single catastrophic event during cancer development.

Cancer is driven by somatically acquired point mutations and chromosomal rearrangements, conventionally thought to accumulate gradually over time. Using next-generation sequencing, we characterize a phenomenon, which we term chromothripsis, whereby tens to hundreds of genomic rearrangements occur in a one-off cellular crisis. Rearrangements involving one or a few chromosomes crisscross back and forth across involved regions, generating frequent oscillations between two copy number states. These genomic hallmarks are highly improbable if rearrangements accumulate over time and instead imply that nearly all occur during a single cellular catastrophe. The stamp of chromothripsis can be seen in at least 2%-3% of all cancers, across many subtypes, and is present in ∼25% of bone cancers. We find that one, or indeed more than one, cancer-causing lesion can emerge out of the genomic crisis. This phenomenon has important implications for the origins of genomic remodeling and temporal emergence of cancer.

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.

The patterns and dynamics of genomic instability in metastatic pancreatic cancer.

Pancreatic cancer is an aggressive malignancy with a five-year mortality of 97-98%, usually due to widespread metastatic disease. Previous studies indicate that this disease has a complex genomic landscape, with frequent copy number changes and point mutations, but genomic rearrangements have not been characterized in detail. Despite the clinical importance of metastasis, there remain fundamental questions about the clonal structures of metastatic tumours, including phylogenetic relationships among metastases, the scale of ongoing parallel evolution in metastatic and primary sites, and how the tumour disseminates. Here we harness advances in DNA sequencing to annotate genomic rearrangements in 13 patients with pancreatic cancer and explore clonal relationships among metastases. We find that pancreatic cancer acquires rearrangements indicative of telomere dysfunction and abnormal cell-cycle control, namely dysregulated G1-to-S-phase transition with intact G2-M checkpoint. These initiate amplification of cancer genes and occur predominantly in early cancer development rather than the later stages of the disease. Genomic instability frequently persists after cancer dissemination, resulting in ongoing, parallel and even convergent evolution among different metastases. We find evidence that there is genetic heterogeneity among metastasis-initiating cells, that seeding metastasis may require driver mutations beyond those required for primary tumours, and that phylogenetic trees across metastases show organ-specific branches. These data attest to the richness of genetic variation in cancer, brought about by the tandem forces of genomic instability and evolutionary selection.

Transflip mutations produce deletions in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is driven by the inactivation of the tumor suppressor genes (TSGs), CDKN2A (P16) and SMAD4 (DPC4), commonly by homozygous deletions (HDs). Using a combination of high density single-nucleotide polymorphism (SNP) microarray and whole genome sequencing (WGS), we fine-mapped novel breakpoints surrounding deletions of CDKN2A and SMAD4 and characterized them by their underlying structural variants (SVs). Only one third of CDKN2A and SMAD4 deletions (6 of 18) were simple interstitial deletions, rather, the majority of deletions were caused by complex rearrangements, specifically, a translocation on one side of the TSG in combination with an inversion on the other side. We designate these as "TransFlip" mutations. Characteristics of TransFlip mutations are: (1) a propensity to target the TSGs CDKN2A and SMAD4 (P < 0.005), (2) not present in the germline of the examined samples, (3) non-recurrent breakpoints, (4) relatively small (47 bp to 3.4 kb) inversions, (5) inversions can be either telomeric or centromeric to the TSG, and (6) non-reciprocal, and non-recurrent translocations. TransFlip mutations are novel complex genomic rearrangements with unique breakpoint signatures in pancreatic cancer. We hypothesize that they are a common but poorly understood mechanism of TSG inactivation in human cancer. © 2015 Wiley Periodicals, Inc.

Targeted pathologic evaluation of bone marrow donors identifies previously undiagnosed marrow abnormalities.

Potential bone marrow donors are screened to ensure the safety of both the donor and recipient. At our institution, potential donors with abnormal peripheral blood cell counts, a personal history of malignancy, or age >60 years are evaluated to ensure that they are viable candidates for donation. Evaluation of the marrow includes morphologic, flow cytometric, and cytogenetic studies. A total of 122 potential donors were screened between the years of 2001 and 2011, encompassing approximately 10% of all donors. Of the screened potential donors, the mean age was 59 years and there were 59 men and 63 women. The donors were screened because of age >60 years (n = 33), anemia (n = 22), cytopenias other than anemia (n = 27), elevated peripheral blood counts without a concurrent cytopenia (n = 20), elevated peripheral blood counts with a concurrent cytopenia (n = 10), history of malignancy (n = 4), abnormal peripheral blood differential (n = 3), prior graft failure (n = 1), history of treatment with chemotherapy (n = 1), and body habitus (n = 1). Marrow abnormalities were detected in 9% (11 of 122) of donors. These donors were screened because of anemia (5 of 22, 23%), age >60 years (2 of 33, 6%), history of malignancy (2 of 4, 50%), elevated peripheral blood counts (1 of 20, 5%), and body habitus (1 of 1, 100%). Abnormalities included plasma cell dyscrasia (n = 3), abnormal marrow cellularity (n = 3), clonal cytogenetic abnormalities (n = 2), low-grade myelodysplastic syndrome (1), a mutated JAK2 V617F allele (n = 1), and monoclonal B cell lymphocytosis (n = 1). Our experience indicates that extended screening of potential donors identifies a significant number of donors with previously undiagnosed marrow abnormalities.

