1p Loss Research Articles

HGG-54. HISTOLOGICAL AND MOLECULAR CHARACTERIZATION OF HIGH-GRADE BRAIN TUMORS SECONDARY TO TOTAL BODY IRRADIATION FOR HEMATOLOGICAL MALIGNANCIES

INTRODUCTIONHematopoietic stem cell transplantation (HSCT) is increasingly used in the treatment of acute leukemias (AL), non-Hodgkin lymphomas and other primary malignancies. However, a growing number of neoplasms secondary to HSCT are reported. The combination of total body irradiation (TBI) and high dose chemotherapy (HD-CT) as conditioning regimen is considered a risk factor. We present four children who survived AL, treated with HSCT and conditioning regimens including TBI/HD-CT. These patients developed a high-grade-brain tumor. We analyzed histological and molecular characteristics of neoplasms.METHODSHistologically, tumors were assessed for: presence of mitosis, necrosis and vascular proliferation; expression of ki67; expression of neuronal and glial markers, p53 and therapeutic targets. We analyzed the DNA methylation profile (DMP) of all tumors using IlluminaEPICarrays and compared it to the brain tumor classifier which allowed to generate the CNVs.RESULTSMorphologically two cases were defined as anaplastic astrocytoma, two cases as glioblastoma. Based on the DMP, all cases were found to belong to the methylation class “glioblastoma, IDH wildtype, subclass midline”, hypermutants, with gain of chromosome 1q and loss of 1p. Two cases showed PDGFRA amplification. All patients were treated with Temozolomide combination therapy +/- Bevacizumab and radiation therapy. At progression three patients were treated with checkpoint inhibitors.CONCLUSIONSThe improvement of the precision medicine is fundamental in the therapeutic decision of brain tumors and even more in neoplasms secondary to antiblastic treatments. DMP and CNV have proven to be useful tools to complement the histological characterization of the reported cases.

TBIO-13. USE OF NEXT GENERATION SEQUENCING TO IDENTIFY MOVE DRIVERS OF CRYPTIC, CLINICALLY AGGRESSIVE BRAIN TUMORS

INTRODUCTIONNext generation sequencing (NGS) is an emerging technology which allows for in-depth analysis of pediatric brain tumors. NGS has particular use in the context of ambiguous or aggressive neoplasms, where it can be leveraged to discover novel drivers, inform pathologic classification, and direct targeted therapies.OBJECTIVEThe objective of this case series was to utilize NGS technology to illuminate the biology of aggressive brain tumors with ambiguous pathologic features and clinically aggressive behavior.METHODSFFPE tumor tissue and matched germline DNA were subjected to whole exome sequencing (WES). Data were analyzed according to the GATK pipeline.RESULTSThe first case is a 6-year-old male who presented with innumerable foci of leptomeningeal nodules throughout the neuroaxis. Original pathology was CNS embryonal tumor. WES identified loss of chromosome 1p and 16q with gain of 1q and amplification of MYC and OTX2 loci (cytogenetic aberrations characteristic of group 3 medulloblastoma) and a deleterious mutation in BCL7B, a known tumor suppressor gene. The second case is a 2-year-old female who presented with a parietal lobe mass diagnosed as high grade neuroepithelial tumor with C11orf95 translocation, but no RELA fusion. WES revealed loss of small region of chromosome 2p and mutations in IDH3G, TRAF2, and JMJD1C, suggesting novel targets for further study.CONCLUSIONSIn both cases, NGS studies were able to shed light on the underlying tumor biology and/or refine the pathologic diagnosis. These data underscore the utility of applying NGS technology to study the biology of pediatric brain tumors.

EPEN-28. NOVEL ONCOGENE AMPLIFICATION IN SPINAL EPENDYMOMA INVOLVING THE MYC LOCUS (8q24)

BACKGROUNDWe report a unique case of spinal ependymoma with classic histology and aggressive clinical behavior which harbored a focal MYC (8q24) amplification. CASE REPORT: A-12-year old male presented with a three months history of back pain and acute onset weakness with ataxia. A spine MRI revealed an avidly enhancing intradural, extramedullary mass occupying the dorsal spinal canal from C6 through T2. The tumor demonstrated mild diffusion restriction and was associated with severe cord compression and mild edema. He underwent gross total resection. Pathological diagnosis was classic grade II ependymoma. Eleven months later, he re-presented with acute onset lower extremity paresthesia and left-handed weakness. Spine MRI demonstrated tumor recurrence extending from C2 through T1-T2 with resultant severe cord compression, again demonstrating avid enhancement and restricted diffusion. He underwent subtotal resection of the mass and focal proton beam irradiation. MOLECULAR CHARACTERISTICS: The patient was enrolled on an institutional comprehensive genomic profiling protocol. The tumor’s copy number profile was complex, including homozygous loss of 17p and notably, amplification of the MYC oncogene. Using fluorescence in situ hybridization, we identified >20 copies of MYC in interphase cells, confirming the gene amplification, while two copies of MYCN (2p24) were seen. DNA methylation further classified this tumor as clustering near posterior fossa group A (score=0.6073) tumors.CONCLUSIONWe report a unique case of an adolescent male with aggressive spinal ependymoma harboring focal MYC amplification. Testing for MYC amplification may be reasonable in newly-diagnosed spinal ependymomas to aid in characterization.

