Highly sensitive fusion detection using plasma cell-free RNA in non-small-cell lung cancers.

ALK, ROS1, and RET kinase fusions are important predictive biomarkers of tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC). Analysis of cell-free DNA (cfDNA) provides a noninvasive method to identify gene changes in tumor cells. The present study sought to use cfRNA and cfDNA for identifying fusion genes. A reliable protocol was established to detect fusion genes using cfRNA and assessed the analytical validity and clinical usefulness in 30 samples from 20 cases of fusion-positive NSCLC. The results of cfRNA-based assays were compared with tissue biopsy and cfDNA-based liquid biopsy (Guardant360 plasma next-generation sequencing [NGS] assay). The overall sensitivity of the cfRNA-based assay was 26.7% (8/30) and that of cfDNA-based assay was 16.7% (3/18). When analysis was limited to the samples collected at chemo-naïve or progressive disease status and available for both assays, the sensitivity of the cfRNA-based assay was 77.8% (7/9) and that of cfDNA-based assay was 33.3% (3/9). Fusion gene identification in cfRNA was correlated with treatment response. These results suggest that the proposed cfRNA assay is a useful diagnostic test for patients with insufficient tissues to facilitate effective administration of first-line treatment and is a useful tool to monitor the progression of NSCLC for consideration of second-line treatments.

Microfluidic-Based Detection of AML-Specific Biomarkers Using the Example of Promyelocyte Leukemia.

A microfluidic assay for the detection of promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) fusion protein was developed. This microfluidic-based system can be used for rapid personalized differential diagnosis of acute promyelocyte leukemia (APL) with the aim of early initiation of individualized therapy. The fusion protein PML-RARα occurs in 95% of acute promyelocytic leukemia cases and is considered as diagnostically relevant. The fusion protein is formed as a result of translocation t(15,17) and is detected in the laboratory by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). Diagnostic methods require many laboratory steps with specialized staff. The developed microfluidic assay includes a sandwich enzyme-linked immunosorbent assay (ELISA) system for PML-RARα on surface of magnetic microparticles in a microfluidic chip. A rapid detection of PML-RARα in cell lysates is achieved in less than one hour. A biotinylated PML-antibody on the surface of magnetic streptavidin coated microparticles is used as capture antibody. The bound translocation product is detected by a RARα antibody conjugated with horseradish peroxidase and the substrate QuantaRed. The analysis is performed in microfluidic channels which involves automated liquid processing with stringent washing and short incubation times. The results of the developed assay show that cell lysates of PML-RARα-positive cells (NB-4) can be clearly distinguished from PML-RARα-negative cells (HL-60, MV4-11).

Recurrent secondary genomic alterations in desmoplastic small round cell tumors.

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is a rare, highly aggressive, translocation-associated soft-tissue sarcoma that primarily affects children, adolescents, and young adults, with a striking male predominance. It is characterized by t(11;22) generating a novel EWSR1-WT1 fusion gene. Secondary genomic alterations are rarely described. METHODS: Tumor tissue from 83 DSRCT patients was assayed by hybrid-capture based comprehensive genomic profiling, FoundationOne® Heme next generation sequencing analysis of 406 genes and RNA sequencing of 265 genes. Tumor mutation burden was calculated from a minimum of 1.4 Mb sequenced DNA. Microsatellite instability status was determined by a novel algorithm analyzing 114 specific loci. RESULTS: Comprehensive genomic profiling identified several genomically-defined DSRCT subgroups. Recurrent genomic alterations were most frequently detected in FGFR4, ARID1A, TP53, MSH3, and MLL3 genes. With the exception of FGFR4, where the genomic alterations predicted activation, most of the alterations in the remaining genes predicted gene inactivation. No DSRCT were TMB or MSI high. CONCLUSIONS: In summary, recurrent secondary somatic alterations in FGFR4, ARID1A, TP53, MSH3, and MLL3 were detected in 82% of DSRCT, which is significantly greater than previously reported. These alterations may have both prognostic and therapeutic implications.

