Chromosomal Copy Number Aberrations Research Articles

NTRK-fused central nervous system tumours: clinicopathological and genetic insights and response to TRK inhibitors

Background Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are found in 1% of gliomas across children and adults. TRK inhibitors are promising therapeutic agents for NTRK-fused gliomas because they are tissue agnostic and cross the blood–brain barrier (BBB). Methods We investigated twelve NGS-verified NTRK-fused gliomas from a single institute, Seoul National University Hospital. Results The patient cohort included six children (aged 1–15 years) and six adults (aged 27–72 years). NTRK2 fusions were found in ten cerebral diffuse low-grade and high-grade gliomas (DLGGs and DHGGs, respectively), and NTRK1 fusions were found in one cerebral desmoplastic infantile ganglioglioma and one spinal DHGG. In this series, the fusion partners of NTRK2 were HOOK3, KIF5A, GKAP1, LHFPL3, SLMAP, ZBTB43, SPECC1L, FKBP15, KANK1, and BCR, while the NTRK1 fusion partners were TPR and TPM3. DLGGs tended to harbour only an NTRK fusion, while DHGGs exhibited further genetic alterations, such as TERT promoter/TP53/PTEN mutation, CDKN2A/2B homozygous deletion, PDGFRA/KIT/MDM4/AKT3 amplification, or multiple chromosomal copy number aberrations. Four patients received adjuvant TRK inhibitor therapy (larotrectinib, repotrectinib, or entrectinib), among which three also received chemotherapy (n = 2) or proton therapy (n = 1). The treatment outcomes for patients receiving TRK inhibitors varied: one child who received larotrectinib for residual DLGG maintained stable disease. In contrast, another child with DHGG in the spinal cord experienced multiple instances of tumour recurrence. Despite treatment with larotrectinib, ultimately, the child died as a result of tumour progression. An adult patient with glioblastoma (GBM) treated with entrectinib also experienced tumour progression and eventually died. However, there was a successful outcome for a paediatric patient with DHGG who, after a second gross total tumour removal followed by repotrectinib treatment, showed no evidence of disease. This patient had previously experienced relapse after the initial surgery and underwent autologous peripheral blood stem cell therapy with carboplatin/thiotepa and proton therapy. Conclusions Our study clarifies the distinct differences in the pathology and TRK inhibitor response between LGG and HGG with NTRK fusions.

EPEN-28. NOVEL TARGET IDENTIFICATION IN RECURRENT POSTERIOR FOSSA EPENDYMOMA GROUP A

Abstract BACKGROUND Posterior fossa ependymoma group A (PF-EPN-A) are an aggressive subgroup of ependymal tumors which occur predominantly in infants and possess a poor prognosis. Treatment is limited to surgical resection and adjuvant radiation with no approved chemotherapies or targeted therapies available. Despite treatment efforts, nearly half of all PF-EPN-A cases will experience tumor recurrence within a decade of initial diagnosis. Our current understanding of relapse is limited to clinical risk stratification based on subtotal resection of the primary tumor, male sex and specific chromosomal copy number aberrations. In an effort to overcome the shortcomings of current literature on PF-EPN-A recurrence, we present a concerted effort to investigate the biology of both recurrent human tissue specimens and patient-derived cell models to identify novel drivers of recurrence. METHODS Utilizing a multi-omics approach, we have conducted transcriptomic (bulk RNA-Seq), proteomic and phosphoproteomic (LC-MS/MS) analyses in order to identify molecular pathways that are enriched in recurrent disease as compared to primaries. We have also performed genome-wide CRISPR essentiality screens to further pinpoint essential genes and pathways with therapeutic potential. RESULTS Through the comparison of primary and recurrent PF-EPN-A samples, we have identified both previously implicated and novel genes and pathways in recurrent disease. Our multi-omic analyses identified the upregulation of genes involved in DNA repair, DNA damage response and cell cycle pathways in recurrent PF-EPN-A. CONCLUSIONS Our combined approach has enabled the identification of novel targets with therapeutic potential in the treatment of recurrent PF-EPN-A. The identified targets will be validated using a combination of drug screening and knockdown approaches in vitro. Top candidates will be further validated in vivo with established recurrent xenograft mouse models. PF-EPN-A is an aggressive disease with limited treatment options and the identification of novel targets with potential therapeutic utility will allow us to better treat this patient population.

