Cancer genome sequencing is essential for understanding tumor evolution and advancing precision medicine.1 However, reference gaps and germline variants obscure detection of small and large somatic variants and methylation in repetitive regions.1-3 It is common for tumor cells to gain or lose chromosome arms due to somatic structural changes that occur inside highly repetitive satellite DNA sequences in the centromeres.4 To identify the full spectrum of somatic variants, including complex rearrangements, we construct and curate near-complete, haplotype-resolved assemblies of the most recent common ancestor of an early-passage broadly-consented hypodiploid pancreatic cancer cell line and matched normal tissues. The tumor assembly completely recapitulates all 35 tumor chromosomes observed with karyotyping, with multiple translocation-induced hybrid chromosomes. The hybrid chromosomes contain putative functional dicentric and fused centromeres, nested foldback inversions causing 14 breakpoints with a haplotype switch in a single event, and centromeric satellite tandem duplications up to 136 kbp. Direct comparison of tumor and normal assembly haplotypes uncovers >7,000 variants altering >1 Mbp of sequence in repetitive regions that have been hidden by reference gaps and germline variants. 44 % of somatic small variants change representation because they alter germline variants on GRCh38, impacting mutational signatures and kataegis/omikli clusters. Most somatic LINE insertions originate from two hypomethylated non-reference germline LINE insertions, highlighting their impact on insertion mutation burden. These assemblies demonstrate that centromeric, acrocentric, and telomeric regions conventionally excluded from analysis harbor extensive somatic and epigenetic changes. Resolving complete tumor genomes enables a deeper understanding of cancer structural plasticity and the endpoints of breakage-fusion-bridge cycles. These assembled, curated paired normal-tumor benchmarks will serve as a critical foundation for developing future algorithms to characterize the most intractable regions of cancer genomes.

Here, we present an optimized high-resolution Hi-C protocol for human breast tissues using the Phase Genomics Proximo Hi-C Kit. We describe steps for liquid nitrogen tissue processing, crosslinking, and quenching, followed by cell lysis and controlled chromatin fragmentation. Proximity ligation captures three-dimensional interactions, followed by reverse crosslinking, DNA purification, and streptavidin bead enrichment. Libraries are prepared on beads, amplified, and cleaned with size selection, producing high-quality material for Illumina sequencing and genome-wide 3D chromatin analysis.For complete details on the use and execution of this protocol, please refer to Choppavarapu et al.1

The Genome in a Bottle Consortium (GIAB), hosted by the National Institute of Standards and Technology (NIST), is developing new matched tumor-normal samples, the first explicitly consented for public dissemination of genomic data and cell lines. Here, we describe a comprehensive genomic dataset from the first individual, HG008, including DNA from an adherent, epithelial-like pancreatic ductal adenocarcinoma (PDAC) tumor cell line and matched normal cells from duodenal and pancreatic tissues. Data for the tumor-normal matched samples comes from seventeen distinct state-of-the-art whole genome measurement technologies, including high depth short and long-read bulk whole genome sequencing (WGS), single cell WGS, Hi-C, and karyotyping. These data will be used by the GIAB Consortium to develop matched tumor-normal benchmarks for somatic variant detection. We expect these data to facilitate innovation for whole genome measurement technologies, de novo assembly of tumor and normal genomes, and bioinformatic tools to identify small and structural somatic variants. This first-of-its-kind broadly consented open-access resource will facilitate further understanding of sequencing methods used for cancer biology.