DIPG-50. A NOVEL ORTHOTOPIC PATIENT-DERIVED XENOGRAFT MODEL OF RADIATION-INDUCED GLIOMA

Diffuse midline glioma (DMG) can arise as a primary tumour but also as a consequence of radiation therapy (RT) in survivors of other paediatric brain tumours. Radiation-associated gliomas are molecularly distinct from primary gliomas and have poorer overall survival. We report a case of radiation-associated DMG following treatment for medulloblastoma, and the development of a matched patient-derived xenograft (PDX) model. A four-year-old boy diagnosed with medulloblastoma was treated with surgical resection, RT and chemotherapy (COG:CCG-99701-Arm B). Eleven years post-diagnosis, the patient relapsed with radiation-associated DMG, participated in a Phase I clinical trial (COG:ACNS0927), and passed away eight months later. Tumour tissue collected at autopsy was intracranially implanted into immunodeficient mice and serially transplanted in vivo. Immunohistochemistry demonstrated both the primary DMG and PDXs expressed PDGFR-alpha and PTEN, were H3K27me3-positive, and had undetectable levels of p53. Sequencing revealed an activating mutation in PI3-kinase (H1047L) and variants of unknown significance in GRK4, FLG, BAZ2A, and CRTC3. DNA methylation array of the PDX demonstrated 1p loss, which is not typically associated with primary DMG, and broad deletion within 9p including CDKN2A/B, MTAP and multiple interferon genes. The methylation profile did not significantly classify with other tumours in the Molecular Neuropathology database (molecularneuropathology.org/mnp). We describe the first reported PDX model of radiation-associated DMG following medulloblastoma, which recapitulates the patient disease and is molecularly distinct from primary DMG. Interrogation of this model through RNA and whole genome sequencing presents a valuable opportunity to better understand and identify novel therapeutic vulnerabilities against this currently incurable disease.

Growth of pancreatic cancers with hemizygous chromosomal 17p loss of MYBBP1A can be preferentially targeted by PARP inhibitors.

Here, we selectively target pancreatic ductal adenocarcinoma (PDAC) cells harboring a hemizygous gene essential for cell growth. MYB binding protein 1A (MYBBP1A), encoding a chromatin-bound protein, is hemizygous in most of the PDAC due to a chromosome 17p deletion that also spans TP53 We find that hemizygous MYBBP1A loss in isogenic PDAC cells promotes tumorigenesis but, paradoxically, homozygous MYBBP1A loss is associated with impaired cell growth and decreased tumorigenesis. Poly-adenosine 5'-diphosphate-ribose polymerase 1 (PARP1) interacts with MYBBP1A and displaces it from chromatin. Small molecules, such as olaparib, that trap PARP1 to chromatin are able to evict the minimal pool of chromatin-bound MYBBP1A protein in MYBBP1A hemizygous cells and impair cell growth, greater than its impact on wild-type cells. Our findings reveal how a cell essential gene with one allele lost in cancer cells can be preferentially susceptible to a specific molecular therapy, when compared to wild-type cells.

Clinical-Pathological, Immunohistochemical, and Genetic Characterization of a Series of Posterior Pituitary Tumors.

Posterior pituitary tumors are supposed to represent the morphological spectrum of a single entity. Herein, we report the clinical-pathological, immunohistochemical, and genetic features of 5 spindle cell oncocytomas (SCOs), 3 pituicytomas, and 1 granular cell tumor (GCT). SCOs had the highest local invasiveness and affected older subjects. The 3 histotypes differed in the content of spindle cells (predominant in pituicytoma and absent in GCT), presence of lymphocytic infiltrate (in SCO and GCT, but not in the pituicytoma) and EMA/GFAP staining (negative in GCT; EMA-positive/GFAP-negative in 4/5 SCO and GFAP-positive in 3/3 pituicytomas). Three SCOs and 1 pituicytoma analyzed with next-generation sequencing had no mutations in 409 genes. However, 1 SCO had previously unreported homozygous deletion of CDKN2A/B and another of SMARCA4, SMARCB1, and NF2. All 3 SCOs had loss of heterozygosity of chromosome 1p, while the pituicytoma had chromosome 19 homozygous loss and chromosomes 10, 13q, and 18q loss of heterozygosity. Since 1p and 13q losses were previously reported in 1 pituicytoma and 1 SCO, respectively, our data demonstrate that posterior pituitary tumors share common genetic alterations. The possibility that posterior pituitary tumors are SMARCA4/SMARCB1-deficient should be kept in mind in the differential diagnosis toward other entities.