Ring Chromosomes in Hematological Malignancies Are Associated with TP53 Gene Mutations and Characteristic Copy Number Variants.

Ring chromosomes (RC) are present in <10% of patients with hematological malignancies and are associated with poor prognosis. Until now, only small cohorts of patients with hematological neoplasms and concomitant RCs have been cytogenetically characterized. Here, we performed a conventional chromosome analysis on metaphase spreads from >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.

Chromosomal abnormalities of adenocarcinoma of the pancreas: identifying early and late changes.

The high level of karyotypic complexity found in epithelial neoplasms hinders the characterization of their cytogenetic evolution. Derivation of such pathways in adenocarcinoma of the pancreas has been particularly limited, because only a few pancreatic carcinomas are resected at an early stage of disease and so the number of primary carcinomas for which analysis of abnormal karyotypes has been reported is small. Here we report the clonal karyotypic abnormalities identified by G-banding analysis of 36 primary pancreatic carcinomas obtained from patients undergoing a Whipple resection with curative intent. The majority of the 36 carcinomas were diploid or triploid (33 of 36; 91%). Numerical alterations were found in all carcinomas for which a complete karyotype was determined. All the chromosomes were involved in gain, loss, or both gain and loss of the entire chromosome, in at least 8 and up to 28 of the carcinomas. Most commonly lost were chromosomes 18 (in 78% of the 36 carcinomas), 17 (56%), 6 (44%), 21 (42%), 22 (42%), Y (36%), and 4 (33%). Gain of chromosome 20 was observed in 10 of the 36 carcinomas. Structural abnormalities were common, resulting in partial chromosomal gains and losses, with a median number of 7 partial imbalances per carcinoma (range, 1-15). Sixteen carcinomas contained double-minute chromosomes, homogeneously staining regions, or both, indicating gene amplification. Pooling data for these 36 carcinomas with the primary carcinomas with karyotypes published in the Mitelman database (http://cgap.nci.nih.gov/Chromosomes/Mitelman), we defined pathways of karyotypic evolution. The most frequent chromosomal imbalances were -18 (67.6%), -10 (34.3%), -4 (31.4%), +20 (31.4%), -15p (23.8%), -14p (22.9%), +2 (21.9%), -5 (21.9%), -13p (20%), +16 (20%), -21p (19%), -17p (19%), +1q (19.0%). Recurrent imbalances identified as occurring early were -1p, -15p, -18, -7q, -8p, -17p, and -5; late recurrent imbalances were +11q, +7q, +6p, -19p, and +2. In contrast to reports from similar analyses in other epithelial carcinomas, we did not find evidence for multiple karyotypic evolutionary pathways.

A break-apart fluorescence in situ hybridization assay for detecting RET translocations in papillary thyroid carcinoma.

At least 15 different translocations have been described activating RET in papillary thyroid carcinomas. A break-apart fluorescence in situ hybridization (FISH) assay should detect many translocations involving the RET gene without requiring knowledge of the partner gene. G-banding and spectral karyotyping was performed to further characterize the papillary carcinoma cell line TPC-1. BAC clones flanking the 5' and 3' regions of RET were labeled with SpectrumRed and biotin detected with avidin-AMCA (blue). In addition to the previously described chromosomal t(1;10;21), TPC-1 was found to have del(7)(q22q31) and der(8)t(8;8)(p21;q11.2). With the BAC probes, TPC-1 interphase nuclei showed the expected signal pattern of one paired red-blue signal as well as separated red and blue signals from the rearranged RET gene in 93% of cells. Interphase nuclei from normal human lymphocytes showed two paired red-blue signals in 97% of cells. TPC-1 cells were found to have the previously described chromosomal rearrangement that involves chromosome 10, with few other cytogenetically detectable genomic alterations. RET rearrangement can be detected by a break-apart BAC FISH probe set in the interphase nuclei of TPC-1 cells. This assay can potentially detect clinically relevant translocations involving RET.