Sarcomas are soft-tissue tumors that pose significant diagnostic challenges for clinical laboratories. With more than 80 subtypes, accurate classification of sarcomas is crucial for providing valuable prognostic and therapeutic guidance. Gene fusions are a hallmark of many sarcoma subtypes, offering pathognomonic markers for diagnosis. Routine pathological diagnosis typically involves hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC), with diagnostic samples most commonly preserved as formalin-fixed, paraffin-embedded (FFPE) specimens. However, FFPE preservation precludes conventional chromosome analysis and presents challenges for molecular tests such as those require high molecular weight DNA (i.e. long read sequencing and optical genome mapping). In addition, current methods, such as fluorescence in situ hybridization (FISH) and targeted RNA-seq panels, have limitations, including the restricted number of genes interrogated, dependence on known gene rearrangement breakpoints or chimera transcripts, and sensitivity to RNA quality, especially in FFPE samples. To address these limitations, we developed a new protocol for genome-wide detection of chromosomal abnormalities using Genomic Proximity Mapping (GPM) technology. GPM is a next-generation cytogenomic technique that employs proximity ligation and low-pass sequencing to identify structural variants and gene fusions across the entire genome. As a DNA-based, genome-wide assay, GPM overcomes the primary constraints of FISH and targeted gene panel by RNA-seq. To evaluate the performance of GPM, we applied it to 12 sarcoma samples that had previously underperformed or failed Anchored Multiplex PCR (AMP) analysis using the FusionPlex solid tumor gene panel. In all cases, GPM not only recapitulated the previous findings but also identified additional pathognomonic fusions in 33% of cases where targeted RNA-seq methods failed due to RNA quality issues or the limited scope of targeted analysis. Overall, GPM detected novel structural variants genome-wide and identified definitive gene fusions were in 8 sarcoma subtypes, achieving a sensitivity >0.9 and specificity of 1. In addition to detecting translocations, inversions, and insertions, GPM can identify novel gene fusions, deletions, duplications/amplifications, and copy-neutral loss of heterozygosity. GPM represents a novel, genome-wide approach to identifying both known and novel gene fusions, as well as other chromosomal abnormalities, in FFPE tissue. It enhances sarcoma classification where other methods fail, addressing the limitations of current cytogenetic and other NGS technologies. Yajuan J. Liu, He Fang, Maika Malig, Emily Reister, Mary Wood, Ivan Liachko, Stephen M. Eacker. Enhanced detection of gene fusions in sarcoma using Genomic Proximity Mapping in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6634.

Introduction: MYC rearrangement (MYC-R), especially those involving the immunoglobulin loci (IGL, IGH, and IGK), are drivers of MM and other malignancies. IGL::MYC has been associated with hyperdiploidy and inferior outcomes. Despite this, MYC is not part of the recommended fluorescence in situ hybridization (FISH) panel at diagnosis. We describe the clinical impact of MYC alterations in newly diagnosed MM (NDMM) and the MYC-R prevalence in systematic MYC assessment by FISH and genomic proximity mapping (GPM). Methods: The Multiple Myeloma Research Foundation CoMMpass (MMRF-NGS cohort) was used to assess the frequency and impact of MYC rearrangements (MYC-R) on progression-free survival (PFS). FISH using a MYC break apart probe was part of the routine FISH panel at our center for MM starting in June 2023 in 133 patients (pts) (WCM-FISH cohort). GPM was performed on CD138+-selected cells from 50 pts who part of a prospective clinical trial of NDMM (NCT01559935 (CarBiRD cohort)) and in 37 pts from the WCM-FISH cohort. MYC-R breakpoints and gain (MYC-g) were identified. PFS was defined as the time from MM diagnosis to progression of disease or death, as determined by the International Myeloma Working Group. High-risk cytogenetics (HR-CG) were defined as any of del17p, t(4;14), t(14;16), or gain1q. Supported by R44CA268681 and WA Care Fund. Results: In the MMRF-NGS cohort, MYC-R was seen in 234/913 pts (25.6%), with most MYC-R having IGH/IGL/IGK (IG::MYC) as a partner in 52% of cases. IG::MYC pts had inferior PFS (25.4 months) when compared to MYC-R with non-IGH/L/K partners (MYC::non-IG) (42.2 months, pairwise p= 0.04) and MYC-negative (neg) (37.9 months, pairwise p= 0.04). There were no differences in PFS between MYC-neg and MYC::non-IG (p = 0.59). In a multivariable model, IG::MYC remained associated with inferior PFS (hazard ratio [HR] 1.39, 1.09-1.7, p = 0.01) after adjusting for age, transplant, triplet vs. duplet induction, and del17p, gain1q, del1p, and del13q status. 24 pts with IG::MYC had sequential samples available, and 22/24 (92%) IG::MYC was present at diagnosis, suggesting IG::MYC-Ris a truncal event and is often present at MM diagnosis. In the WCM-FISH cohort, MYC alterations were present in 44/133 pts with MM (33%). MYC-R was present in 35 pts (26%), while MYC-g was present in 13 (9.8%). Pts with MYC-R had significantly shorter PFS compared to MYC-neg (15.6 vs. 35.7 months, p = 0.017). Other variables associated with PFS included ISS, R-ISS, lactate dehydrogenase, albumin, del17p, and HR-CG. In the multivariable model, only MYC-R was associated with inferior PFS (HR 2.82, 95% CI 1.12-7.06, p = 0.02). When pts were stratified by MYC-R and HR-CG status, pts with MYC -R and no HR-CG had inferior PFS compared to MYC-neg/no HR-CG pts (p = 0.001) and had comparable PFS to HR-CG alone. Most MYC-R cases were not further assessed by standard FISH for, with MYC::IGH being the only partner genes identified (3/35 cases). In the CarBiRD cohort, GPM identified 17/50 (34%) pts with MYC alterations, including 12 pts (24%) with MYC-R. The MYC partner was identified in all cases, with 8 pts having IG::MYC. MYC-R, were associated with inferior PFS when compared to MYC-neg (HR 2.1, 1.06 – 4.4, p = 0.03), mainly driven MYC::IG cases, which had inferior PFS when compared to non-MYC-R pts (HR 4.27, 1.82 – 10.1, p < 0.001). In the pts who had GPM and FISH available (87 pts), GPM identified MYC alteration in 32/87 (37%) of cases, including MYC-R in 27/87 (31%) of pts. 48% of MYC-R involved the IGL, IGK or IGH (6, 4, and 3 cases, respectively). GPM identified the breakpoint in all cases, also identifying complex alterations involving several translocations or inversions in the same patient in addition to inversions of chromosome 8q. GPM detected all cases of t(11;14), t(4;14), MYC-R, and t(14;16) identified by FISH, in addition to 3 pts with t(11;14), t(14;16), and MYC-R that FISH missed due to atypical breakpoints. MYC alterations were associated with a higher rate of del1p, hyperdiploidy, and del17p, while mutually exclusive from t(11;14). Conclusion: MYC-R is common in NDMM and is associated with inferior PFS independent of other genomic variables. MYC FISH should be considered part of the standard risk stratification. MYC partner may have important prognostic implications. GPM provides higher resolution and can locate other recurrent abnormalities in MM, in addition to characterizing complex MYC alterations and breakpoints.