A novel NAP1L4/NUTM1 fusion arising from translocation t(11;15)(p15;q12) in a myeloid neoplasm with eosinophilia and rearrangement of PDGFRA highlights an unusual clinical feature and therapeutic reaction.

NUT midline carcinoma (NMC) is an aggressive neoplasm and mainly involved in the head and neck area. The defining genetic hallmark on these tumors is that testis-specific nuclear gene (NUTM1) fuses to bromodomain protein family member 4 gene (BRD4), resulting in the formation of BRD4-NUTM1 transcript. Here, we report a case with myeloid neoplasm complicating with eosinophilia (MLN-Eo) and rearrangement of PDGFRA, which co-exists with a new nucleosome assemble protein 1-like 4 gene (NAP1L4) NAP1L4-NUTM1 fusion. The patient have unusually clinical features and therapeutic reaction to imatinib mesylate. The cloned NAP1L4-NUTM1 gene structure is also determined.

Characterizing false-positive fluorescence in situ hybridization results by mate-pair sequencing in a patient with chronic myeloid leukemia and progression to myeloid blast crisis.

Traditional cytogenetic testing methodologies, including conventional chromosome analysis and fluorescence in situ hybridization (FISH), are invaluable for the detection or recurrent genetic abnormalities in various hematologic malignancies. However, technological advances, including a novel next-generation sequencing technique termed mate-pair sequencing (MPseq), continue to revolutionize the field of cytogenetics by enabling the characterization of structural variants at a significantly higher resolution compared to traditional methodologies. To illustrate the power of MPseq, we present a 27-year-old male diagnosed with chronic myeloid leukemia in myeloid blast crisis with multiple chromosomal abnormalities observed in all 20 metaphases from a peripheral blood specimen, including t(9;22)(q34;q11.2) and t(4;11)(q12;p15). Suspicious of a novel NUP98/PDGFRA fusion [t(4;11)(q12;p15)], break-apart FISH probe sets for the PDGFRA (4q12) and NUP98 (11p15.4) gene regions were performed and were both positive in approximately 86% of 200 interphase nuclei. However, subsequent MPseq testing revealed breakpoints located within the NUP98 gene and within an intergenic region (4q12) located between the CHIC2 and PDGFRA genes, indicating this 4;11 translocation does not result in the predicted NUP98/PDGFRA gene fusion as inferred from FISH and conventional chromosome results. This case demonstrates the clinical utility of MPseq, particularly for characterizing novel gene fusion events which may ultimately identify a false-positive FISH result.

Comparison of four next generation sequencing platforms for fusion detection: Oncomine by ThermoFisher, AmpliSeq by illumina, FusionPlex by ArcherDX, and QIAseq by QIAGEN.

As fusion detection NGS techniques are adopted by clinical labs, assay performance comparison is urgently needed. We compared four fusion-detection assay platforms on a pilot cohort of 24 prostate cancer samples: (1) Oncomine Comprehensive panel v3; (2) AmpliSeq comprehensive panel v3; (3) The solid tumor panel of FusionPlex; and (4) The human oncology panel of QIAseq. The assays were compared for the detection of different types of fusion based on whether the partner gene or the breakpoints are known. All assays detected fusion with known gene partners and known breakpoint, represented by TMPRSS2-ERG. A fusion with known partners but unknown breakpoint, TMPRSS2-ETV4, was reported by OCAv3 and FusionPlex, but not by AICv3 because the specific breakpoint was not in the manifest, nor by QIAseq since the panel did not target the exact exons involved. For fusion with unknown partners, FusionPlex identified the largest number of ETV1 fusions because it had the highest exon coverage for ETV1. Among these, SNRPN-ETV1 and MALAT1-ETV1, were novel findings. To determine reportability of low-level calls of highly prevalent fusions, such as TMPRSS2-ERG, we propose the use of percent fusion reads over total number of reads per sample instead of the fusion read count.

Feasibility and clinical utility of a pan-solid tumor targeted RNA fusion panel: A single center experience.