Accessing Genomic Alternations in Chronic Lymphocytic Leukemia Using an NGS-Based Comprehensive Genomic Profiling Assay

Background: Next generation sequencing (NGS) is an integral component in the characterization of hematologic malignancies, including chronic lymphocytic leukemia (CLL). Fluorescence in situ hybridization (FISH) and conventional cytogenetics (CC) are cost effective and are currently the gold standard for detecting copy number abnormalities (CNAs) in hematologic malignancies. NGS is emerging as a comprehensive assay that can detect CNAs while surveying the whole genome for single nucleotide variants and loss of heterozygosity (CN-LOH). Identifying CNA events in addition to mutations and RNA fusions may help identify and characterize the highly complex genetic landscape of hematologic malignancies. Methods: A custom total nucleic acid (TNA) NGS panel was designed which consists of mutation profiles of 297 genes, transcriptome profile of 213 genes, and genomic backbones of 14 chromosomes to identify unbalanced abnormalities. Two-hundred seventy CLL patients were included in the study (abnormalities detected in 236 cases in total: 61 cases by CC; 230 cases by FISH; and 53 cases by both CC and FISH, and no abnormalities detected in 34 cases by both FISH and CC). Mutation profiles including SNVs, indels, and structural changes were interrogated with a custom bioinformatic pipeline which utilized PureCN and CNVkit algorithms to identify structural changes. NGS results were compared to results of CC and FISH. CNA detection of sex chromosome and balanced rearrangement including translocation and inversion was excluded from the analysis Results: CNAs were detected by NGS in 56 of 61 cases (91%) reported by CC and in 178 of 230 cases (77%) detected by FISH. Seventy-seven CNAs detected by CC and 202 CNAs detected by FISH were identified by NGS. NGS failed to detect 13q deletion, detected by FISH in 48 cases. Abnormalities not detected by neither cytogenetics nor FISH were detected by NGS in 108 (gain) and 32 (loss) cases. In addition, we observed abnormalities in 9 of 34 cases by NGS reported as normal by both FISH and cytogenetics. CN-LOH was detected in 9% of cases predominantly on 13q, 17p and 22q. In addition to trisomy 12, gains of 20p and 20q were observed in each 72 (30%) and 43 (18%) cases. CN gains of 7p, 8q, and 17q were also observed in 12%, 12%, and 7% of cases, respectively. Oncogenic driver mutations in KRAS (p.G12D) and (p.G13D) were observed in four and five cases with CN gains, respectively. IKZF3, a recurrent hotspot pathogenic mutation in CLL and a potential prognostic marker that may positively regulate MYC, was detected in five patients with CN gains. CN loss of 11q, 2q, 13q, 3p, 17p, 21q, and 6q were among the most common chromosomes with CN loss (Figure 1). Notably, LOH of RB1, DLEU7, COG3, and FOX1 genes on 13q, of TP53, WRAP53, SLC52A1, CTC1, and ABR genes on 17p and of PRDM1, EPHA7, and CASP8AP2 genes on 6q were observed. Identifying cases with 13q14 deletions that include RB1 could change the CLL patient management due to the aggressive clinical course. Recurrent loss of function mutations in KMT2C (p.E2798Gfs*11), NOTCH1 (p.P2514Rfs*4), and TP53 (p.H179R) in 7q, 9q, and 17p were observed. Identifying both CN loss combined with loss of function mutations in tumor suppressors could help improve patient care. Conclusions: Abnormalities detected by cytogenetics were mostly detected by NGS, but NGS offers a higher resolution including CN events of various length, LOH events, and single gene mutations. CNAs detected at higher resolution is useful in identifying patients with 13q14 loss that include/exclude RB1 which may affect patient management. However, an accurate detection of the CNA could be affected in part by a baseline established by a panel of normal and the depth of coverage. Differences in sensitivity of methodologies can also be attributed to in vitro proliferation and tissue culture conditions utilized for CC analysis. CC and FISH can identify clones with multiple abnormalities as well as clonal evolution. Comprehensive genomic profile including high resolution copy number changes and mutational profiles, detectable by NGS, may provide better profiling for a patient for clinical management. Disclosures Jung: NeoGenomics: Current Employment. Thangavelu:NeoGenomics: Current Employment. Nam:NeoGenomics: Current Employment. Bender:NeoGenomics: Current Employment. Agersborg:NeoGenomics: Current Employment. Weiss:Bayer: Other: speaker; Genentech: Other: Speaker; Merck: Other: Speaker; NeoGenomics: Current Employment. Funari:NeoGenomics: Current Employment.

Deciphering the clonal relationship between glandular and squamous components in adenosquamous carcinoma of the lung using whole exome sequencing

Adenosquamous carcinoma of the lung (ASC) is a rare subtype of non-small cell lung cancer, consisting of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) components. ASC shows morphological characteristics of classic LUAD and LUSC but behaves more aggressively. Although ASC can serve as a model of lung cancer heterogeneity and transdifferentiation, its genomic background remains poorly understood. In this study, we sought to explore the genomic landscape of macrodissected LUAD and LUSC components of three ASC using whole exome sequencing (WES). Identified truncal mutations included the pan-cancer tumor-suppressor gene TP53 but also EGFR, BRAF, and MET, which are characteristic for LUAD but uncommon in LUSC. No truncal mutation of classical LUSC driver mutations were found. Both components showed unique driver mutations that did not overlap between the three ASC. Mutational signatures of truncal mutations differed from those of the branch mutations in their descendants LUAD and LUSC. Most common signatures were related to aging (1, 5) and smoking (4). Truncal chromosomal copy number aberrations shared by all three ASC included losses of 3p, 15q and 19p, and an amplified region in 5p. Furthermore, we detected loss of STK11 and SOX2 amplification in ASC, which has previously been shown to drive transdifferentiation from LUAD to LUSC in preclinical mouse models. Conclusively, this is the first study using WES to elucidate the clonal evolution of ASC. It provides strong evidence that the LUAD and LUSC components of ASC share a common origin and that the LUAD component appears to transform to LUSC.

