Theragnostic chromosomal rearrangements in treatment-naive pancreatic ductal adenocarcinomas obtained via endoscopic ultrasound.

A crucial mutational mechanism in malignancy is structural variation, in which chromosomal rearrangements alter gene functions that drive cancer progression. Herein, the presence and pattern of structural variations were investigated in twelve prospectively acquired treatment-naïve pancreatic cancers specimens obtained via endoscopic ultrasound (EUS). In many patients, this diagnostic biopsy procedure and specimen is the only opportunity to identify somatic clinically relevant actionable alterations that may impact their care and outcome. Specialized mate pair sequencing (MPseq) provided genome-wide structural variance analysis (SVA) with a view to identifying prognostic markers and possible therapeutic targets. MPseq was successfully performed on all specimens, identifying highly rearranged genomes with complete SVA on all specimens with > 20% tumour content. SVA identified chimeric fusion proteins and potentially immunogenic readthrough transcripts, change of function truncations, gains and losses of key genes linked to tumour progression. Complex localized rearrangements, termed chromoanagenesis, with broad pattern heterogeneity were observed in 10 (83%) specimens, impacting multiple genes with diverse cellular functions that could influence theragnostic evaluation and responsiveness to immunotherapy regimens. This study indicates that genome-wide MPseq can be successfully performed on very limited clinically EUS obtained specimens for chromosomal rearrangement detection and potential theragnostic targets.

Optimizing clinical cytology touch preparations for next generation sequencing.

Intraoperative diagnosis is routinely performed on cytology touch preparations (TPs) from core needle biopsies (CNBs). Current interest promotes their utility as an important source of patient tissue for clinical genomic testing. Herein we present whole genome structural variant analysis (SVA) from mate-pair sequencing (MPseq) and whole exome sequencing (WES) mutation calling in DNA directly whole genome amplified (WGA) from TPs. Chromosomal copy changes and somatic DNA junction detection from MPseq of TPs were highly consistent with associated CNBs and bulk resected tissues in all cases. While increased frequency coverage noise from limitations of amplification of limited sample input was significant, this was effectively compensated by natural tumor enrichment during the TP process, which also enhanced variant detection and loss of heterozygosity evaluations from WES. This novel TP methodology enables expanded utility of frequently limited CNB for both clinical and research genomic testing.

Suppression of Somatic Expansion Delays the Onset of Pathophysiology in a Mouse Model of Huntington's Disease.

Huntington's Disease (HD) is caused by inheritance of a single disease-length allele harboring an expanded CAG repeat, which continues to expand in somatic tissues with age. The inherited disease allele expresses a toxic protein, and whether further somatic expansion adds to toxicity is unknown. We have created an HD mouse model that resolves the effects of the inherited and somatic expansions. We show here that suppressing somatic expansion substantially delays the onset of disease in littermates that inherit the same disease-length allele. Furthermore, a pharmacological inhibitor, XJB-5-131, inhibits the lengthening of the repeat tracks, and correlates with rescue of motor decline in these animals. The results provide evidence that pharmacological approaches to offset disease progression are possible.

Genomic rearrangements in sporadic lymphangioleiomyomatosis: an evolving genetic story.

Sporadic lymphangioleiomyomatosis is a progressive pulmonary cystic disease resulting from the infiltration of smooth muscle-like lymphangioleiomyomatosis cells into the lung. The migratory/metastasizing properties of the lymphangioleiomyomatosis cell together with the presence of somatic mutations, primarily in the tuberous sclerosis complex gene (TSC2), lead many to consider this a low-grade malignancy. As malignant tumors characteristically accumulate somatic structural variations, which have not been well studied in sporadic lymphangioleiomyomatosis, we utilized mate pair sequencing to define structural variations within laser capture microdissected enriched lymphangioleiomyomatosis cell populations from five sporadic lymphangioleiomyomatosis patients. Lymphangioleiomyomatosis cells were confirmed in each tissue by hematoxylin eosin stain review and by HMB-45 immunohistochemistry in four cases. A mutation panel demonstrated characteristic TSC2 driver mutations in three cases. Genomic profiles demonstrated normal diploid coverage across all chromosomes, with no aneuploidy or detectable gains/losses of whole chromosomal arms typical of neoplastic diseases. However, somatic rearrangements and smaller deletions were validated in the two cases which lacked TSC2 driver mutations. Most significantly, one of these sporadic lymphangioleiomyomatosis cases contained two different size deletions encompassing the entire TSC1 locus. The detection of a homozygous deletion of TSC1 driving a predicted case of sporadic lymphangioleiomyomatosis, consistent with the common two-hit TSC2 mutation model, has never been reported for sporadic lymphangioleiomyomatosis. However, while no evidence of the hereditary tuberous sclerosis complex disease was reported for this patient, the potential for mosaicism and sub-clinical phenotype cannot be ruled out. Nevertheless, this study demonstrates that somatic structural rearrangements are present in lymphangioleiomyomatosis disease and provides a novel method of genomic characterization of sporadic lymphangioleiomyomatosis cells, aiding in defining cases with no detected mutations by conventional methodologies. These structural rearrangements could represent additional pathogenic mechanisms in sporadic lymphangioleiomyomatosis disease, potentially affecting response to therapy and adding to the complex genetic story of this rare disease.