Gene fusions are caused by chromosomal rearrangements and encode fusion proteins that can act as oncogenic drivers in cancers. Traditional methods for detecting oncogenic fusion transcripts include fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). However, these methods are limited in scalability and pose significant technical and interpretational challenges. Next-generation sequencing (NGS) is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for cancer patients. We present our experience with the validation of a custom-designed Archer Anchored Multiplex PCR (AMP™) technology-based NGS technology, "NYU FUSION-SEQer" using RNA sequencing. We examine both analytical performance and clinical utility of the panel using 75 retrospective validation samples and 84 prospective clinical samples of solid tumors. Our panel showed robust sequencing performance with strong enrichment for target regions. The lower limit of detection was 12.5% tumor fraction at 125 ng of RNA input. The panel demonstrated excellent analytic accuracy, with 100% sensitivity, 100% specificity and 100% reproducibility on validation samples. Finally, in the prospective cohort, the panel detected fusions in 61% cases (n = 51), out of which 41% (n = 21) enabling diagnosis and 59% (n = 30) enabling treatment and prognosis. We demonstrate that the fusion panel can accurately, efficiently and cost-effectively detect the majority of known fusion genes, novel clinically relevant fusions and provides an excellent tool for discovery of new fusion genes in solid tumors.

ESMO recommendations on the standard methods to detect NTRK fusions in daily practice and clinical research.

BACKGROUND: NTRK1, NTRK2 and NTRK3 fusions are present in a plethora of malignancies across different histologies. These fusions represent the most frequent mechanism of oncogenic activation of these receptor tyrosine kinases, and biomarkers for the use of TRK small molecule inhibitors. Given the varying frequency of NTRK1/2/3 fusions, crucial to the administration of NTRK inhibitors is the development of optimal approaches for the detection of human cancers harbouring activating NTRK1/2/3 fusion genes. MATERIALS AND METHODS: Experts from several Institutions were recruited by the European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) to review the available methods for the detection of NTRK gene fusions, their potential applications, and strategies for the implementation of a rational approach for the detection of NTRK1/2/3 fusion genes in human malignancies. A consensus on the most reasonable strategy to adopt when screening for NTRK fusions in oncologic patients was sought, and further reviewed and approved by the ESMO TR and PM WG and the ESMO leadership. RESULTS: The main techniques employed for NTRK fusion gene detection include immunohistochemistry, fluorescence in situ hybridization (FISH), RT-PCR, and both RNA-based and DNA-based next generation sequencing (NGS). Each technique has advantages and limitations, and the choice of assays for screening and final diagnosis should also take into account the resources and clinical context. CONCLUSION: In tumours where NTRK fusions are highly recurrent, FISH, RT-PCR or RNA-based sequencing panels can be used as confirmatory techniques, whereas in the scenario of testing an unselected population where NTRK1/2/3 fusions are uncommon, either front-line sequencing (preferentially RNA-sequencing) or screening by immunohistochemistry followed by sequencing of positive cases should be pursued.

Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes.

The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) assay, called "the Eprobe leukemia assay," for major and minor BCR-ABL1, RUNX1-RUNX1T1, and various isoforms of PML-RARA. This assay is comprised of Eprobes that are exciton-controlled hybridization-sensitive fluorescent oligonucleotides. Melting curve analyses were performed on synthetic quantitative standard RNAs with strict quality control. Quantification capacity was evaluated by comparison with TaqMan RT-qPCR using 67 primary leukemia patient samples. The lower limit of detection and the limit of quantification of this assay were less than 31.3 copies/reaction and 62.5 copies/reaction, respectively. This assay correctly detected the fusion genes in samples with 100% sensitivity and specificity. The specificity of the reactions was confirmed by melting curve analyses. The assay detected low-level expression of minor BCR-ABL1 co-expressed with major BCR-ABL1. These results illustrate the feasibility and high accuracy of the Eprobe leukemia assay, even for minimal residual disease monitoring.

Dermatofibrosarcoma protuberans with a novel COL6A3-PDGFD fusion gene and apparent predilection for breast.