Mucinous carcinoma with micropapillary features is morphologically, clinically and genetically distinct from pure mucinous carcinoma of breast

Micropapillary features are seen in pure mucinous carcinoma of breast (PMC), which is termed mucinous carcinoma with micropapillary features (MPMC). However, whether MPMC can be identified as a morphologically, clinically or genetically distinct entity from PMC remains controversial. In this study, a retrospective review of 161 cases of breast mucinous carcinoma was conducted to assess the clinicopathologic features, prognostic implications, and genomic alterations of MPMC and PMC. MPMCs were identified in 32% of mucinous carcinomas showing an excellent interobserver agreement (ICC = 0.922). MPMCs occurred at a younger age and exhibited higher nuclear grade, more frequent lymph nodal metastasis, lymphovascular invasion, and HER2 amplification compared with PMCs. Survival analyses revealed that MPMCs show decreased progression-free survival compared with PMCs in both unmatched and matched cohorts. A similar outcome of distant disease-free survival was observed only in the unmatched cohort. However, no statistical difference in recurrence score was observed between MPMC and PMC using a 21-gene assay. Notably, both MPMCs and PMCs displayed low mutation burden, common mutations affecting TTN, GATA3, SF3B1, TP53, recurrent 6q14.1-q27 losses, and 8p11.21-q24.3 gains. GATA3, TP53, and SF3B1 were recurrently mutated in MPMCs, while PIK3CA mutations were exclusively detected in PMCs. Moreover, MPMCs harbored 17q and 20q gains as well as 17p losses, while PMCs displayed gains at 6p. PI3K-Akt, mTOR, ErbB, and focal adhesion pathways were more frequently deregulated in MPMCs than in PMCs, which may responsible for the aggressive tumor behavior of MPMCs. Our findings suggest that MPMC is morphologically, clinically, and genetically distinct from PMC.

The p53-inducible CLDN7 regulates colorectal tumorigenesis and has prognostic significance

Most colorectal cancer (CRC) are characterized by allele loss of the genes located on the short arm of chromosome 17 (17p13.1), including the tumor suppressor p53 gene. Although important, p53 is not the only driver of chromosome 17p loss. In this study, we explored the biological and prognostic significance of genes around p53 on 17p13.1 in CRC. The Cancer Genome Atlas (TCGA) were used to identify differentially expressed genes located between 1000 kb upstream and downstream of p53 gene. The function of CLDN7 was evaluated by both in vitro and in vivo experiments. Quantitative real-time PCR, western blot, and promoter luciferase activity, immunohistochemistry were used to explore the molecular drivers responsible for the development and progression of CRC. The results showed that CLDN7, located between 1000 kb upstream and downstream of p53 gene, were remarkably differentially expressed in tumor and normal tissues. CLDN7 expression also positively associated with p53 level in different stages of the adenoma-carcinoma sequence. Both in vitro and in vivo assays showed that CLDN7 inhibited cell proliferation in p53 wild type CRC cells, but had no effects on p53 mutant CRC cells. Mechanistically, p53 could bind to CLDN7 promoter region and regulate its expression. Clinically, high CLDN7 expression was negatively correlated with tumor size, invasion depth, lymphatic metastasis and AJCC III/IV stage, but was positively associated with favorable prognosis of CRC patients. Collectively, our work uncovers the tumor suppressive function for CLDN7 in a p53-dependent manner, which may mediate colorectal tumorigenesis induced by p53 deletion or mutation.

Clinical, Genomic, and Pharmacological Study of MYCN-Amplified RB1 Wild-Type Metastatic Retinoblastoma.

Simple SummaryWe present two cases of a subtype of retinoblastoma, a rare ocular tumor in children, presenting without the typical mutation in the RB1 gene but showing amplification of the transcription factor MYCN frequently reported in pediatric malignancies. Even though previous reports suggested that this tumor sybtype could present metastases, patients with metastatic disease were not reported. Our cases had metastasis to the orbit and lymph nodes and poor sensitivity to standard chemotherapy but no dissemination to the central nervous system, as described for patients with mutations in the RB1. We were able to grow the cells of one of our patients in vitro, perform comprehensive genomic analysis that showed previously not reported mutations and other chromosomal alterations. In an animal model, we could reproduce the clinical dissemination and we identified an innovative active drug combination that could help for the treatment of these children with poor prognosis.An uncommon subgroup of unilateral retinoblastomas with highly aggressive histological features, lacking aberrations in RB1 gene with high-level amplification of MYCN (MCYNRB1) has only been described as intra-ocular cases treated with initial enucleation. Here, we present a comprehensive clinical, genomic, and pharmacological analysis of two cases of MCYNRB1 with orbital and cervical lymph node involvement, but no central nervous system spread, rapidly progressing to fatal disease due to chemoresistance. Both patients showed in common MYCN high amplification and chromosome 16q and 17p loss. A somatic mutation in TP53, in homozygosis by LOH, and high chromosomal instability leading to aneuploidy was identified in the primary ocular tumor and sites of dissemination of one patient. High-throughput pharmacological screening was performed in a primary cell line derived from the lymph node dissemination of one case. This cell line showed resistance to broad spectrum chemotherapy consistent with the patient’s poor response but sensitivity to the synergistic effects of panobinostat–bortezomib and carboplatin–panobinostat associations. From these cells we established a cell line derived xenograft model that closely recapitulated the tumor dissemination pattern of the patient and served to evaluate whether triple chemotherapy significantly prolonged survival of the animals. We report novel genomic alterations in two cases of metastatic MCYNRB1 that may be associated with chemotherapy resistance and in vitro/in vivo models that serve as basis for tailoring therapy in these cases.