Genetic alterations of TP53 and OTX2 indicate increased risk of relapse in WNT medulloblastomas

This study aimed to re-evaluate the prognostic impact of TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers in WNT-activated medulloblastoma (WNT-MB). In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC, and TP53 were analyzed by DNA sequencing. Chromosomal copy-number aberrations were assessed by molecular inversion probe technology (MIP), SNP6, or 850k methylation array hybridization. Prognostic impact was evaluated in 120 patients with follow-up data from the HIT2000 medulloblastoma trial or HIT registries. CTNNB1 mutations were present in 92.2%, and APC mutations in 6.8% of samples. One CTNNB1 wild-type tumor gained WNT activation due to homozygous FBXW7 deletion. Monosomy 6 was present in 78.6%, and more frequent in children than adults. 16.1% of tumor samples showed TP53 mutations, of those 60% with nuclear positivity for the p53 protein. Loss of heterozygosity at the TP53 locus (chromosome 17p13.1) was found in 40.7% (11/27) of TP53 mutant tumor samples and in 12.6% of TP53 wild-type cases (13/103). Patients with tumors harboring TP53 mutations showed significant worse progression-free survival (PFS; 5-year-PFS 68% versus 93%, p = 0.001), and were enriched for chromosomes 17p (p = 0.001), 10, and 13 losses. Gains of OTX2 (14q22.3) occurred in 38.9% of samples and were associated with poor PFS and OS (5-year-PFS 72% versus 93%, p = 0.017 resp. 5-year-OS 83% versus 97%, p = 0.006). Multivariable Cox regression analysis for PFS/OS identified both genetic alterations as independent prognostic markers. Our data suggest that patients with WNT-MB carrying TP53 mutations or OTX2 gains (58.1%) are at higher risk of relapse. Eligibility of these patients for therapy de-escalation trials needs to be debated.

JS04.4.A Beyond β-catenin: Genetic alterations ofTP53 andOTX2 and older age indicate increased risk of relapse in WNT medulloblastomas

Abstract Background This genetic analysis of WNT-activated medulloblastomas (WNT-MBs) aimed to re-evaluate the prognostic impact of age, TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers. Material and Methods In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC and TP53 were analyzed by Sanger and/or NGS panel sequencing. Chromosomal copy number aberrations were assessed by high-resolution, genome-wide molecular inversion probe technology (MIP), SNP6 array, and/or 850k methylation bead-array hybridization. Association with prognosis was evaluated in 133 patients with follow-up data from the HIT2000 medulloblastoma trial, HIT registries, and the NOA-07 trial. Results CTNNB1 mutations were present in 92.2% of the samples. APC mutations were found in 6.8% (13 samples). One CTNNB1 wildtype tumor gained WNT-activation due to a homozygous deletion of FBXW7. Monosomy 6 was present in 78.6%, and more frequent in children compared to adolescents/adults (≥16 years). Adolescents/adults showed worse overall survival (OS; p=0.009) compared to children, but not worse progression-free survival (PFS; p=0.106). With an age cut-off at 18 years, no survival difference was found. Also adolescents alone (16-20 years) had worse OS (p=0.003) compared to children, whereas in patients ≥21 (n=12 adults with PFS/OS data) no tumor progression/relapse occurred. Only one adult died due to therapy-related complications. WNT-MB patients with tumors harboring TP53 mutations (24/133, 18.1%) showed significant worse PFS (p=0.001), which was also found in children and adolescents individually (p=0.004, resp. p=0.017). Gains of the OTX2 locus on chromosome 14q found in 40.2% (35/87) of samples were independent of TP53 mutations and also associated with poor PFS and OS (p=0.034, resp. p=0.016). Individual analyses of OTX2 gains within age groups showed only worse OS in children (p=0.012). Multivariate Cox regression analysis for PFS identified both genetic alterations, but not age, as independent prognostic markers. For OS, multivariate analysis found OTX2 gains and older age as independent prognostic markers. Conclusion Our data suggest that adolescent patients with WNT-MB and those patients carrying TP53 mutations or OTX2 gains - independent of age - are at higher risk of relapse. Eligibility of these patients for therapy de-escalation trials needs to be debated.