Dermatofibrosarcoma protuberans is a locally aggressive superficial mesenchymal neoplasm. It typically occurs in adulthood, and has been reported to have a slight male predilection. Tumors have a characteristic histopathologic appearance, including: storiform architecture, infiltrative "honeycomb" growth within subcutaneous adipose tissue, and immunoreactivity for CD34. Virtually all molecularly characterized cases to date have been found to harbor a COL1A1-PDGFB fusion product. Following identification of an index patient with a novel COL6A3-PDGFD fusion gene, we undertook a molecular investigation, using a combination of RNA sequencing and fluorescence in situ hybridization (FISH), to assess the prevalence of PDGFD rearrangement in dermatofibrosarcoma protuberans (N = 63). Three additional patients were found to have balanced PDGFD rearrangements. Interestingly, all 4 tumors arose on the breast of females. As a result, we subsequently examined 16 additional cases of primary breast dermatofibrosarcoma protuberans, identifying 2 additional tumors with PDGFD rearrangement. The morphology and immunophenotype of all 6 cases was analogous to those with the canonical COL1A1-PDGFB fusion; none of the cases showed fibrosarcomatous transformation. This study illustrates that the COL6A3-PDGFD fusion product is rare in dermatofibrosarcoma protuberans, and associated with an apparent predilection for breast. An awareness of this variant is important for pathologists, as it will not be detected using conventional reverse transcription polymerase chain reaction or FISH-based diagnostic assays for dermatofibrosarcoma protuberans.

Co-fuse: a new class discovery analysis tool to identify and prioritize recurrent fusion genes from RNA-sequencing data.

Recurrent oncogenic fusion genes play a critical role in the development of various cancers and diseases and provide, in some cases, excellent therapeutic targets. To date, analysis tools that can identify and compare recurrent fusion genes across multiple samples have not been available to researchers. To address this deficiency, we developed Co-occurrence Fusion (Co-fuse), a new and easy to use software tool that enables biologists to merge RNA-seq information, allowing them to identify recurrent fusion genes, without the need for exhaustive data processing. Notably, Co-fuse is based on pattern mining and statistical analysis which enables the identification of hidden patterns of recurrent fusion genes. In this report, we show that Co-fuse can be used to identify 2 distinct groups within a set of 49 leukemic cell lines based on their recurrent fusion genes: a multiple myeloma (MM) samples-enriched cluster and an acute myeloid leukemia (AML) samples-enriched cluster. Our experimental results further demonstrate that Co-fuse can identify known driver fusion genes (e.g., IGH-MYC, IGH-WHSC1) in MM, when compared to AML samples, indicating the potential of Co-fuse to aid the discovery of yet unknown driver fusion genes through cohort comparisons. Additionally, using a 272 primary glioma sample RNA-seq dataset, Co-fuse was able to validate recurrent fusion genes, further demonstrating the power of this analysis tool to identify recurrent fusion genes. Taken together, Co-fuse is a powerful new analysis tool that can be readily applied to large RNA-seq datasets, and may lead to the discovery of new disease subgroups and potentially new driver genes, for which, targeted therapies could be developed. The Co-fuse R source code is publicly available at https://github.com/sakrapee/co-fuse .

An enzyme-free and label-free surface plasmon resonance biosensor for ultrasensitive detection of fusion gene based on DNA self-assembly hydrogel with streptavidin encapsulation.

In this research, an enzyme-free and label-free surface plasmon resonance (SPR) biosensing strategy has been developed for ultrasensitive detection of fusion gene based on the heterogeneous target-triggered DNA self-assembly aptamer-based hydrogel with streptavidin (SA) encapsulation. In the presence of target, the capture probes (Cp) immobilized on the chip surface can capture the PML/RARα, forming a Cp-PML/RARα duplex. After that, the aptamer-based network hydrogel nanostructure is formed on the gold surface via target-triggered self-assembly of X shaped polymers. Subsequently, the SA can be encapsulated into hydrogel by the specific binding of SA aptamer, forming the complex with super molecular weight. Thus, the developed strategy achieves dramatic enhancement of the SPR signal. Using PML/RARα "S" subtype as model analyte, the developed biosensing method can detect target down to 45.22 fM with a wide linear range from 100 fM to 10 nM. Moreover, the high efficiency biosensing method shows excellent practical ability to identify the clinical PCR products of PML/RARα. Thus, this proposed strategy presents a powerful platform for ultrasensitive detection of fusion gene and early diagnosis and monitoring of disease.