Genetic and epigenetic profiling indicates the proximal tubule origin of renal cancers in end-stage renal disease.

End‐stage renal disease (ESRD) patients on dialysis therapy have a higher incidence of renal cell carcinomas (RCCs), which consist of 2 major histopathological types: clear‐cell RCCs (ESRD‐ccRCCs) and acquired cystic disease (ACD)‐associated RCCs. However, their genetic and epigenetic alterations are still poorly understood. Here, we investigated somatic mutations, copy number alterations (CNAs), and DNA methylation profiles in 9 ESRD‐ccRCCs and 7 ACD‐associated RCCs to identify their molecular alterations and cellular origins. Targeted sequencing of 409 cancer‐related genes, including VHL, PBRM1, SETD2, BAP1, KDM5C, MET, KMT2C (MLL3), and TP53, showed ESRD‐ccRCCs harbored frequent VHL mutations, while ACD‐associated RCCs did not. CNA analysis showed that ESRD‐ccRCCs had a frequent loss of chromosome 3p while ACD‐associated RCCs had a gain of chromosome 16. Beadarray methylation analysis showed that ESRD‐ccRCCs had methylation profiles similar to those of sporadic ccRCCs, while ACD‐associated RCCs had profiles similar to those of papillary RCCs. Expression analysis of genes whose expression levels are characteristic to individual segments of a nephron showed that ESRD‐ccRCCs and ACD‐associated RCCs had high expression of proximal tubule cell marker genes, while chromophobe RCCs had high expression of distal tubule cell/collecting duct cell marker genes. In conclusion, ESRD‐ccRCCs and ACD‐associated RCCs had mutation and methylation profiles similar to those of sporadic ccRCCs and papillary RCCs, respectively, and these 2 histopathological types of RCCs were indicated to have originated from proximal tubule cells of the nephron.

Age Dependency of the Prognostic Impact of Tumor Genomics in Localized Resectable MYCN-Nonamplified Neuroblastomas. Report From the SIOPEN Biology Group on the LNESG Trials and a COG Validation Group.

PURPOSEFor localized, resectable neuroblastoma without MYCN amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome.PATIENTS AND METHODSDiagnostic samples were obtained from 317 tumors, International Neuroblastoma Staging System stages 1/2A/2B, from 3 cohorts: Localized Neuroblastoma European Study Group I/II and Children’s Oncology Group. Genomic data were analyzed using multi- and pangenomic techniques and fluorescence in-situ hybridization in 2 age groups (cutoff age, 18 months) and were quality controlled by the International Society of Pediatric Oncology European Neuroblastoma (SIOPEN) Biology Group.RESULTSPatients with stage 1 tumors had an excellent outcome (5-year event-free survival [EFS] ± standard deviation [SD], 95% ± 2%; 5-year overall survival [OS], 99% ± 1%). In contrast, patients with stage 2 tumors had a reduced EFS in both age groups (5-year EFS ± SD, 84% ± 3% in patients < 18 months of age and 75% ± 7% in patients ≥ 18 months of age). However, OS was significantly decreased only in the latter group (5-year OS ± SD in < 18months and ≥ 18months, 96% ± 2% and 81% ± 7%, respectively; P = .001). In < 18months, relapses occurred independent of segmental chromosome aberrations (SCAs); only 1p loss decreased EFS (5-year EFS ± SD in patients 1p loss and no 1p loss, 62% ± 13% and 87% ± 3%, respectively; P = .019) but not OS (5-year OS ± SD, 92% ± 8% and 97% ± 2%, respectively). In patients ≥ 18 months, only SCAs led to relapse and death, with 11q loss as the strongest marker (11q loss and no 11q loss: 5-year EFS ± SD, 48% ± 16% and 85% ± 7%, P = .033; 5-year OS ± SD, 46% ± 22% and 92% ± 6%, P = .038).CONCLUSIONGenomic aberrations of resectable non–MYCN-amplified stage 2 neuroblastomas have a distinct age-dependent prognostic impact. Chromosome 1p loss is a risk factor for relapse but not for diminished OS in patients < 18 months, SCAs (especially 11q loss) are risk factors for reduced EFS and OS in those > 18months. In older patients with SCA, a randomized trial of postoperative chemotherapy compared with observation alone may be indicated.