Genomic and microenvironmental landscape of stage I follicular lymphoma, compared with stage III/IV

AbstractAlthough the genomic and immune microenvironmental landscape of follicular lymphoma (FL) has been extensively investigated, little is known about the potential biological differences between stage I and stage III/IV disease. Using next-generation sequencing and immunohistochemistry, 82 FL nodal stage I cases were analyzed and compared with 139 FL stage III/IV nodal cases. Many similarities in mutations, chromosomal copy number aberrations, and microenvironmental cell populations were detected. However, there were also significant differences in microenvironmental and genomic features. CD8+ T cells (P = .02) and STAT6 mutations (false discovery rate [FDR] <0.001) were more frequent in stage I FL. In contrast, programmed cell death protein 1–positive T cells, CD68+/CD163+ macrophages (P < .001), BCL2 translocation (BCL2trl+) (P < .0001), and KMT2D (FDR = 0.003) and CREBBP (FDR = 0.04) mutations were found more frequently in stage III/IV FL. Using clustering, we identified 3 clusters within stage I, and 2 clusters within stage III/IV. The BLC2trl+ stage I cluster was comparable to the BCL2trl+ cluster in stage III/IV. The two BCL2trl– stage I clusters were unique for stage I. One was enriched for CREBBP (95%) and STAT6 (64%) mutations, without BLC6 translocation (BCL6trl), whereas the BCL2trl– stage III/IV cluster contained BCL6trl (64%) with fewer CREBBP (45%) and STAT6 (9%) mutations. The other BCL2trl– stage I cluster was relatively heterogeneous with more copy number aberrations and linker histone mutations. This exploratory study shows that stage I FL is genetically heterogeneous with different underlying oncogenic pathways. Stage I FL BCL2trl– is likely STAT6 driven, whereas BCL2trl– stage III/IV appears to be more BCL6trl driven.

MEDB-61. Genetic alterations ofTP53 andOTX2 indicate increased risk of relapse in WNT medulloblastomas

PURPOSE: This genetic analysis of WNT-activated medulloblastomas (WNT-MBs) aimed to re-evaluate the prognostic impact of TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers in a retrospective cohort of patients treated according to the protocols of the HIT medulloblastoma studies. PATIENTS AND METHODS: In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC, and TP53 were analyzed by Sanger and/or NGS panel sequencing. Chromosomal copy number aberrations (CNAs) were assessed by high-resolution, genome-wide molecular inversion probe technology (MIP), SNP6 array, and/or 850k methylation bead-array hybridization. Complete clinical data were available from 120 patients. RESULTS: Patients with WNT-MBs had a female predominance (1.4:1) and a median age of 13 years (range 3-69 years). CTNNB1 mutations were present in 92.2% of the samples, APC mutations in 6.8%. One CTNNB1 wildtype tumor gained WNT-activation due to a homozygous deletion of FBXW7. Monosomy 6 was present in 78.6%, but more frequent in children compared to adults. 16.1% of the tumor samples showed TP53 mutations, of those 60% with nuclear positivity for the p53 protein. A loss of heterozygosity at the TP53 locus on chromosome 17p13.1 was found in 40.7% (11/27) of TP53 mutant tumor samples and in 18.5% of the whole cohort (24/130 cases). Patients with tumors harboring TP53 mutations showed significant worse progression-free survival (PFS; p=0.001), but not overall survival (OS) and were enriched for chromosomes 17p (p=0.001), 10, and 13 losses. Gains of the OTX2 locus on chromosome 14q were also associated with poor PFS and OS (p=0.017 resp. p=0.006). Multivariate Cox regression analysis identified both genetic alterations as independent prognostic markers for PFS and OS. CONCLUSION: For ongoing and future de-escalation trials for patients with WNT medulloblastomas, we recommend to exclude patients with tumors carrying TP53 mutations or OTX2 gains.