2'-Fluoro ribonucleic acid modified DNA dual-probe sensing strategy for enzyme-amplified electrochemical detection of double-strand DNA of PML/RARα related fusion gene.

In the study, a novel sensing strategy based on dual-probe mode, which involved two groups of 2'-fluoro ribonucleic acid (2'-F RNA) modified probes, was designed for the detection of synthetic target double-strand DNA (dsDNA) of PML/RARα fusion genes in APL. And each pair of probes contained a thiolated capture probe (C1 or C2) immobilized on one of electrode surfaces in the dual-channel electrochemical biosensor and a biotinylated reporter probe (R1 or R2). The two groups of 2'-F RNA modified probes were separately complementary with the corresponding strand (Sa or Sb) from target dsDNA in order to prevent renaturation of target dsDNA. Through flanking target dsDNA, two "sandwitch" complexes (C1/Sa/R1 and C2/Sb/R2) were separately shaped by capture probes (C1 and C2) and free reporter probes (R1 and R2) in hybridization solution on the surfaces of different electrodes after the thermal denaturation. The biotin-modified enzyme which produced the measurable electrochemical current signal was localized to the surface by affinity binding between biotin with streptavidin. Under the optimal condition, the biosensor was able to detect 84 fM target dsDNA and showed a good specificity in PBS hybridization solution. Otherwise, the investigations of the specificity and sensitivity of the biosensor were carried out further in the mixed hybridization solution containing different kinds of mismatch sequences as interference background. It can be seen that under a certain interference background, the method still exhibited excellent selectivity and specificity for the discrimination between the fully-complementary and the mismatch sequences. The results of our research laid a good basis of further detection research in practical samples.

Routine genetic testing of lung cancer specimens derived from surgery, bronchoscopy and fluid aspiration by next generation sequencing.

After the development of EGFR tyrosine kinase inhibitors (TKIs), genetic testing of EGFR became required for effective treatment of lung cancer. Initially, the testing was conducted separately for each mutated region. However, many EGFR mutations have since been identified that determine the efficacy of EGFR-TKIs. Therefore, genetic testing of EGFR by next generation sequencing (NGS) may be a suitable strategy for lung cancer. Here we examined the applicability of the NGS method in regard to sensitivity, time and cost. A total of 939 specimens were obtained from 686 lung cancer patients at our hospital. DNA and RNA were simultaneously extracted from specimens derived from surgery, bronchoscopy, and fluid aspiration. Specimens included cerebrospinal fluid, pleural effusion, abdominal fluid, and pericardial effusion. From RNA, target regions (EGFR, KRAS, ALK fusion and RET fusion) were enriched by RT-PCR and sequenced with MiSeq. From DNA, PCR or PCR-RFLP conventional methods were performed. NGS and conventional methods were carried out routinely per week. Among the total 939 specimens, 38 specimens could not be examined with NGS. Among these, 34 specimens were analyzed by conventional testing with simultaneously extracted DNA. The remaining four specimens could not be tested with either method. Compared with the conventional method, the concordance rate of mutations was 99% (892/901), excluding specimens with NGS failure. The time period required from processing of specimens to results was 4 days, and the cost per sample was sufficiently low. In conclusion, the genetic testing with NGS method was useful for lung cancer treatment. The cost, sensitivity and time were able to tolerate routine examinations.

Pediatric, Adolescent, and Young Adult Thyroid Carcinoma Harbors Frequent and Diverse Targetable Genomic Alterations, Including Kinase Fusions.