Abstract NG02: Genomic evolution of cancer models: Perils and opportunities

Abstract NG02: Genomic evolution of cancer models: Perils and opportunities

Abstract 820: Genomics based personalized oncology of cancer of unknown primary

Abstract Cancers of unknown primary site (CUPs) represent a heterogeneous group of metastatic tumors, which accounts for 3-5% of malignancies. Due to the poor prognosis and limited local and systemic treatment options, there is an urgent need for improvement of molecularly driven treatment strategies. We investigated the molecular profile and clinical course of 70 patients enrolled in a prospective precision oncology registry trial conducted by the National Center for Tumor Diseases (NCT) Heidelberg/Dresden and the German Cancer Consortium (DKTK) that addresses younger adults with advanced-stage cancer across histologies as well as patients with rare tumors (NCT/DKTK MASTER). Molecular analyses included whole genome sequencing (WGS, n=29), whole exome sequencing (WES, n=41) and transcriptome analysis (n=55). All patients were diagnosed with CUP-syndrome, 61/70 (87.1%) of diagnoses fulfilled the ESMO Clinical Practice Guidelines. Progression free survival (PFS) of the first treatment based on MASTER (PFS2) was compared to the PFS of the last prior systemic treatment (PFS1) in each individual patient. Within the coding sequence, we identified 0 to 1386 nonsynonymous point mutations (SNVs, median=41) and 0 to 38 insertions/deletions (indels, median=3) per sample. Hypermutation (≥100 SNVs and indels) was observed in 14 samples. Mutations of TP53 and KRAS were significantly enriched. Analysis of copy-number changes (CNVs) was performed in 51 samples (27 WGS and 24 WES) and revealed complex CNV profiles in most cases. Gains and losses involved single arms or whole chromosomes. Gains in chromosome 8q, 1q and 7 and losses in chromosome 6q and 17p occurred in more than 40% of the patients. Fusions of EML4-ALK and FGFR2 were found in three and six cases, respectively. In one case, pathological reevaluation for NUT midline carcinoma was recommended based on a NUTM1-MXI1 fusion. In total, pathological reevaluation based on characteristic genetic events was recommended in five cases. Germline analysis of 70 cases revealed five pathogenic variants (ACMG Class 5) in CHEK2, BRCA1, CDKN2A, NBN and ERCC3. In addition, one likely pathogenic variant (ACMG Class 4) was found in FH. The molecular tumor board recommended targeted therapy in 56/70 (80.0%) patients which could be applied in 20/56 (35.7%) cases. The molecularly driven treatment approaches translated into a median PFS2/1 ratio of 2.25 (n=17). Median PFS1 was 89 days (range 31-304, n=17) compared to a median PFS2 of 180 days (range 50-805, n=17). For three patients in which PFS1 could not be determined median PFS2 was 305 days (range 182-336). We demonstrate that a comprehensive molecular analysis of CUPs provides clinically relevant information and additional, molecularly stratified treatment approaches in many cases. These targeted therapies can be highly beneficial even in heavily pretreated patients. Citation Format: Maximilian Werner, Lino Möhrmann, Małgorzata Oleś, Andreas Mock, Arne Jahn, Simon Kreutzfeldt, Sebastian Uhrig, Martina Fröhlich, Barbara Hutter, Daniela Richter, Gina Rüter, Ivan Jelas, Rainer Hamacher, Johanna Falkenhorst, Sebastian Wagner, Christian Brandts, Melanie Börries, Anna Illert, Klaus Metzeler, Benedikt Westphalen, Alexander Desuki, Thomas Kindler, Albrecht Stenzinger, Evelin Schröck, Benedikt Brors, Peter Horak, Christoph Heining, Stefan Fröhling, Hanno Glimm. Genomics based personalized oncology of cancer of unknown primary [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 820.

Abstract 6606: Develop dual-targeted CAR-T cells to achieve RCC cures

Abstract Clear cell renal cell carcinoma (ccRCC) is the major type of RCC and is among the 10 most common cancers in both men and women. ccRCC is characterized by the loss of chromosome 3p, where the von-Hippel-Lindau (VHL) gene is found. Its gene product, pVHL suppresses the hypoxia inducible factor (HIF) transcription factors and the deletion of VHL leads to HIF hyperactivation, upregulating the expression of many downstream genes involved in angiogenesis, metabolism, and cell-cycle regulation. Two gene products, carbonic anhydrase IX (CAIX) and CD70 which we are pursuing as therapeutic targets are downstream genes of the HIF-1α pathway and thus commonly overexpressed in ccRCC. Despite the development of checkpoint blockade inhibitors (CBIs) for the treatment of this disease, curative therapies are rare. Chimeric Antigen Receptor (CAR)-T cells are a new type of “living drug”, in which T cells are engineered to express a single chain variable antibody fragment (scFv) linked to an intracellular signaling motif that includes CD3ζ (z) activation domain (first generation), with CD28 or 41BB (second generation), or both (third generation) costimulatory domains. CAR-T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR-T cells, the suppressive tumor microenvironment (TME), and on-target off-tumor toxicities resulting from the sharing of CAR-T target epitopes on healthy tissues. To translate CAR-T cell therapy to ccRCC, we developed dual-targeted CAR-T cells against both CAIX and CD70, in which dual CAR can target double positive (CAIX+ CD70+) and single positive (CAIX+ CD70-, CAIX- CD70+) tumor cell populations to address this heterogeneity. By IHC staining, we demonstrated that these two target antigens are overexpressed in ccRCC and represent circa 90% of the tumor cell population regardless of disease stages. Here we build on our previous findings and demonstrate that 41BB CAR and CD4/CD8 CAR-T cell mixtures exhibit the superior efficacy and persistence in our ccRCC orthotopic NSG-SGM3 mouse model compared to other CAR constructs and CD8 alone. A panel of dual-targeted CARs have been encoded in our 41BB CAR construct and assessed in vitro and in vivo by using Celigo imaging cytometry assay and humanized ccRCC orthotopic NSG-SGM3 mouse model respectively. Dual-targeted anti-CAIX/CD70 CD4/8 CAR-T cells outperformed singly-targeted CAR-T cells with elevated efficacy. In summary, anti-CAIX/CD70 CAR-T cells hold a great promise to achieve ccRCC cures. Citation Format: Yufei Wang, Marion Grimaud, Alicia Buck, Atef Fayed, Matthew Chang, Rebecca Jennings, Maura Sticco-Ivins, Miriam Ficial, Leo L. Chan, Sabina Signoretti, Quan Zhu, Wayne A. Marasco. Develop dual-targeted CAR-T cells to achieve RCC cures [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6606.