Head and neck squamous cell carcinoma (HNSCC) encompass a heterogenous classification of solid tumors that account for ∼5% of the total new cancer cases globally. HNSCC presents as metastatic disease at estimated rates of 4-25%, most commonly in bone, liver and lung. Brain metastasis (BM) is uncommon and is particularly deadly. There is a need to better understand biomarkers that may presage brain metastasis in hopes of identifying those patients requiring aggressive treatment to head-off metastatic disease. Furthermore, identification of common biomarkers may elucidate therapeutic strategies to treat this disease. To compliment ongoing targeted sequencing efforts, we have applied high-resolution cytogenomic analysis using the genomic proximity mapping (GPM) platform, CytoTerra, to investigate chromosomal abnormalities associated with BM HNSCC. GPM uses proximity ligation and short-read sequencing to detect alterations in genomic structure. This technology is compatible with formalin-fixed paraffin-embedded tissue samples because it uses formalin fixation to capture genomic proximity information. 15 µm molecular curls were used as inputs for library preparation from 2 primary tumors of BM HNSCC and 3 samples of non-BM HNSCC. Chromosomal abnormalities were detected using the cloud-based CytoTerra analytic platform which utilizes a suite of AI-based and bioinformatic tools. GPM libraries with sufficient QC statistics were generated from all five samples. Evaluation of HNSCC genomes revealed a wide array of structural alterations commonly observed in cancer, including balanced and unbalanced translocations, inversions, chromothripsis, deletions, duplications, and copy-neutral loss of heterozygosity. Notably structural rearrangements involving the JAK1 and EGFR loci were observed in BM HNSCC samples. In addition we identified a novel NRG1::FAM110B fusion in one case of BM HNSCC. None of these rearrangement involved the disruption of the coding sequence of the gene and therefore would escape detection through standard panel sequencing. Non-BM HNSCC showed similar classes of chromosomal abnormalities and included a deletion of KDM6A. Variants identified in serial sampling of neighboring HNSCC lesions in non-BM HNSCC demonstrated a high degree of overlap, demonstrating reproducibility. Uniquely identified variants allow inferences on tumor evolution between the two lesions. GPM with the CytoTerra next generation cytogenomic platform successfully identified chromosome abnormalities in FFPE HNSCC samples. BM HNSCC samples demonstrated structural alterations in known and targetable oncogenes suggesting that CytoTerra can be a useful tool for novel biomarker discovery in solid tumors, including HNSCC. Emily Reister, Maika Malig, Mary Wood, Alexander Muratov, Ivan Liachko, Stephen Eacker, Ida Deichaite. Discovery of biomarkers of brain metastasis using Genomic Proximity Mapping (GPM) on formalin-fixed paraffin-embedded head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 716.

