CDK4 Amplification Research Articles

BIOM-43. THE GENOMIC, TRANSCRIPTOMIC, AND EPIGENOMIC LANDSCAPE OF ISOCITRATE DEHYDROGENASE WILD TYPE GLIOBLASTOMA ACROSS THE AGE CONTINUUM

Abstract BACKGROUND Older age is a poor prognostic factor for glioblastoma (GBM) patients. We tested whether the intrinsic molecular landscape of the tumor may contribute to this poor prognosis. METHODS In accordance with the 2021 WHO classification scheme, we included only isocitrate dehydrogenase (IDH) wild type GBM. Based on published literature, we defined older as age > 65. RNA expression, gene amplification, tumor mutational burden (TMB) and mutational profiles were analyzed in three unique datasets: Tempus (n = 1,410), Caris (n = 1,432), and TCGA (n = 557). Comparison were made between < 65 and ³ 65 year olds using Pearson’s Chi-squared tests, Fisher’s exact tests, or Wilcoxon rank-sum where appropriate. RESULTS From our evaluable gene sets, TERT promoter mutations were more prevalent in patients ³ 65 years old (Caris 82.64 vs 77.27%, p = 0.016; Tempus 58.0 vs 49.0%, p = 0.002). There were no significant differences in PDCD1, CD274, CD3E, TNFRSF18, CD40, CD8A, TNFRSF4, CTLA4, HAVCR2, TNFSF9, CD274, or CDKN2A; PDL-1 (by IHC); dMMR/MSI-H, TMB; CDK6 amplification, EGFR amplification, EGFR, EGFRvIII, EGFR fusions, MET fusions, PTEN, TP53, or NF-1. MGMT promoter methylation (Caris data) was more common in the older group (49.73 v 34.14%, p < 0.001). TGCA data demonstrated that gene expression, TMB, and methylation did not change significantly with age. Additionally, PCOLCE2 and SLC10A4 were differentially methylated, and missense mutations, of any type, were more common in the older group (p=0.006). CONCLUSION Despite worse survival outcomes for older patients with IDHwt GBM as compared to younger counterparts, the molecular landscape is similar at the genomic, transcriptomic and epigenomic levels. The key exception is TERT promoter mutations that are more common in older GBM patients. Poorer survival is therefore not likely to be attributable solely to intratumoral factors.

Comprehensive Molecular Characterization of Soft Tissue Sarcoma for Prediction of Pazopanib-Based Treatment Response.

Even though pazopanib, a multitargeted tyrosine kinase inhibitor, has been approved for refractory soft tissue sarcoma (STS), little is known about the molecular determinants of the response to pazopanib. We performed integrative molecular characterization to identify potential predictors of pazopanib efficacy. We obtained fresh pre-treatment tumor tissue from 35 patients with advanced STS receiving pazopanib-based treatment. Among those, 18 (51.4%) received pazopanib monotherapy, and the remaining 17 (48.6%) received pazopanib in combination with durvalumab, programmed death-ligand 1 blockade. Whole-exome and transcriptome sequencing were performed for each tumor and patient germline DNA. Of the 35 patients receiving pazopanib-based treatment, nine achieved a partial response (PR), resulting in an objective response rate (ORR) of 27.3%, and the median progression-free survival (PFS) was 6.0 months. Patients with CDK4 amplification (copy ratio tumor to normal > 2) exhibited shorter PFS (3.7 vs. 7.9 months, p=2.09×10-4) and a poorer response (ORR; 0% vs. 33.3%) compared to those without a gene amplification (copy ratio ≤ 2). Moreover, non-responders demonstrated transcriptional activation of CDK4 via DNA amplification, resulting in cell cycle activation. In the durvalumab combination cohort, seven of the 17 patients (41.2%) achieved a PR, and gene expression analysis revealed that durvalumab responders exhibited high immune/stromal cell infiltration, mainly comprising natural killer cells, compared to non-responders as well as increased expression of CD19, a B-cell marker. Despite the limitation of heterogeneity in the study population and treatment, we identified possible molecular predictors of pazopanib efficacy that can be employed in future clinical trials aimed at evaluating therapeutic strategies.

Efficacy and potential resistance mechanisms of afatinib in advanced non-small cell lung cancer patients with EGFR G719X/L861Q/S768I.