Immune evolution from preneoplasia to invasive lung adenocarcinomas and underlying molecular features

The mechanism by which anti-cancer immunity shapes early carcinogenesis of lung adenocarcinoma (ADC) is unknown. In this study, we characterize the immune contexture of invasive lung ADC and its precursors by transcriptomic immune profiling, T cell receptor (TCR) sequencing and multiplex immunofluorescence (mIF). Our results demonstrate that anti-tumor immunity evolved as a continuum from lung preneoplasia, to preinvasive ADC, minimally-invasive ADC and frankly invasive lung ADC with a gradually less effective and more intensively regulated immune response including down-regulation of immune-activation pathways, up-regulation of immunosuppressive pathways, lower infiltration of cytotoxic T cells (CTLs) and anti-tumor helper T cells (Th), higher infiltration of regulatory T cells (Tregs), decreased T cell clonality, and lower frequencies of top T cell clones in later-stages. Driver mutations, chromosomal copy number aberrations (CNAs) and aberrant DNA methylation may collectively impinge host immune responses and facilitate immune evasion, promoting the outgrowth of fit subclones in preneoplasia into dominant clones in invasive ADC.

Molecular Basis and Clinical Features of Neuroblastoma.

Neuroblastoma, a neoplasm of the sympathetic nervous system, originates from neuroblastoma stem cells during embryogenesis. It exhibits unique clinical features including a tendency for spontaneous regression of tumors in infants and a high frequency of metastatic disease at diagnosis in patients aged over 18 months. Genetic risk factors and epigenetic dysregulation also play a significant role in the development of neuroblastoma. Over the past decade, our understanding of this disease has advanced considerably. This has included the identification of chromosomal copy number aberrations specific to neuroblastoma development, risk groups, and disease stage. However, high-risk neuroblastoma remains a therapeutic challenge for pediatric oncologists. New therapeutic approaches have been developed, either as alternatives to conventional chemotherapy or in combination, to overcome the dismal prognosis. Particularly promising strategies are targeted therapies that directly affect cancer cells or cancer stem cells while exhibiting minimal effect on healthy cells. This review summarizes our understanding of neuroblastoma biology and prognostic features and focuses on novel therapeutic strategies for this intractable disease.

Chromosome 20 loss is characteristic of breast implant–associated anaplastic large cell lymphoma

AbstractBreast implant–associated anaplastic large cell lymphoma (BIA-ALCL) is a very rare type of T-cell lymphoma that is uniquely caused by a single environmental stimulus. Here, we present a comprehensive genetic analysis of a relatively large series of BIA-ALCL (n = 29), for which genome-wide chromosomal copy number aberrations (CNAs) and mutational profiles for a subset (n = 7) were determined. For comparison, CNAs for anaplastic lymphoma kinase (ALK)− nodal anaplastic large cell lymphomas (ALCLs; n = 24) were obtained. CNAs were detected in 94% of BIA-ALCLs, with losses at chromosome 20q13.13 in 66% of the samples. Loss of 20q13.13 is characteristic of BIA-ALCL compared with other classes of ALCL, such as primary cutaneous ALCL and systemic type ALK+ and ALK− ALCL. Mutational patterns confirm that the interleukin-6–JAK1–STAT3 pathway is deregulated. Although this is commonly observed across various types of T-cell lymphomas, the extent of deregulation is significantly higher in BIA-ALCL, as indicated by phosphorylated STAT3 immunohistochemistry. The characteristic loss of chromosome 20 in BIA-ALCL provides further justification to recognize BIA-ALCL as a separate disease entity. Moreover, CNA analysis may serve as a parameter for future diagnostic assays for women with breast implants to distinguish seroma caused by BIA-ALCL from other causes of seroma accumulation, such as infection or trauma.

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.

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.