Introduction Cytogenetics encompasses a suite of diagnostics assays that provides diagnostic and prognostic information that can help guide care for acute myeloid leukemias (AML) patients. Routine cytogenetic workup for AML includes karyotyping and fluorescence in situ hybridization (FISH) for known recurrent variants. The workup can be extended to include chromosome genome array testing (CGAT) but because all 3 of these tests have a non-overlapping set of limitations that can necessitate multiple, often iterative, rounds of testing in complex cases and may miss information in cases with cryptic fusions or small copy number alterations. Therefore, there is an unmet need for an accurate, fast and reliable test that can provide a solution to these limitations. In this study, we evaluate Genomic Proximity Mapping (GPM), a novel method that captures much of the same information as karyotyping, FISH, and CGAT in a single assay. Methods GPM uses proximity ligation sequencing to capture ultra-long range contiguity information from conventional short-read sequencing. In brief, 200,000-500,000 cells from bone marrow, peripheral blood, or aphersis samples are crosslinked with formalin prior to library preparation. Because sequences that are closer on a chromosome are more likely to physically interact and be crosslinked, GPM can use the frequency pairwise of crosslinked sequence interactions to determine the structure of chromosomes. Results In order to evaluate its performance, we benchmarked GPM against standard-of-care cytogenetics conducted on newly diagnosed 166 AML cases in a multicenter, retrospective study utilizing real-world patient samples. There was a strong concordance between GPM and cytogenetic findings, with 100% of European LeukemiaNet (ELN) specified cytogenetic risk variants identified by cytogenetic being identified by GPM. However, GPM also identified additional variants of known clinical significance not observed by standard-of-care cytogenetics. To determine if GPM findings had clinical benefits for risk stratification, we compared overall survival (OS) of patients, risk-stratified using ELN 2022 criteria, using either cytogenetics or GPM to determine the cytogenetic abnormalities. Separation of favorable, intermediate, and adverse risk categories were statistically significant when variants identified either by cytogenetics or GPM were used for risk categorization (GPM, P = 0.0006; cytogenetics P = 0.0009 for GPM and cytogenetics, Mantel-Cox log rank test). However, Kaplan-Meier analysis demonstrated that GPM showed more separation in intermediate (median OS 2.1 years for GPM and 2.4 years for cyto), and adverse (median OS 1.1 yrs for GPM and 0.9 years for cytogenetics) categories. The ELN-directed risk based on karyotype and FISH presentation was revised for 14 (8.4%) out of 166 cases with available data based on GPM assay results. Discussion and Conclusion These findings argue that GPM can be used as a single assay to accurately stratify AML patient risk and may provide additional benefits over cytogenetics, yielding more accurate risk stratification.

Brain metastasis (BM) is a rare but severe complication of head and neck squamous cell carcinoma (HNSCC), with limited knowledge of molecular characteristics and immunogenicity. We analyzed 61 cases of HNSCC-BM from three academic institutions (n = 24) and Foundation Medicine Inc (FMI, n = 37). A subset of cases underwent next-generation sequencing, multiple immunofluorescence, and proximity ligation sequencing. Gene enrichment analysis compared alterations in FMI BM samples (n = 37) with local samples (n = 4082). Demographics included: median age of 59 years, 75