Integrated analysis of the genomic instability of PTEN in clinically insignificant and significant prostate cancer.

Patients with clinically insignificant prostate cancer remain a major over-treated population. PTEN loss is one of the most recurrent alterations in prostate cancer associated with an aggressive phenotype, however, the occurrence of PTEN loss in insignificant prostate cancer has not been reported and its role in the separation of insignificant from significant prostate cancer is unclear. An integrated analysis of PTEN loss was, therefore, performed for structural variations, point mutations and protein expression in clinically insignificant (48 cases) and significant (76 cases) prostate cancers treated by radical prostatectomy. Whole-genome mate pair sequencing was performed on tumor cells isolated by laser capture microdissection to characterize PTEN structural alterations. Fluorescence in situ hybridization probes were constructed from the sequencing data to detect the spectrum of these PTEN alterations. PTEN loss by mate pair sequencing and fluorescence in situ hybridization occurred in 2% of insignificant, 13% of large volume Gleason score 6, and 46% of Gleason score 7 and higher cancers. In Gleason score 7 cancers with PTEN loss, PTEN alterations were detected in both Gleason pattern 3 and 4 in 57% of cases by mate pair sequencing, 75% by in situ hybridization and 86% by immunohistochemistry. PTEN loss by sequencing was strongly associated with TMPRSS2-ERG fusion, biochemical recurrence, PTEN loss by in situ hybridization and protein loss by immunohistochemistry. The complex nature of PTEN rearrangements was unveiled by sequencing, detailing the heterogeneous events leading to homozygous loss of PTEN. PTEN point mutation was present in 5% of clinically significant tumors and not in insignificant cancer or high-grade prostatic intraepithelial neoplasia. PTEN loss is infrequent in clinically insignificant prostate cancer, and is associated with higher grade tumors. Detection of PTEN loss in Gleason score 6 cancer in a needle biopsy specimen indicates a higher likelihood of clinically significant prostate cancer.

Chromosomal rearrangements and copy number abnormalities of TP63 correlate with p63 protein expression in lung adenocarcinoma.

The TP63 gene encodes a member of the p53 family of transcription factors. Although TP53 is a well-known tumor suppressor gene, the role of p63 in tumorigenesis is controversial. Our group recently identified novel chromosomal rearrangements involving TP63 in approximately 6% of peripheral T-cell lymphomas, which correlated with a p63+/p40- immunohistochemical profile. As a subset of lung adenocarcinomas are p63+/p40-, we undertook the current study to examine the presence of TP63 rearrangements and correlate with p63/p40 expression. Next-generation sequencing was used to identify genomic rearrangements of TP63 in 37 adenocarcinomas. Confirmatory fluorescence in-situ hybridization (FISH) using a break-apart probe to the TP63 gene region and immunohistochemistry for p63 and p40 were performed on adenocarcinomas with TP63 rearrangements identified by mate-pair sequencing. Immunohistochemistry for p63 and p40 was performed on 45 additional adenocarcinomas, and FISH was performed on all adenocarcinomas with p63 positivity. TP63 rearrangement was identified in two adenocarcinoma specimens from a single patient. The rearrangement resulted in a complex rearrangement of 3q that fused B3GALNT1 at the 3' intron to TP63. FISH confirmed the rearrangement in both tumors. Immunohistochemistry staining for p63 was diffuse (>80% cells+) and p40 was negative. Of the 44 additional adenocarcinomas, 13 (30%) showed p63 expression; p40 was negative in all cases. No case showed rearrangement of TP63 by a break-apart FISH. However, extra copies of the intact TP63 locus were seen in the p63-positive areas of all 12 cases, with copy numbers ranging from three to seven. We have identified a novel chromosomal rearrangement involving TP63 in a p63+/p40- lung adenocarcinoma. Break-apart FISH testing can be used to diagnose this finding. Immunohistochemistry for p63 was not specific for this rearrangement, as nearly 33% of adenocarcinomas expressed p63. Additional copies of the intact TP63 locus were also a common finding and correlated with immunohistochemistry positivity for p63.