Percentage genome change and chromosome 7q amplification predict sorafenib response in advanced hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) may arise from genomic instability and has dismal outcome. Sorafenib is the first-line treatment for advanced stage HCC, but its therapeutic efficacy is less than 50%. Biomarkers for predicting the therapeutic efficacy of sorafenib administration to patients with advanced HCC are required. Here, we evaluated the role of chromosomal copy number aberrations (CNAs) in patients with advanced HCC who were treated with sorafenib along with their drug response. MethodsThe response to sorafenib treatment of twenty-three HCC patients who developed advanced recurrence after partial hepatectomy was analyzed using the modified Response Evaluation Criteria in Solid Tumors (mRECIST). Formalin fixed paraffin embedded (FFPE) tissue specimens obtained after tumor resection were analyzed using the Affymetrix OncoScan® FFPE assay. ResultsFrom the 23 patients analyzed in this study, 7 (30.4%) had complete/partial response to sorafenib (CR/PR), 7 (30.4%) had stable disease (SD), and 9 (39.1%) had progressive disease (PD). The mean genome-wide percentage of genome change acquisition via the OncoScan platform was 19.8% for patients with CR/PR/SD and 50.02% in the PD group (p = 0.055). Percentage of genome change above 33% was associated with adverse outcomes for sorafenib treatment in the time-to-progression analysis (p = 0.007) and overall survival (p = 0.096). Among these CNAs, amplification of chromosome 7q, containing the multidrug resistance gene ATP Binding Cassette Subfamily B Member 1 (ACBC1), significantly associated with poor overall survival (p = 0.004) and time-to-progression (p < 0.001). ConclusionsHigher percentage genome change and amplification of chromosome 7q in advanced HCC is associated with sorafenib resistance.

Prognosis, Biology, and Targeting of TP53 Dysregulation in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological cancer and is characterized by genetic features including translocations, chromosomal copy number aberrations, and mutations in key oncogene and tumor suppressor genes. Dysregulation of the tumor suppressor TP53 is important in the pathogenesis of many cancers, including MM. In newly-diagnosed MM patients, TP53 dysregulation occurs in three subsets: monoallelic deletion as part of deletion of chromosome 17p (del17p) (~8%), monoallelic mutations (~6%), and biallelic inactivation (~4%). Del17p is an established high-risk feature in MM and is included in current disease staging criteria. Biallelic inactivation and mutation have also been reported in MM patients but are not yet included in disease staging criteria for high-risk disease. Emerging clinical and genomics data suggest that the biology of high-risk disease is complex, and so far, traditional drug development efforts to target dysregulated TP53 have not been successful. Here we review the TP53 dysregulation literature in cancer and in MM, including the three segments of TP53 dysregulation observed in MM patients. We propose a reverse translational approach to identify novel targets and disease drivers from TP53 dysregulated patients to address the unmet medical need in this setting.

Large-Scale Analysis of Relapsed/Refractory Multiple Myeloma Genome Reveals Increased Prevalence of High-Risk Molecular Features and Oncogenic Drivers