The European Leukemia Network (ELN) has established criteria for patient risk stratification based on cytogenetic abnormalities in observed in acute myeloid leukemia (AML). Risk stratification based on cytogenetic analysis is commonly used to guide therapy selection and overall patient care. Proximity ligation sequencing (PLS) is a next generation cytogenomic method that uses short-read NGS to capture ultra-long-range genomic contiguity and detect chromosome abnormalities including those in the ELN risk stratification. To test the utility of PLS in evaluating cytogenetic risk in AML, we performed a retrospective study of AML cases gathered from clinical archives. In all, libraries from 95 samples were prepared using the OncoTerra PLS library preparation kit and sequenced on one of three platforms: Illumina, Element, or Singular short-read sequencing platforms. Libraries sequenced across all three platforms passed QC metrics for library performance. Patient risk based on variants identified by the OncoTerra PLS analytic platform and the reported standard-of-care cytogenetics were assessed following the ELN 2022 guidelines. The predictive power of OncoTerra and standard-of-care cytogenetics were evaluated based on the overall survival of patients segregated into favorable, intermediate, and adverse risk categories. Statistical comparison of Kaplan-Meier analysis between OncoTerra and standard-of-care cytogenetics demonstrated that PLS significantly improved the segregation of patient outcomes across risk groups. These findings show that PLS has the potential significantly improve cytogenetic risk stratification within the context of established ELN risk variants for AML using short-read sequencing platforms.

Cytogenetics encompasses a suite of diagnostics assays that provides critical diagnostic and prognostic information that can help guide care for acute myeloid leukemias (AML) patients. Routine cytogenetic workup for AML includes karyotyping and fluorescence in situ hybridization (FISH) for known recurrent variants. The workup can be extended to include chromosome genome array testing (CGAT) but because all 3 of these tests have a non-overlapping set of limitations that can necessitate multiple, often iterative, rounds of testing in complex cases and may miss information in cases with cryptic fusions or small copy number alterations. Therefore, there is an unmet need for an accurate, fast and reliable test that can provide a solution to these limitations. In this study, we evaluate Genomic Proximity Mapping (GPM), a novel method that captures much of the same information as karyotyping, FISH, and CGAT in a single assay. GPM uses proximity ligation sequencing to capture ultra-long range contiguity information from conventional short-read sequencing. In brief, 200,000-500,000 cells from bone marrow, peripheral blood, or aphersis samples are crosslinked with formalin prior to library preparation. Because sequences that are closer on a chromosome are more likely to physically interact and be crosslinked, GPM can use the frequency pairwise of crosslinked sequence interactions to determine the structure of chromosomes. In order to evaluate its performance, we benchmarked GPM against standard-of-care cytogenetics conducted on newly diagnosed 166 AML cases in a multicenter, retrospective study utilizing real-world patient samples. There was a strong concordance between GPM and cytogenetic findings, with 100% of European LeukemiaNet (ELN) specified cytogenetic risk variants identified by cytogenetic being identified by GPM. However, GPM also identified additional variants of known clinical significance not observed by standard-of-care cytogenetics. To determine if GPM findings had clinical benefits for risk stratification, we compared overall survival (OS) of patients, risk-stratified using ELN 2022 criteria, using either cytogenetics or GPM to determine the cytogenetic abnormalities. Separation of favorable, intermediate, and adverse risk categories were statistically significant when variants identified either by cytogenetics or GPM were used for risk categorization (GPM, P = 0.0006; cytogenetics P = 0.0009 for GPM and cytogenetics, Mantel-Cox log rank test). However, Kaplan-Meier analysis demonstrated that GPM greater separation in intermediate (median OS 2.1 years for GPM and 2.4 years for cyto), and adverse (median OS 1.1 yrs for GPM and 0.9 years for cytogenetics) categories. The ELN-directed risk based on karyotype and FISH presentation was revised for 14 (8.4%) out of 166 cases with available data based on GPM assay results. These findings argue that GPM can be used as a single assay to accurately stratify AML patient risk and may provide additional benefits over cytogenetics, yielding more accurate risk stratification. Support: NIH/NCI U10CA180888, U10CA180819, U24CA196175, R44CA278140

Background:Cytogenetic analysis encompasses a suite of standard-of-care diagnostic testing methods that is routinely applied in cases of acute myeloid leukemia (AML) to assess chromosomal changes that are clinically relevant for risk classification and treatment decisions. Objective:In this study, we assess the use of Genomic Proximity Mapping (GPM) for cytogenomic analysis of AML diagnostic specimens for detection of cytogenetic risk variants included in the European Leukemia Network (ELN) risk stratification guidelines. Methods:Archival patient samples (N=48) from the Fred Hutchinson Cancer Center leukemia bank with historical clinical cytogenetic data were processed for GPM and analyzed with the CytoTerra® cloud-based analysis platform. Results:GPM showed 100% concordance for all specific variants that have associated impacts on risk stratification as defined by ELN 2022 criteria, and a 72% concordance rate when considering all variants reported by the FH cytogenetic lab. GPM identified 39 additional variants, including variants of known clinical impact, not observed by cytogenetics. Conclusions:GPM is an effective solution for the evaluation of known AML-associated risk variants and a source for biomarker discovery.