Epigenomics may begin to explain in vitro differential response to hypomethylating agents in MMR-D hypermethylated endometrial cancer.

This work examines differences in chromatin accessibility, methylation, and response to DNA hypomethylating agents between mismatch repair-deficient and non-mismatch repair-deficient endometrial cancer. Next-generation sequencing of a stage 1B, grade 2 endometrioid endometrial cancer tumor revealed microsatellite instability and a variant of unknown significance in POLE along with global and MLH1 hypermethylation. Inhibition of viability by decitabine in the study and comparison tumors was minimal, as shown by an inhibitory effect of 0 and 17.9, respectively. Conversely, the inhibitory effect of azacitidine on the study tumor was more pronounced, at 72.8 versus 41.2. In vitro, mismatch repair-deficient endometrial cancer with MLH1 hypermethylation respond better to DNA methyltransferase inhibition by azacytidine (DNA/RNA inhibition), than to decitabine (DNA-only inhibition). Additional large studies are needed to substantiate our findings.

Renin Production by Juxtaglomerular Cell Tumors and Clear Cell Renal Cell Carcinoma and the Role of Angiotensin Signaling Inhibitors.

OBJECTIVE: To profile juxtaglomerular cell tumors (JXG) and histologic mimics by analyzing renin expression; to identify non-JXG renin-producing tumors in The Cancer Genome Atlas (TCGA) data sets; and to define the prevalence of hypertension (HTN) and patient outcomes with angiotensin signaling inhibitor (ASI) use in tumors of interest. PATIENTS AND METHODS: Thirteen JXGs and 10 glomus tumors (GTs), a histologic mimic, were evaluated for clinicopathologic features; TCGA data were analyzed to identify non-JXG renin-overexpressing tumors. An institutional registry was queried to determine the incidence of HTN, the use of ASIs in hypertensive patients, and the impact of ASIs on outcomes including progression-free survival (PFS) in a tumor type with high renin expression (clear cell renal cell carcinoma [CC-RCC] diagnosed between January 1, 2005, and December 31, 2012). RESULTS: We found an association between renin production and HTN in JXG compared with GT. Analysis of TCGA data found that a subset of CC-RCCs overexpress renin relative to 29 other tumor types. Furthermore, analysis of our institutional registry revealed a high prevalence (64%) of HTN among 1203 patients treated with radical or partial nephrectomy for nonmetastatic CC-RCC. On multivariable Cox regression, patients with HTN treated with ASIs (34%) had improved PFS (hazard ratio, 0.76; 95% CI, 0.57 to 1.00; P=.05) compared with patients with HTN not treated with ASIs (30%). CONCLUSION: The identification of renin expression in a subset of CC-RCC may provide a biologic rationale for the high prevalence of HTN and improved PFS with ASI use in hypertensive patients with nonmetastatic CC-RCC.

Personalized tumor-specific DNA junctions to detect circulating tumor in patients with endometrial cancer.