Introduction Multiple myeloma (MM) is a heterogeneous disease defined by genetic lesions including translocations, chromosomal copy number aberrations (CNA), and mutations. Large-scale analyses of genomic data from newly-diagnosed patients (ndMM) have defined key oncogenic drivers beyond previously known primary events. In relapsed/refractory MM (rrMM), analyses have been completed on small numbers of patient samples for mutational profiles; however, a large-scale analysis of genomic and transcriptomic data has not been performed. In this initial analysis, we describe the rrMM genomic landscape including prevalence of key translocations, oncogenic/tumor suppressor mutational drivers, and chromosomal CNA. Methods We generated whole genome sequencing (WGS) and whole transcriptome expression (RNA-seq) from 485 rrMM patient samples derived from clinical trials: NCT01712789/CC-4047-MM-010 (N=236), NCT02045017/CC-4047-MM-013 (N=17), NCT02773030/CC-220-MM-001 (N=45) and NCT01421524/CC-122-ST-001MM2 (N=10). Somatic single nucleotide variants (SNVs) and indels were derived from WGS from 308 samples using GATK/MuTect2 and annotated using ANNOVAR. Clonal and subclonal CNA were identified using Sclust. Translocations were determined using Manta. Oncogenic/tumor suppressor drivers were identified using cDriver, which utilizes recurrence and functional consequence and cancer cell fraction (CCF) of each mutation. Results Key translocations included: t(11;14) [76/308 (25%)], t(8;14) [79/308 (25%)], t(4;14) [44/308 (14%)], t(14;16) [24/308 (8%)], t(6;14) CCND3 [18/308 (6%)], t(14;20) [17/308 (5.5%)], and t(6;14) [IRF4 17/308 (5.5%)]. Hyperdiploidy was detected in 140/308 (46%) patients. Key chromosomal CNA included del14q 134/308 (44%), del13q 131/308 (43%), del8p 113/308 (37%), del17p 53/308 (17%), amplification of 1q (≥4 copies) 45/308 (15%), and del1q 31/308 (10%). Compared to samples from ndMM patients, we saw an increase of del17p (17% vs. 8%) and t(11;14) (25% vs. 15%) in rrMM. Further, out of the 53 del17p patients, 45 (85%) were high CCF (&amp;gt;0.55) versus 63/107 (59%) reported in ndMM (Thakurta et al, Blood. 2019) Double Hit patients (biallelic inactivation of TP53 or amplification of 1q on a background of ISS3) was detected in 12% patients which is significantly higher than ndMM (6%, p &amp;lt; 0.05) (Walker et al, Leukemia. 2018) We identified a total of 107 driver genes (FDR&amp;lt;0.05). Of these, 23 were known cancer genes according to the COSMIC Cancer Gene Census (CGC) of which 19 were Tier1 such as TP53, DNMT3A and SETD2. Further, 19/107 driver genes were previously identified in ndMM from the Myeloma Genome Project including IRF4, TRAF3, NFKB2 and FGFR3. The top ten ranking driver genes in rrMM were DIS3, FAM46C, IGLL5, KMT2B, TRAF3, SP140, MALRD1, TP53, L1TD1 and PRKCD. Among these, novel driver genes such as IGLL5 (N=19, 6.2%; medianCCF=1; Immunoglobulin Lambda-Like Polypeptide 5) were also identified. We examined if loss-of-function (LOF) and gain-of-function (GOF) variants result in different sets of drivers, thus identifying putative tumor suppressor genes (TSG) and oncogenes (ONC) respectively. We identified a total of 72 and 43 ONCs and TSGs, respectively (FDR &amp;lt; 0.05). The top ten ranking ONCs included driver genes such as UBR4 and IRF4. Among the top ten ranking TSGs were novel driver genes such as TDG and SMARCA4 as well as known ndMM driver genes such as UBR5 and CDKN1B. We confirmed that FGFR3 driver mutations were associated with t(4;14) and IRF4 were associated with t(11;14) (p &amp;lt; 0.05) in our rrMM population. Further analysis will be focused on association of significant mutations with CNA and translocation groups, impact on clinical outcomes in genetic subsets, delineating the effect of multiple lines of therapy on tumor clonality, genetic architecture of resistance patterns, and the role of oncogenic drivers. Conclusions We have established and analyzed the largest molecular rrMM dataset with associated clinical outcome data. These analyses are revealing the evolution of genetic drivers of resistance to therapy and will assist in identification of subsets of poor prognostic groups (eg, Double Hit) and new molecular subsets of rrMM where novel targeted therapies could be developed. Disclosures Towfic: Celgene Corporation: Employment, Equity Ownership. Ansari-Pour:Celgene Corporation: Consultancy. Ortiz:Celgene Corporation: Employment, Equity Ownership. Gooding:Celgene Corporation: Research Funding. Rozelle:Celgene Corporation: Other: Contractor for Celgene. Zadorozhny:Celgene Corporation: Other: Contractor for Celgene. Mavrommatis:Celgene Corporation: Employment, Equity Ownership. Lata:Celgene Corporation: Employment, Equity Ownership. Stong:Celgene Corporation: Employment, Equity Ownership. Kamalakaran:Celgene Corporation: Employment, Equity Ownership. Tsai:Celgene Corporation: Employment, Equity Ownership. Flynt:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene: Employment, Equity Ownership.

Clonality analysis of pulmonary tumors by genome-wide copy number profiling.