6036 Background: BM is a rare complication of HNSCC that carries a high rate of morbidity and poor prognosis. Clinical risk factors, molecular characteristics, and the immunogenicity of HNSCC BM are not well defined, leaving a critical knowledge gap in this field. We performed one of the largest multi-institutional analyses summarizing the clinical, molecular, and immunologic profile of 61 cases of BM-HNSCC. Methods: We conducted a pooled analysis of the clinical characteristics pertaining to BM-HNSCC from 3 academic institutions (n=24). Next-generation sequencing (NGS) and immune profiling (IP) of primary and BM specimens was conducted on a subset of cases (n=19 and n=16, respectively); there were 3 paired samples for NGS and 0 for IP. Four samples (2 BM and 2 non-BM) were submitted to Phase Genomics, Inc for evaluation of structural variants in BM genomes by proximity ligation sequencing (PLS). These results were complimented by a comparative analysis of genomic alterations in an additional cohort of BM (n=37) and local samples (n=4082) submitted for NGS at Foundation Medicine, Inc (FMI). Statistical comparisons were done using Fisher’s exact testing of 2x2 contingency tables with p-values controlled for FDR by the Benjamini-Hochberg procedure. Results: Clinical features were as follows: median age at diagnosis 59 years, 75% male, 55% current/former smokers, 75% oropharyngeal primary, and 84% HPV+ or p16+. The most frequently altered genes in BM specimens (62% HPV/p16+) were ATM (54%), KMT2A (54%), PTEN (46%), RB1 (46%), and TP53 (46%). BM and non-BM samples demonstrated significant levels of structural rearrangement ranging from 9 to 90 variants by PLS. IP identified lower densities of CD8+, PD1+, PDL1+, and FOXP3+ cells in BMs compared to primary tumors. PDL1 combined positive scores were <1% in 12/13 unpaired samples (92%; 10 BM and 2 primary). The FMI BM-HNSCC cohort (51% HPV+) identified CDKN2A (40.5%), TP53 (37.8%), and PIK3CA (27.0%) as the most frequently altered genes. Enrichment analysis of the FMI cohort showed MAP2K2 alterations significantly enriched in BM (11.8% vs 6.4%, P=0.005) and TSC1 alterations significantly enriched in the local site (67.3% vs 37.8%, P=0.008). HPV+ was also significantly enriched in the BM cohort (51.25% vs 26.11%, P=0.001). Overall survival from BM diagnosis was 6m (range 0-27m). Conclusions: HNSCC patients with BM have higher-than-expected proportions of oropharyngeal primary site and HPV/p16-positivity. The most frequent molecular alterations in BM samples are also commonly found in non-BM HNSCC, including targetable PIK3CA alterations. MAP2K2 alterations were significantly enriched in BM compared to non-BM samples, which warrants further investigation. BM samples also tended to have lower markers of immunogenicity. This latter finding could have important clinical implications when considering immunotherapy or immune-modulating drugs.

Abstract Cytogenetics forms the cornerstone of diagnostic genetic testing in leukemia, providing both prognostic and diagnostic information to help guide treatment. Currently, standard-of-care cytogenetics in acute myeloid leukemia (AML) is composed of a combination of karyotyping, FISH testing, and in some cases chromosomal microarray analysis, each test offering a unique combination of advantages and weaknesses. A particular challenge for this combination of tests is the delivery of results in a timely and comprehensive manner. To overcome these limitations, we have developed OncoTerra, a proximity-ligation-sequencing based method which can rapidly detect all classes of variants detected by cytogenetics tests in a single assay. We applied OncoTerra to a cohort of more than 100 samples from a SWOG AML trial (S0106), benchmarking sensitivity and specificity of the variants. We observed an overall >0.95 sensitivity and a specificity of 1 for the variants identified by cytogenetics and identified numerous cryptic variants. Overall, we identified variants that changed the diagnostic risk stratification of 15% of the patient involved in the trial based on the ELN 2022 criteria. This translated into better overall prediction of outcomes of patients in the trial and has identified both known and novel variants missed by standard-of-care tools. Citation Format: Stephen Eacker, Maika Malig, Mary Wood, Alexander Muratov, Olga Sala-Torra, Ivan Liachko, Cecilia Yeung, Jerald Radich. Retrospective analysis of acute myeloid leukemia using next generation cytogenomics identifies variants missed at diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5069.