INTRODUCTION: There are no reliable blood biomarkers for monitoring endometrial cancer patients in the current clinical practice. Circulating tumor DNA (ctDNA) is emerging as a promising non-invasive method to measure tumor burden, define prognosis and monitor disease status in many solid cancers. In this pilot study, we investigated if unique tumor-specific DNA junctions can be used to detect ctDNA levels in patients with endometrial cancer. METHODS: Chromosomal rearrangements in primary tumors of eleven patients with high-grade or advanced stage endometrial cancer were determined by whole-genome Mate-Pair sequencing. Identified unique tumor-specific junctions were evaluated in pre- and six-week post-surgery patient plasma using individualized quantitative polymerase chain reaction (qPCR) assays. The relationship between clinicopathological features and detection of ctDNA was investigated. RESULTS: CtDNA was detected in 60% (6/10) of cases pre-surgery and in 27% (3/11) post-surgery. The detection of ctDNA pre-surgery was consistent with clinical indicators of aggressive disease such as advanced stage (80% - 4/5), lymphatic spread of disease (100% - 3/3), serous histology (80% - 4/5), deep myometrial invasion (100% - 3/3), lympho-vascular space invasion (75% - 3/4). All patients in which ctDNA was detected post-surgically had type II endometrial cancer. DISCUSSION: This pilot study demonstrates the feasibility of using personalized tumor-specific junction panels for detecting ctDNA in the plasma of endometrial cancer patients. Larger studies and longer follow-up are needed to validate the potential association between pre-surgical ctDNA detection and the presence of cancers with aggressive pathologic tumor characteristics or advanced stage observed in this study.

Mate-pair sequencing identifies a cryptic BMPR2 mutation in hereditary pulmonary arterial hypertension.

Current guidelines suggest screening all patients with idiopathic pulmonary arterial hypertension for genetic aberrations, particularly mutations in Bone Morphogenic Protein Receptor Type II (BMPR2), the gene most commonly implicated in the pathogenesis of PAH. Herein, we present a novel technique used to identify a pathogenic germline BMPR2 alteration in a 36-year-old female and family members with hereditary pulmonary arterial hypertension who each screened negative by standard cytogenetics and molecular genetics testing.

Integration of Comprehensive Genomic Analysis and Functional Screening of Affected Molecular Pathways to Inform Cancer Therapy.

OBJECTIVE: To select optimal therapies based on the detection of actionable genomic alterations in tumor samples is a major challenge in precision medicine. METHODS: We describe an effective process (opened December 1, 2017) that combines comprehensive genomic and transcriptomic tumor profiling, custom algorithms and visualization software for data integration, and preclinical 3-dimensiona ex vivo models for drug screening to assess response to therapeutic agents targeting specific genomic alterations. The process was applied to a patient with widely metastatic, weakly hormone receptor positive, HER2 nonamplified, infiltrating lobular breast cancer refractory to standard therapy. RESULTS: Clinical testing of liver metastasis identified BRIP1, NF1, CDH1, RB1, and TP53 mutations pointing to potential therapies including PARP, MEK/RAF, and CDK inhibitors. The comprehensive genomic analysis identified 395 mutations and several structural rearrangements that resulted in loss of function of 36 genes. Meta-analysis revealed biallelic inactivation of TP53, CDH1, FOXA1, and NIN, whereas only one allele of NF1 and BRIP1 was mutated. A novel ERBB2 somatic mutation of undetermined significance (P702L), high expression of both mutated and wild-type ERBB2 transcripts, high expression of ERBB3, and a LITAF-BCAR4 fusion resulting in BCAR4 overexpression pointed toward ERBB-related therapies. Ex vivo analysis validated the ERBB-related therapies and invalidated therapies targeting mutations in BRIP1 and NF1. Systemic patient therapy with afatinib, a HER1/HER2/HER4 small molecule inhibitor, resulted in a near complete radiographic response by 3 months. CONCLUSION: Unlike clinical testing, the combination of tumor profiling, data integration, and functional validation accurately assessed driver alterations and predicted effective treatment.

Targeting HER2 in patient-derived xenograft ovarian cancer models sensitizes tumors to chemotherapy.

Ovarian cancer is the most lethal gynecologic malignancy. About 75% of ovarian cancer patients relapse and/or develop chemo-resistant disease after initial response to standard-of-care treatment with platinum-based therapies. HER2 amplifications and overexpression in ovarian cancer are reported to vary, and responses to HER2 inhibitors have been poor. Next generation sequencing technologies in conjunction with testing using patient-derived xenografts (PDX) allow validation of personalized treatments. Using a whole-genome mate-pair next generation sequencing (MPseq) protocol, we identified several high grade serous ovarian cancers (HGS-OC) with DNA alterations in genes encoding members of the ERBB2 pathway. The efficiency of anti-HER2 therapy was tested in three different PDX lines with the identified alterations and high levels of HER2 protein expression. Treatment responses to pertuzumab or pertuzumab/trastuzumab were compared in each PDX line WITH standard carboplatin and paclitaxel combination treatment. In all three PDX models, HER2-targeted therapy resulted in significant inhibition of tumor growth compared with untreated controls. However, the responses in each case were inferior to those to chemotherapy, even for chemo-resistant lines. When chemotherapy and HER2-targeted therapy were administered together, a significant regression of tumor was observed after 6 weeks of treatment compared with chemotherapy alone. Post-treatment analysis of these tissues revealed that inhibition of the ERBB2 pathway occurred at the level of phosphorylation and expression of downstream targets. In conclusion, while targeting of presumably activated ERBB2 pathway alone in HGS-OC results in a modest treatment benefit, a combination therapy including both chemotherapy drugs and HER2 inhibitors provides a far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to HER2-targeted therapy.