Multiple tumors in patients are frequently diagnosed, either synchronous or metachronous. The distinction between a second primary and a metastasis is important for treatment. Chromosomal DNA copy number aberrations (CNA) patterns are highly unique to specific tumors. The aim of this study was to assess genome-wide CNA-patterns as method to identify clonally related tumors in a prospective cohort of patients with synchronous or metachronous tumors, with at least one intrapulmonary tumor. In total, 139 tumor pairs from 90 patients were examined: 35 synchronous and 104 metachronous pairs. Results of CNA were compared to histological type, clinicopathological methods (Martini-Melamed-classification (MM) and ACCP-2013-criteria), and, if available, EGFR- and KRAS-mutation analysis. CNA-results were clonal in 74 pairs (53%), non-clonal in 33 pairs (24%), and inconclusive in 32 pairs (23%). Histological similarity was found in 130 pairs (94%). Concordance between histology and conclusive CNA-results was 69% (74 of 107 pairs: 72 clonal and two non-clonal). In 31 of 103 pairs with similar histology, genetics revealed non-clonality. In two out of four pairs with non-matching histology, genetics revealed clonality. The subgroups of synchronous and metachronous pairs showed similar outcome for the comparison of histological versus CNA-results. MM-classification and ACCP-2013-criteria, applicable on 34 pairs, and CNA-results were concordant in 50% and 62% respectively. Concordance between mutation matching and conclusive CNA-results was 89% (8 of 9 pairs: six clonal and two non-clonal). Interestingly, in one patient both tumors had the same KRAS mutation, but the CNA result was non-clonal. In conclusion, although some concordance between histological comparison and CNA profiling is present, arguments exist to prefer extensive molecular testing to determine whether a second tumor is a metastasis or a second primary.

Establishment and Genetic Landscape of Precancer Cell Model Systems from the Head and Neck Mucosal Lining.

Head and neck squamous cell carcinomas (HNSCC) develop in fields of genetically altered cells. These fields are often dysplastic and a subset can be recognized as (erythro)leukoplakia, but most are macroscopically invisible. There is a lack of adequate treatment options to eradicate these fields, whereas they underlie the development of primary tumors as well as part of the local relapses. Unfortunately, there are almost no representative cellular models available to identify suitable treatment options. To this end, clinical biopsy specimens (n = 98) were cultured from normal appearing mucosa of the surgical margins of patients with primary HNSCCs (n = 32) to generate precancer cell culture models. This collection was extended with six previously established precancer cell cultures. Genetic analysis was performed on cultures with an extended life span (≥20 population doublings), the previously established cultures, and some randomly selected cultures. In total, cancer-associated changes were detected in 18 out of 34 (53%) cultures analyzed, which appeared to be independent of life span. A variety of genetic changes were identified, including somatic mutations as well as chromosomal copy-number aberrations (CNA). Loss of CDKN2A/p16Ink4A and mutations in TP53/p53 were most prominent. Remarkably, in some of these precancer cell cultures only chromosomal CNAs were detected, and none of the frequently occurring driver mutations. IMPLICATIONS: The precancer cell cultures, characterized herein, form a representative collection of field models that can be exploited to identify and validate new therapeutic strategies to prevent primary HNSCCs and local relapses.

Autologous cell lines from circulating colon cancer cells captured from sequential liquid biopsies as model to study therapy-driven tumor changes

Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However, CTC investigation is hampered by their low number, making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy, and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype, stem-cell like characteristics, angiogenesis potential, an osteomimetic signature and the capacity to escape from the immune system. Moreover, they showed changes in mRNA and protein expression (e.g., DEFA6, ABCB1 and GAL), whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits, treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time.

P3.03-06 Differentially Expressed microRNAs in Lung Adenocarcinoma Invert Effects of Copy Number Aberrations of Prognostic Genes

Significant associations between chromosomal copy number aberrations (CNAs) and differential gene expression have been found across many cancers. However, significantly downregulated genes have been often found to reside within chromosomal regions with increased number of copies and vice versa, creating a paradoxical signal. This phenomenon was usually ignored as a noise, but can potentially be a consequence of interference of other regulatory mechanisms controlling mRNA transcription. To explore existence of such paradoxes in lung adenocarcinoma (LUAD), we performed integrative analysis of 1,937 tumour and normal tissue samples, comprising copy number aberrations, gene expression and microRNA expressions data and conducted meta-analysis of 9 microRNA expression studies. We identified and validated 75 “paradoxical” genes whose differential expression consistently contrasted with aberrations of their copy numbers. Of these, 41 genes (p < 0.001) are prognostic and form a clinically relevant signature. Interestingly, differential expression of 19 microRNAs that are frequently deregulated in LUAD, explains observed paradoxes. Our results show that deregulation of paradoxical genes is crucial in LUAD and their expression pattern is maintained epigenetically, defying gene copy number status. Our work highlights importance of large integrative analysis of diverse biological data and the need to examine phenomena that contrast the established knowledge.