Outdoor cultivation is commonly used to produce algal biomass for a variety of bioproducts including food, feed, fuel, pharmaceuticals, and nutraceuticals. Outdoor cultivation ponds are highly susceptible to pest pressures that may lead to periods of low productivity or even entire loss of the algal crop. Therefore, there is a need for rapid, real-time tracking of pests for early intervention to mitigate crop loss. Herein, we describe the development of a field deployable, low-cost qPCR assay for detecting both known and novel pests of a farmed eukaryotic alga species, Nannochloropsis sp. We performed a proximity guided metagenome deconvolution approach (ProxiMetaTM) to discover novel pests that temporally correspond to periods of reduced pond productivity. This approach provided high-quality metagenome assemblies that were used to design qPCR probes to detect specific pests of interest. The portable qPCR assay, designed to be deployed at remote field locations, enables low-cost surveillance with a rapid (2 h) turn-around time. Frequent sampling allows for early detection and prompts intervention strategies to remedy infected ponds to minimize crop loss. The qPCR assay was used to successfully detect a known predatory bacterium within the order Bdellovibrionales both in the lab and at a remote field location. Furthermore, we assembled the genome of two novel, site-specific pests in the Saprospiraceae family and successfully designed qPCR probes that differentially detected their presence in two different pond locations. Ultimately, this assay has the potential to monitor multiple pests simultaneously and tailor targets to match likely pest infections that differ across geographical locations, helping to mitigate crop loss on a large scale.

Abstract Activating KRAS mutations, homologous recombination deficiency (HRD), deficient mismatch repair (dMMR), and other prognostic or predictive biomarkers are seen in fewer than half of pancreatic ductal adenocarcinoma (PDAC) cases. Structural variants (SV), such as gene fusions, translocations, deletions, duplications, etc, are known to play a role in oncogenesis, metastasis, and/or treatment resistance. In solid tumors, SV discovery and validation has been limited, often due to a fresh or live tissue requirement in cytogenomic analyses. Recent innovations in processing formalin-fixed paraffin-embedded (FFPE) samples for “next-generation” chromatin capture and long-range sequencing platforms (i.e. Hi-C) have overcome this barrier. Hi-C is a genome-wide technology which surveys rearrangements, generating genomic contiguity data from highly fragmented chromatin. Unlike targeted (e.g. hybrid capture) assays, Hi-C can survey the landscape of genomic aberrations in an unbiased manner. Archived FFPE has previously been an obstacle to Hi-C analysis of solid tumors. We present here, to our knowledge, the first Hi-C analysis of FFPE PDAC samples using a previously described, proprietary Hi-C platform. Thirty-one samples from 20 unique patients diagnosed and treated at the University of Colorado Comprehensive Cancer Center between 2014-2022 were processed and analyzed. Twenty-two samples were surgical resections paired with later biopsies of metastatic recurrences. Nine were unpaired surgical resections or metastatic biopsies. Seven of these cases had somatic KRAS mutations, five had germline mutations in DNA repair genes, and none had evidence of dMMR or MSI-high. Fifty percent of samples had sufficient tumor content for processing and successful library generation. Notably, two paired samples from 2014 were successfully processed and sequenced demonstrating the applicability of this technology to archival samples. In successfully analyzed samples, the vast majority of SVs observed in metastases were also seen in paired primary tumors. Several duplications and deletions recurred across two or more patients. Furthermore, at least three SVs from this cohort were observed in other tumor types from a separate cohort analysis using this technology. Cases with relatively more SVs demonstrated a longer time to metastatic recurrence than cases with fewer SVs. Cases with germline-mutated cancer-predisposing genes demonstrated a similar SV signature and time to metastatic recurrence as some cases with or without unknown germline and somatic tumor mutation status. Overall, our findings show that this novel platform can successfully profile archived FFPE PDAC samples. Some paired samples show a shared SV signature suggesting the presence of metastatic clones at the time of initial diagnosis. Following these results, we plan to process and analyze a larger cohort of PDAC cases enriched for wild-type KRAS and HRD carriers. We plan to compare our findings to Hi-C results from other tumors and further focus on any SVs recurrent across tumor types. Citation Format: Abhishek Pandey, Stephen Eacker, Adrie Van Bokhoven, Kathleen Torko, Christopher H Lieu. Proximity ligation sequencing reveals novel and recurrent structural genomic variants in FFPE pancreatic ductal adenocarcinoma samples [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A03.