Shared and unique genomic structural variants of different histological components within testicular germ cell tumours identified with mate pair sequencing.

Post-pubertal testicular germ-cell tumours (TGCTs) can present with a variety of distinct histologies which are nevertheless lineage related and often co-occurring. The exact lineage relationships and developmental pathways leading to the different histologies is debated. In order to investigate the relationship of histologic populations, mate-pair sequencing (MPseq) and exome sequencing (ExomeSeq) were conducted on different histological populations within the same tumour. Ten TGCTs with 1-3 histologic types/tumour were sequenced. Junctions of somatic chromosomal rearrangements were identified on a per genome basis, with germ cell neoplasia in situ possessing the least (median 1, range 0-4) and embryonal carcinoma the most (median 8.5, range 6-12). Copy number variation revealed gains and losses, including isoform 12p (i12p) (10/10 samples), and chromosomes 7, 8, and 21 gains (7/10 samples). Mapping of shared junctions within a tumour revealed lineage relationships, but only i12p was shared between patients. ExomeSeq from two cases demonstrated a high level of copy-neutral loss of heterozygosity. Parallel assessment of separate histologies within a single TGCT demonstrated cumulative and divergent changes, suggesting the importance of parallel sequencing for detection of relevant biomarkers.

Using Genomics to Differentiate Multiple Primaries From Metastatic Lung Cancer.

INTRODUCTION: Genomic technologies present a promising mechanism of resolving the clinical dilemma of distinguishing independent primary tumors from intrapulmonary metastases in NSCLC. We evaluated the utility of discordant mapping somatic junctions from chromosomal rearrangements in diagnosing metastatic disease compared to the current standard histologic review. MATERIAL AND METHODS: Mate-pair sequencing was performed on DNA extracted from 76 distinct tumors from 37 cases of multiple lung cancers. Discordant mapping junctions and chromosomal copy levels were assessed for each tumor. Blood-derived DNA was available on 22 of these cases for germline assessments. A lung cancer next-generation sequencing panel was additionally performed on tumor pairs from 17 patients. RESULTS: Whereas mate-pair sequencing was able to classify lineage in all tumor pairs, histologic review appeared to misclassify lineage in 9 of 33 (27%) same-histology tumor pair comparisons. Based on disagreement between the reviewing pathologists, histopathologic lineage was classified as indeterminate in seven cases. In two cases where pathologists agreed on a metastatic call, no shared junctions were found suggesting independent primaries. Although germline junctions passing algorithmic filters were common, on average less than three were present and all had predictable structures of small focal rearrangements or transposons. Evaluation of shared chromosomal copy changes and driver mutations through a lung cancer next-generation sequencing panel, while informative, were nondefinitive in calling lineage in all cases. CONCLUSIONS: The highly unique nature and prevalence of chromosomal rearrangement in lung cancers provide a useful and definitive technique for calling lineage in multifocal lung cancer.

Large Chromosomal Rearrangements Yield Biomarkers to Distinguish Low-Risk From Intermediate- and High-Risk Prostate Cancer.