Abstract 1493: The clonal relationship between adenomatous and squamous components in adenosquamous carcinoma of the lung

Abstract Background: Adenosquamous carcinoma (ASC) is a rare dichotomous malignancy of non-small-cell lung cancer (NSCLC) consisting of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) components. Although ASC has morphologically distinct characteristics of LUAD and LUSC, it behaves more aggressively than either subtype alone and is associated with worse prognosis. To date, not much is known about the molecular makeup of this tumor. In this study, we performed whole exome sequencing to investigate the evolutionary and clonal relationship between the two components of ASC. Methods: FFPE tissues of three ASCs were examined. After staining with the lineage markers TTF-1 and p40 to visualize LUAD and LUSC, respectively, the two components were macrodissected and subjected to whole exome sequencing separately. Results: The six separately analyzed tumor areas harbored a median of 73 (range 54 - 255) non-synonymous somatic mutations and a median mutation burden of 3.5 mutations/Mb (range 2.3 - 11.6). The LUSC and LUAD components of the three ASCs shared 46 - 299 mutations, accounting for 45%-87% of the mutations. Clonality analysis shows that most of driver mutations were clonal, further supporting the monoclonal origin of the two components. We detected 19 shared cancer gene mutations including TP53 p.Gln192* and p.His179Arg. An additional TP53 mutation p.Gly108Asp was found in LUAD of one patient. We further identified truncal mutations in EGFR, BRAF and MET, which are characteristic for LUAD but uncommon in LUSC. Furthermore,AMER1 and KMT2D, present in 9% and 12% of LUAD patients but not yet described in LUSC, were truncally mutated in two patients. No truncal mutations were found in typical LUSC driver genes such as FAT1 or PTEN. Shared chromosomal copy number aberrations between the two components were detected in all patients and included losses of 3p, 15q and 19p, and an amplified region in 5p, encompassing TERT. Focal deletions that occurred in all samples, such as in chromosome 3p are common in both classical LUAD and LUSC, whereas deletion in 15q and 19p occur in &amp;gt;90% of LUAD but in only 20% of LUSC. Identification of genomic SOX2amplification and STK11 loss in two of the three LUSC components, each, support previous pre-clinical evidence from mouse models suggesting a role of these genes in transdifferentiation from LUSC to ASC. Phylogenetic and mutational signature analyses showed that the trunks of all three ASCs were dominated by ageing and smoking-related signatures 1 and 4. However, the contributions of signatures changed markedly in the LUAD and LUSC branches. Conclusion: Our study investigated two macrodissected components, LUAD and LUSC, from three patients with ASC. Shared mutations and copy number aberrations suggested a LUAD-like genetic profile and demonstrated a clear monoclonal relationship between the two components indicative of a common ancestor. However, there were also major differences between the distinct components representing diverse evolutionary trajectories. Citation Format: Luca Roma, Arthur Krause, Salvatore Piscuoglio, Charlotte K. Ng, Lukas Bubendorf. The clonal relationship between adenomatous and squamous components in adenosquamous carcinoma of the lung [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1493.

Sporadic oncocytic tumors with features intermediate between oncocytoma and chromophobe renal cell carcinoma: comprehensive clinicopathological and genomic profiling