Cytogenetic methods are a cornerstone of the modern diagnostic workflow for acute myeloid leukemia and related disorders. The goal of these methods is to identify structural genomic aberrations that drive the cancer or otherwise act as an indicator of patient risk. Despite their success, any one cytogenetic test is limited either in resolution, ability to provide unbiased survey of the genome, or ability to detect balanced aberrations. To address these limitations, we have applied proximity ligation sequencing (PLS) to characterize structural variants (SV) in AML genomes. PLS captures ultra-long-range sequence information without high-molecular-weight DNA and requires a modest (5-7x) sequence coverage of the genome. Applying this method to AML diagnostic specimens, we find that PLS identifies cytogenetically defined translocations (reciprocal and non-reciprocal) and inversions with specificity and sensitivity >0.95. The high resolution of PLS (<10kb) detected non-canonical variants missed by standard methods as well as structural variants below limits of detection by karyotyping, identifying variants in over half the patients previously determined to have a normal karyotype (Table 1). This included previously described variants of clinical significance as well as a recurrent inversion not previously described in AML. These observations highlight the effectiveness of PLS to provide high-resolution insights into known clinically relevant variants and discover variants missed by traditional methodologies.

Chromosomal rearrangements can generate oncogenic gene fusions, a rich class of targets for precision diagnosis and therapeutics. However, detecting these gene fusions using RNA sequencing can be challenging, particularly when dealing with FFPE tissues, due to issues of RNA stability and quality. Additionally, RNA sequencing cannot detect promoter/enhancer juxtaposition of genes, limiting the scope of fusion detection efforts. Moreover, current DNA-based methods such as long-read sequencing and optical genome mapping are not suitable for FFPE samples due to the input requirement of high-molecular-weight DNA. In the current study, we evaluate the utility of proximity ligation sequencing (Hi-C, 3D genomics) as a novel method to identify chromosomal aberrations in neoplasia in the clinical setting. We analyzed 24 tumor samples, including fresh/frozen and FFPE specimens that had been previously characterized using other methods such as RNA-seq, karyotyping, and/or FISH. Our results showed that that Hi-C detected both gene juxtapositions and chimeric gene fusions resulting from balanced and unbalanced chromosomal rearrangements with 100% concordance with previously applied methods. Furthermore, Hi-C can detect copy number alterations and copy neutral loss of heterozygosity simultaneously in a single assay. For example, Hi-C detected IGH::BCL2, PAX5::MYC, and 11q interstitial gain and terminal loss in a high grade B-cell lymphoma. In conclusion, this study demonstrated that Hi-C/3D genomics can precisely detect all types of structural and copy number variants in a single assay with sequence resolution for various sample types including FFPE tissues. Various methods for gene rearrangement detection will be compared and discussed.

Cytogenetic diagnostics including karyotyping, FISH, and chromosome microarray are regularly applied in cases of suspected leukemia, including acute myeloid leukemia (AML). Each of these three tests can identify non-overlapping sets of chromosome aberrations known to have prognostic or diagnostic value. Because each test addresses different classes of prognostic variants, all three tests are often performed in series or in parallel, adding time and expense to the diagnostic process. Here we describe OncoTerra, a platform based on proximity ligation sequencing (PLS), that can be used to identify variants commonly assayed by karyotyping, FISH, and array in a single assay. OncoTerra uses a specialized library preparation chemistry that requires no specialized equipment. Following Illumina sequencing, data is analyzed on a secure cloud platform. In a pilot study of 48 AML patient samples, OncoTerra identified variants previously observed by cytogenetics and additional variants that were previously identified. These variants were composed of translocations, inversions, and copy number variants either too small or too complicated to be accurately identified by cytogenetics. Importantly, additional variants identified by OncoTerra changed risk categories for 25% of patients based on ELN 2022 standards. These findings support deployment of OncoTerra as a time- and cost-effective, high-resolution cytogenomic solution to improve patient care.