OBJECTIVE: To test the hypothesis that chromosomal rearrangements (CRs) can distinguish low risk of progression (LRP) from intermediate and high risk of progression (IHRP) to prostate cancer (PCa) and if these CRs have the potential to identify men with LRP on needle biopsy that harbor IHRP PCa in the prostate gland. PATIENTS AND METHODS: Mate pair sequencing of amplified DNA from pure populations of Gleason patterns in 154 frozen specimens from 126 patients obtained between August 14, 2001, and July 15, 2011, was used to detect CRs including abnormal junctions and copy number variations. Potential CR biomarkers with higher incidence in IHRP than in LRP to cancer and having significance in PCa biology were identified. Independent validation was performed by fluorescence in situ hybridization in 152 specimens from 124 patients obtained between February 12, 2002, and July 12, 2008. RESULTS: The number of abnormal junctions did not distinguish LRP from IHRP. Loci corresponding to genes implicated in PCa were more frequently altered in IHRP. Integrated analysis of copy number variations and microarray data yielded 6 potential markers that were more frequently detected in Gleason pattern 3 of a Gleason score 7 of PCa than in Gleason pattern 3 of a Gleason score 6 PCa. Five of those were cross-validated in an independent sample set with statistically significant areas under the receiver operating characteristic curves (AUCs) (P≤.01). Probes detecting deletions in PTEN and CHD1 had AUCs of 0.87 (95% CI, 0.77-0.97) and 0.73 (95% CI, 0.60-0.86), respectively, and probes detecting gains in ASAP1, MYC, and HDAC9 had AUCs of 0.71 (95% CI, 0.59-0.84), 0.82 (95% CI, 0.71-0.93), and 0.77 (95% CI, 0.66-0.89), respectively (for expansion of gene symbols, use search tool at www.genenames.org). CONCLUSION: Copy number variations in regions encompassing important PCa genes were predictive of cancer significance and have the potential to identify men with LRP PCa by needle biopsy who have IHRP PCa in their prostate gland.

Neoantigenic Potential of Complex Chromosomal Rearrangements in Mesothelioma.

INTRODUCTION: Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Whereas other carcinogen-related tumors are associated with a high tumor mutation burden, mesothelioma is not. We sought to resolve this discrepancy. METHODS: We used mate-pair (n = 22), RNA (n = 28), and T cell receptor sequencing along with in silico predictions and immunologic assays to understand how structural variants of chromosomes affect the transcriptome. RESULTS: We observed that inter- or intrachromosomal rearrangements were present in every specimen and were frequently in a pattern of chromoanagenesis such as chromoplexy or chromothripsis. Transcription of rearrangement-related junctions was predicted to result in many potential neoantigens, some of which were proven to bind patient-specific major histocompatibility complex molecules and to expand intratumoral T cell clones. T cells responsive to these predicted neoantigens were also present in a patient's circulating T cell repertoire. Analysis of genomic array data from the mesothelioma cohort in The Cancer Genome Atlas suggested that multiple chromothriptic-like events negatively impact survival. CONCLUSIONS: Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel immunotherapeutic strategies and the selection of patients to receive immunotherapies.

SVAtools for junction detection of genome-wide chromosomal rearrangements by mate-pair sequencing (MPseq).

Mate-pair sequencing (MPseq), using long-insert, paired-end genomic libraries, is a powerful next-generation sequencing-based approach for the detection of genomic structural variants. SVAtools is a set of algorithms to detect both chromosomal rearrangements and large (>10 kb) copy number variants (CNVs) in genome-wide MPseq data. SVAtools can also predict gene disruptions and gene fusions, and characterize the genomic structure of complex rearrangements. To illustrate the power of SVAtools' junction detection methods to provide comprehensive molecular karyotypes, MPseq data were compared against a set of samples previously characterized by traditional cytogenetic methods. Karyotype, FISH and chromosomal microarray (CMA), performed for 29 patients in a clinical laboratory setting, collectively revealed 285 breakpoints in 87 rearrangements. The junction detection methods of SVAtools detected 87% of these breakpoints compared to 48%, 42% and 57% for karyotype, FISH and CMA respectively. Breakpoint resolution was also reported to 1 kb or less and additional genomic rearrangement complexities not appreciable by standard cytogenetic techniques were revealed. For example, 63% of CNVs detected by CMA were shown by SVAtools' junction detection to occur secondary to a rearrangement other than a simple deletion or tandem duplication. SVAtools with MPseq provides comprehensive and accurate whole-genome junction detection with improved breakpoint resolution, compared to karyotype, FISH, and CMA combined. This approach to molecular karyotyping offers considerable diagnostic potential for the simultaneous detection of both novel and recurrent genomic rearrangements in hereditary and neoplastic disorders.

Quantification of Somatic Chromosomal Rearrangements in Circulating Cell-Free DNA from Ovarian Cancers.