Morphology, clinical behavior, and genomic profiles of renal oncocytoma (RO) and its malignant counterpart chromophobe renal cell carcinoma (ChRCC) are distinctly different. However, there is a substantial group of sporadic oncocytic tumors with peculiar hybrid phenotypes as well as a perplexing degree of morphologic and immunohistochemical overlap between classic RO and ChRCC with eosinophilic cytoplasm. The aim of this study is to provide detailed characterization of these hybrid tumors.Thirty-eight sporadic oncocytic neoplasms with ambiguous morphology from two institutions were reviewed by 4 pathologists. CKIT positivity was used as a selection criterion. We correlated CK7 and S100A1 immunostaining and detailed morphologic features with cytogenetic profiles. DNA from the formalin-fixed paraffin-embedded tissues was extracted and analyzed using cytogenomic microarray analysis (CMA) to evaluate copy number alterations (CNA) and ploidy. CMA categorized cases into 3 groups: RO (N=21), RO variant (N=7), and ChRCC (N=10). Cytogenetic RO had either no CNA (48%) or loss of chromosome 1p, X, or Y (52%). RO variant had additional chromosomal losses [-9q, -14 (n=2), -13] and chromosomal gains [+1q (n=2), +4, +7 (n=2), +13, +19, +20, and +22]. ChRCCs were either hypodiploid with numerous monosomies (40%) or hypotetraploid with multiple relative losses (60%). RO, RO variant, and ChRCC groups differed significantly in tumor architecture (p<0.01), stroma (p=0.013), presence of nuclear wrinkling, perinuclear halos, and well-defined cell borders in >5% of cells (p<0.01), focal cell clearing (p=0.048) and CK7 expression (p<0.02). Pathologic prediction of the cytogenetic subtype using only two categories (benign RO or malignant ChRCC) would overcall or undercall up to 40% of tumors that were ChRCC based on cytogenetics. This finding provides the rationale for an intermediate diagnostic category of the so-called hybrid tumors (hybrid oncocytic/chromophobe tumor [HOCT]). HOCT was a heterogeneous group enriched for cytogenetic RO variant. Other HOCTs have a profile of either RO or ChRCC. The genomic profile allows classification of oncocytic tumors with ambiguous morphology into RO, RO variant, and ChRCC. Several architectural and cytologic features combined with CK7 expression are significantly associated with cytogenetic RO, RO variant, or ChRCC tumors. Doubled hypodiploidy by whole-genome endoduplication is a common phenomenon in eosinophilic ChRCC.

Monosomal karyotype and chromosome 17p loss or TP53 mutations in decitabine-treated patients with acute myeloid leukemia

TP53 aberrations reportedly predict favorable responses to decitabine (DAC) in acute myeloid leukemia (AML). We evaluated clinical features and outcomes associated with chromosome 17p loss or TP53 gene mutations in older, unfit DAC-treated AML patients in a phase II trial. Of 178 patients, 25 had loss of 17p in metaphase cytogenetics; 24 of these had a complex (CK+) and 21 a monosomal karyotype (MK+). In analyses in all patients and restricted to CK+ and MK+ patients, 17p loss tended to associate with higher rates of complete remission (CR), partial remission (PR), or antileukemic effect (ALE). Despite favorable response rates, there was no significant OS difference between patients with or without loss of 17p in the entire cohort or in the CK+ and MK+ cohort. TP53 mutations were identified in eight of 45 patients with material available. Five of the eight TP53-mutated patients had 17p loss. TP53-mutated patients had similar rates of CR/PR/ALE but shorter OS than those with TP53 wild type (P = 0.036). Moreover, patients with a subclone based on mutation data had shorter OS than those without (P = 0.05); only one patient with TP53-mutated AML had a subclone. In conclusion, 17p loss conferred a favorable impact on response rates, even among CK+ and MK+ patients that however could not be maintained. The effect of TP53 mutations appeared to be different; however, patient numbers were low. Future research needs to further dissect the impact of the various TP53 aberrations in HMA-based combination therapies. The limited duration of favorable responses to HMA treatment in adverse-risk genetics AML should prompt physicians to advance allografting for eligible patients in a timely fashion.

Association of heterogeneous MYCN amplification with clinical features, biological characteristics and outcomes in neuroblastoma: A report from the Children's Oncology Group

PurposeMYCN amplification (MNA) is associated with poor outcomes in neuroblastoma. Less is known about heterogeneous MNA within a tumour. We compared clinical characteristics, biologic features and clinical outcomes of patients with heterogeneous MNA to patients with either homogeneous MNA or MYCN wild-type tumours. Patients and methodsIn this retrospective cohort study, we categorized patients as having tumours with MYCN wild-type, homogeneous MNA (>20% amplified tumour cells) or heterogeneous MNA (≤20% amplified tumour cells). We used chi-squared or Fisher's exact tests to compare features between groups. We used log-rank tests and Cox models to compare event-free survival (EFS) and overall survival (OS) between groups. ResultsMYCN status and heterogeneity status (if amplified) could be ascertained in diagnostic tumour samples from 5975 patients, including 57 (1%) with heterogeneous MNA, 981 (16.4%) with homogeneous MNA, and 4937 (82.6%) with MYCN wild-type tumours. Multiple clinical and biological features differed between patients with heterogeneous vs. homogeneous MNA, including enrichment for thoracic primary sites and paucity of 1p loss of heterozygosity with heterogeneous MNA (p < 0.0001). Importantly, EFS and OS were not significantly different between patients with heterogeneous vs. homogeneous MNA. Further, EFS and OS for patients with heterogeneous MNA were significantly inferior to patients with wild-type MYCN. ConclusionAlthough neuroblastomas with heterogeneous MNA demonstrate significantly different biological and clinical patterns compared with homogeneous MNA, prognosis is similar between the two groups. These results support current practice that treats patients with heterogeneous MNA similarly to patients with homogeneous MNA.