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

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.

Cytogenetic study of malignant triton tumor: a case report.

Malignant triton tumor (MTT) is a highly malignant neoplasm, classified as a variant of malignant peripheral nerve sheath tumor (MPNST) with rhabdomyoblastic differentiation. Few cytogenetic studies of MTT have been reported using conventional cytogenetic analysis. Here, we report a comprehensive cytogenetic study of a case of MTT using G-banding, Spectral Karyotyping(), and fluorescence in situ hybridization (FISH) for specific regions. A complex hyperdiploid karyotype with multiple unbalanced translocations was observed: 48 approximately 55,XY,der(7)add(7)(p?)dup(7)[2],der(7) t(7;20)(p22;?)ins(20;19)[5],der(7)ins(8;7)(?;p22q36)t(3;8)t(8;20)[15],-8[5],-8[19],r(8)dup(8), +der(8)r(8;22)[4],-9[9],der(11)t(11;20)(p15;?)ins(20;19)[22],der(12)t(8;12)(q21;p13)[21],der(13) t(3;13)(q25;p11),-17,-19,der(19)t(17;19)(q11.2;q13.1),-20,-22,+4 approximately 7r[cp24]/46,XY[13]. The 1995 International System for Human Cytogenetic Nomenclature was followed where possible. Note that breakpoints were frequently omitted where only SKY information was known for a small part of an involved chromosome. Our analysis revealed some breakpoints in common with those previously reported in MTT, MPNST, and rhabdomyosarcoma, namely 7p22, 7q36, 11p15, 12p13, 13p11.2, 17q11.2, and 19q13.1. FISH showed high increase of copy number for MYC and loss of a single copy for TP53.

Establishment and characterization of a new cell line, A99, from a primary small cell carcinoma of the pancreas.

OBJECTIVES: Small cell carcinoma (SCC) of the pancreas is a rare malignancy with a poor prognosis. We established and characterized a primary human pancreatic SCC cell line, designated A99. METHODS: Cancer tissue was obtained from the liver metastasis of an SCC of the pancreas and xenografted into nude mice. The first-pass xenograft was then used to establish a cultured cell line called A99. Cellular morphology, immunohistochemical properties, tumorigenic potential, and genetic alterations of this new line were characterized. RESULTS: A99 cells grew consistently in culture, formed colonies in soft agar, and grew as subcutaneous xenografts when inoculated into nude mice. A99 cells were positive for pancytokeratin, synaptophysin, chromogranin A, neuron-specific enolase, CD57 (Leu7), CD56, protein gene product 9.5, thyroid transcription factor 1, Smad4, p53, and p16, but not for CD99, PDX-1, or retinoblastoma protein. Sequencing analysis revealed homozygous point mutations of KRAS and TP53. Cytogenetic analysis revealed complex chromosomal rearrangements including marker chromosomes. CONCLUSIONS: A99 is the first cell line reported to be derived from a primary SCC of the pancreas. The establishment of this cell line may serve as a useful model system for studying the cell biology of this rare cancer or for evaluating novel targeted agents in preclinical models.

A de novo complex karyotype with two independent balanced translocations and a double inversion of chromosome 6 presenting with multiple congenital anomalies.

We report a 4-year-old female with a de novo complex karyotype with multiple chromosomal rearrangements and a distinctive phenotype. Her medical history is significant for having been a twin born at 35 weeks gestation, breech presentation, with feeding problems and poor growth as an infant, gastroesophageal reflux disease, peripheral pulmonic stenosis, omphalocele, high myopia, and severe mental retardation. She is small for her age with microcephaly, posteriorly sloping forehead, shallow orbits, long palpebral fissures, prominent nose, wide mouth, absent uvula, kyphosis, brachydactyly, bridged palmar crease, and hypertonia. Peripheral blood lymphocytes revealed a karyotype of 46,XX,t(1;12)(p22.3;q21.3),inv(6)(p24q23),t(7;18)(q11.2;q21.2) in all cells. Parental karyotypes and that of her twin were normal. Spectral Karyotyping (SKY) and fluorescence in situ hybridization (FISH) with whole chromosome paints for chromosomes 1, 6, 7, 12, and 18 did not reveal additional rearrangements. Prometaphase G-banding analysis suggested that the "inverted" chromosome 6 might contain a cryptic rearrangement. Although no deletion nor duplication was detected using metaphase comparative genomic hybridization (CGH), multicolor high resolution banding (mBAND) demonstrated a double inversion of chromosome 6, resulting in a final karyotype as above but including der(6)(pter --> p23::q21 --> q22.3::q21 --> p23::q22.3 --> qter).