Recently, the use of a liquid biopsy has shown promise in monitoring tumor burden. While point mutations have been extensively studied, chromosomal rearrangements have demonstrated greater tumor specificity. Such rearrangements can be identified in the tumor and subsequently detected in the plasma of patients using quantitative PCR (qPCR). In this study we used a whole-genome mate-pair protocol to characterize a landscape of genomic rearrangements in the primary tumors of ten ovarian cancer patients. Individualized tumor-specific primer panels of aberrant chromosomal junctions were identified for each case and detected by qPCR within the cell-free DNA. Selected chromosomal junctions were detected in pre-surgically drawn blood in eight of the ten patients. Of these eight, three demonstrated the continued presence of circulating tumor DNA (ctDNA) post-surgery, consistent with their documented presence of disease, and in five ctDNA was undetectable in the post-surgical blood collection, consistent with their lack of detectable disease. The ctDNA fraction was calculated using a novel algorithm designed for the unique challenges of quantifying ctDNA using qPCR to allow observations of real-time tumor dynamics. In summary, a panel of individualized junctions derived from tumor DNA could be an effective way to monitor cancer patients for relapse and therapeutic efficacy using cfDNA.

Integrated Genomic Analysis of Pancreatic Ductal Adenocarcinomas Reveals Genomic Rearrangement Events as Significant Drivers of Disease.

Many somatic mutations have been detected in pancreatic ductal adenocarcinoma (PDAC), leading to the identification of some key drivers of disease progression, but the involvement of large genomic rearrangements has often been overlooked. In this study, we performed mate pair sequencing (MPseq) on genomic DNA from 24 PDAC tumors, including 15 laser-captured microdissected PDAC and 9 patient-derived xenografts, to identify genome-wide rearrangements. Large genomic rearrangements with intragenic breakpoints altering key regulatory genes involved in PDAC progression were detected in all tumors. SMAD4, ZNF521, and FHIT were among the most frequently hit genes. Conversely, commonly reported genes with copy number gains, including MYC and GATA6, were frequently observed in the absence of direct intragenic breakpoints, suggesting a requirement for sustaining oncogenic function during PDAC progression. Integration of data from MPseq, exome sequencing, and transcriptome analysis of primary PDAC cases identified limited overlap in genes affected by both rearrangements and point mutations. However, significant overlap was observed in major PDAC-associated signaling pathways, with all PDAC exhibiting reduced SMAD4 expression, reduced SMAD-dependent TGFß signaling, and increased WNT and Hedgehog signaling. The frequent loss of SMAD4 and FHIT due to genomic rearrangements strongly implicates these genes as key drivers of PDAC, thus highlighting the strengths of an integrated genomic and transcriptomic approach for identifying mechanisms underlying disease initiation and progression.

Identification of independent primary tumors and intrapulmonary metastases using DNA rearrangements in non-small-cell lung cancer.

PURPOSE: Distinguishing independent primary tumors from intrapulmonary metastases in non-small-cell carcinoma remains a clinical dilemma with significant clinical implications. Using next-generation DNA sequencing, we developed a chromosomal rearrangement-based approach to differentiate multiple primary tumors from metastasis. METHODS: Tumor specimens from patients with known independent primary tumors and metastatic lesions were used for lineage test development, which was then applied to multifocal tumors. Laser capture microdissection was performed separately for each tumor. Genomic DNA was isolated using direct in situ whole-genome amplification methodology, and next-generation sequencing was performed using an Illumina mate-pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction. RESULTS: A total of 41 tumor samples were sequenced (33 adenocarcinomas [ADs] and eight squamous cell carcinomas [SQCCs]), with a range of three to 276 breakpoints per tumor identified. Lung tumors predicted to be independent primary tumors based on different histologic subtype did not share any genomic rearrangements. In patients with lung primary tumors and paired distant metastases, shared rearrangements were identified in all tumor pairs, emphasizing the patient specificity of identified breakpoints. Multifocal AD and SQCC samples were reviewed independently by two pulmonary pathologists. Concordance between histology and genomic data occurred in the majority of samples. Discrepant tumor samples were resolved by genome sequencing. CONCLUSION: A diagnostic lineage test based on genomic rearrangements from mate-pair sequencing demonstrates promise for distinguishing independent primary from metastatic disease in lung cancer.