Abstract 4579: The genetic heterogeneity and the molecular evolution of systemic metastatic castration resistant prostate cancer during therapy

Abstract Background: Circulating tumour cells (CTCs) have a significant prognostic impact in metastatic castration-resistant prostate cancer (mCRPC) and provide direct access to the systemic disease. Here, we performed genome-wide copy number analysis of CellSearchTM detected CTCs to follow the genomic evolution of mCRPC during systemic therapy. Methods: Blood samples were collected from 13 patients with mCRPC before, during and after two different targeted therapy regimes. Single CTCs were isolated from CellSearchTM cartridges using the DEPArray™ system and the MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using Ampli1™ WGA kit. Amplification products were analysed by array-based comparative genomic hybridization (aCGH) and low-pass sequencing (LPS) using Ampli1™ LowPass kit to detect somatic chromosomal copy number aberrations (CNAs) and explore the degree of genomic heterogeneity. Results: We analysed &amp;gt;300 CTCs for CNAs by aCGH and/or LPS. Although most CTCs displayed CNAs typical for mCRPC, we identified three genomic CTC-groups across all cells with the EpCAMpos/CKpos/DAPIpos/CD45neg phenotype: CTCs with typical mCRPC CNAs (Type A; 80%), extremely aberrant CTCs (Type B; 11%), CNA-negative/low CTCs (Type C; 9%). The occurrence of Type B and C was almost mutually exclusive. At baseline, we noted different levels of CTC-heterogeneity and different CTC-aberration levels between the different patients. Interestingly, CTCs of patients with a relevant decrease in CTC-count under PARP-inhibition (CTC-responder) displayed significantly elevated CNAs at baseline compared to CTC-non-responders. During therapy, we could observe clonal changes among all patients. Conclusions: Our data show that CTC-analysis enables the dissection of clinical relevant intra-patient heterogeneity and clonal evolution under therapy in mCRPC patients. Comprehensive genomic monitoring during therapy might help to tailor therapies more effectively and may pinpoint to molecular mechanism of therapeutic resistance. Citation Format: Rui PL Neves, Streit LRF Anna, Katharina Raba, Elina-Katharina Bongers, Bianca Behrens, Guus van Dalum, Penny Flohr, Joaquin Mateo, Semini Sumanasuriya, Mateus Crespo, Berni Ebbs, Gemma Fowler, Pasquale Rescigno, Suzanne Carreira, Maryou Lambros, Edoardo Petrini, Marianna Garonzi, Nicolò Manaresi, Johann de Bono, Nikolas H. Stoecklein. The genetic heterogeneity and the molecular evolution of systemic metastatic castration resistant prostate cancer during therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4579.

Immunohistochemical and molecular analysis of spitzoid neoplasms with pulverocyte subclones

Clonal naevi are characterized by a focal proliferation of pigmented melanocytes in an otherwise banal naevus. These subclones are often composed of aggregates of larger, epithelioid melanocytes with nuclear atypia and dusty-grey cytoplasmic pigmentation, which are referred to as 'pulverocytes', and this finding may lead to a misdiagnosis of malignant melanoma (MM). To characterize the significance of subclones of dusty-grey pigmented epithelioid melanocytes within spitzoid neoplasms. We studied the histological and molecular features of a series of 20 spitzoid neoplasms with pulverocyte subclones encountered in our practice, including both atypical Spitz tumours (ASTs) and invasive MMs. Pulverocytes were predominantly dermal, and the percentage of subclones ranged from 2% to 40%, with a median of 10% in ASTs and 25% in lesions we classified as MM. In cases with > 10% subclones, there was an increased odds of fluorescence in situ hybridization positivity (OR = 12; 95% CI 1.2-293.4; P = 0.03) and an increased odds of homozygous 9p21 deletion (OR = 3.6; 95 CI 0.28-89.82; P = 0.33), although the latter did not reach statistical significance. We consider spitzoid lesions with a small subclone population to be a variant of a clonal naevus with indolent behaviour, whereas lesions with larger pulverocyte populations are more likely to have chromosomal copy number aberrations and in some cases may represent malignant transformation.