BACKGROUND: Thyroid carcinoma, which is rare in pediatric patients (age 0-18 years) but more common in adolescent and young adult (AYA) patients (age 15-39 years), carries the potential for morbidity and mortality. METHODS: Hybrid-capture-based comprehensive genomic profiling (CGP) was performed prospectively on 512 consecutively submitted thyroid carcinomas, including 58 from pediatric and AYA (PAYA) patients, to identify genomic alterations (GAs), including base substitutions, insertions/deletions, copy number alterations, and rearrangements. This PAYA data series includes 41 patients with papillary thyroid carcinoma (PTC), 3 with anaplastic thyroid carcinoma (ATC), and 14 with medullary thyroid carcinoma (MTC). RESULTS: GAs were detected in 93% (54/58) of PAYA cases, with a mean of 1.4 GAs per case. In addition to BRAF V600E mutations, detected in 46% (19/41) of PAYA PTC cases and in 1 of 3 AYA ATC cases, oncogenic fusions involving RET, NTRK1, NTRK3, and ALK were detected in 37% (15/41) of PAYA PTC and 33% (1/3) of AYA ATC cases. Ninety-three percent (13/14) of MTC patients harbored RET alterations, including 3 novel insertions/deletions in exons 6 and 11. Two of these MTC patients with novel alterations in RET experienced clinical benefit from vandetanib treatment. CONCLUSION: CGP identified diverse clinically relevant GAs in PAYA patients with thyroid carcinoma, including 83% (34/41) of PTC cases harboring activating kinase mutations or activating kinase rearrangements. These genomic observations and index cases exhibiting clinical benefit from targeted therapy suggest that young patients with advanced thyroid carcinoma can benefit from CGP and rationally matched targeted therapy. The Oncologist 2017;22:255-263 IMPLICATIONS FOR PRACTICE: The detection of diverse clinically relevant genomic alterations in the majority of pediatric, adolescent, and young adult patients with thyroid carcinoma in this study suggests that comprehensive genomic profiling may be beneficial for young patients with papillary, anaplastic, or medullary thyroid carcinoma, particularly for advanced or refractory cases for which clinical trials involving molecularly targeted therapies may be appropriate.

Reverse transcription-polymerase chain reaction molecular testing of cytology specimens: Pre-analytic and analytic factors.

The introduction of molecular testing into cytopathology laboratory practice has expanded the types of samples considered feasible for identifying genetic alterations that play an essential role in cancer diagnosis and treatment. Reverse transcription-polymerase chain reaction (RT-PCR), a sensitive and specific technical approach for amplifying a defined segment of RNA after it has been reverse-transcribed into its DNA complement, is commonly used in clinical practice for the identification of recurrent or tumor-specific fusion gene events. Real-time RT-PCR (quantitative RT-PCR), a technical variation, also permits the quantitation of products generated during each cycle of the polymerase chain reaction process. This review addresses qualitative and quantitative pre-analytic and analytic considerations of RT-PCR as they relate to various cytologic specimens. An understanding of these aspects of genetic testing is central to attaining optimal results in the face of the challenges that cytology specimens may present. Cancer Cytopathol 2017;125:11-19. © 2016 American Cancer Society.

InFusion: Advancing Discovery of Fusion Genes and Chimeric Transcripts from Deep RNA-Sequencing Data.

Analysis of fusion transcripts has become increasingly important due to their link with cancer development. Since high-throughput sequencing approaches survey fusion events exhaustively, several computational methods for the detection of gene fusions from RNA-seq data have been developed. This kind of analysis, however, is complicated by native trans-splicing events, the splicing-induced complexity of the transcriptome and biases and artefacts introduced in experiments and data analysis. There are a number of tools available for the detection of fusions from RNA-seq data; however, certain differences in specificity and sensitivity between commonly used approaches have been found. The ability to detect gene fusions of different types, including isoform fusions and fusions involving non-coding regions, has not been thoroughly studied yet. Here, we propose a novel computational toolkit called InFusion for fusion gene detection from RNA-seq data. InFusion introduces several unique features, such as discovery of fusions involving intergenic regions, and detection of anti-sense transcription in chimeric RNAs based on strand-specificity. Our approach demonstrates superior detection accuracy on simulated data and several public RNA-seq datasets. This improved performance was also evident when evaluating data from RNA deep-sequencing of two well-established prostate cancer cell lines. InFusion identified 26 novel fusion events that were validated in vitro, including alternatively spliced gene fusion isoforms and chimeric transcripts that include intergenic regions. The toolkit is freely available to download from http:/bitbucket.org/kokonech/infusion.

Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers.

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.