Afatinib is the only currently approved EGFR-tyrosine kinase inhibitors for advanced non-small cell lung cancer (NSCLC) patients with EGFR G719X/L861Q/S768I. However, there are limited real-world data concerning the benefits and resistance mechanisms of afatinib in patients with these nonclassical mutations. To fill this gap, the present study was conducted. All NSCLC patients treated with afatinib were screened, and patients with EGFR G719X/L861Q/S768I were enrolled into the analysis. Either tumor tissue or blood specimens were detected by the commercial next-generation sequencing (NGS) panels or amplification-refractory mutation system (ARMS)-polymerase chain reaction (PCR) to figure out the mutation genotype. A total of 106 advanced NSCLC patients with EGFR G719X/L861Q/S768I received afatinib treatment. The benefits of afatinib exhibited heterogeneity in different mutation genotypes. Notably, at baseline, NGS testing was performed in 59 patients, and TP53 was the most frequently coexisting mutation. Patients with TP53 mutations obtained fewer survival benefits than those with TP53 wild-type. A total of 68 patients ultimately experienced progression, and 27 patients received NGS testing to clarify the potential resistance mechanisms. EGFR-T790M, CDK4 amplification, FGFR1 amplification, PIK3CA, MET amplification, RET fusions, HER2, and BRAF mutations were identified in three (11.1%), three (11.1%), three (11.1%), three (11.1%), three (11.1%), one (3.7%), one (3.7%), and one (3.7%) of the cases, respectively. Five patients underwent ARMS-PCR testing for detecting EGFR-T790M mutation, and only one patient was T790M-positive. The present study elucidated the differential benefits of afatinib within different mutation genotypes and first revealed the spectrum of potential resistance mechanisms in patients with EGFR G719X/L861Q/S768I. The results of this study may provide practical clinical information that can guide optimal treatment in this setting.

880P Phase II trial of the cyclin dependent kinase 4/6 inhibitor SHR6390 in patients with advanced head and neck mucosal melanoma harboring CDK4 amplification

Mucosal melanoma (MM) is a rare but devastating subtype of melanoma with 5-year survival rates ranging from only 16% to 25%. Our previous studies have demonstrated robust anti-tumor effects of CDK 4/6 inhibitors in head and neck MM (HNMM) patients (pts) derived xenograft models with CDK4 amplification. We performed a phase II study to determine the efficiency and safety of SHR6390, a CDK4/6 inhibitor, in pts with HNMM harboring CDK4 amplification.

Comprehensive Analysis of Genomic Alterations in Hepatoid Adenocarcinoma of the Stomach and Identification of Clinically Actionable Alterations.

Simple SummaryHepatoid adenocarcinoma of the stomach (HAS) is a subset of gastric cancer (GC) histologically characterized by hepatocellular carcinoma-like foci with or without alpha-fetoprotein secretion, which is easily misdiagnosed. Genomic alterations and potential targets for this population are still largely unknown. Additionally, treatment regimens of HAS are mainly based on GC guidelines, which is not reasonable for diseases with great heterogeneity. The present study comprehensively depicts the genomic features of HAS, and they are significantly different from GC, AFP-producing GC (AFPGC), and liver hepatocellular carcinoma (LIHC). Multiple aggressive behavior-related amplificated or deleted regions in HAS are firstly reported. Moreover, reliable and practicable clinically actionable alterations for HAS are identified, providing evidence for making personalized therapy based on the genomic characteristics of HAS instead of GC.Hepatoid adenocarcinoma of the stomach (HAS) is a rare malignancy with aggressive biological behavior. This study aimed to compare the genetic landscape of HAS with liver hepatocellular carcinoma (LIHC), gastric cancer (GC), and AFP-producing GC (AFPGC) and identify clinically actionable alterations. Thirty-eight cases of HAS were collected for whole-exome sequencing. Significantly mutated genes were identified. TP53 was the most frequently mutated gene (66%). Hypoxia, TNF-α/NFκB, mitotic spindle assembly, DNA repair, and p53 signaling pathways mutated frequently. Mutagenesis mechanisms in HAS were associated with spontaneous or enzymatic deamination of 5-methylcytosine to thymine and defective homologous recombination-related DNA damage repair. However, LIHC was characteristic of exposure to aflatoxin and aristolochic acid. The copy number variants (CNVs) in HAS was significantly different compared to LIHC, GC, and AFPGC. Aggressive behavior-related CNVs were identified, including local vascular invasion, advanced stages, and adverse prognosis. In 55.26% of HAS patients there existed at least one clinically actionable alteration, including ERBB2, FGFR1, CDK4, EGFR, MET, and MDM2 amplifications and BRCA1/2 mutations. MDM2 amplification with functional TP53 was detected in 5% of HAS patients, which was proved sensitive to MDM2 inhibitors. A total of 10.53% of HAS patients harbored TMB > 10 muts/Mb. These findings improve our understanding of the genomic features of HAS and provide potential therapeutic targets.

Characterization of MET Exon 14 Skipping Alterations (in NSCLC) and Identification of Potential Therapeutic Targets Using Whole Transcriptome Sequencing

IntroductionGenomic alterations in the juxtamembrane exon 14 splice sites in NSCLC lead to increased MET stability and oncogenesis. We present the largest cohort study of MET Exon 14 (METex14) using whole transcriptome sequencing. MethodsA total of 21,582 NSCLC tumor samples underwent complete genomic profiling with next-generation sequencing of DNA (592 Gene Panel, NextSeq, whole exome sequencing, NovaSeq) and RNA (NovaSeq, whole transcriptome sequencing). Clinicopathologic information including programmed death-ligand 1 and tumor mutational burden were collected and RNA expression for mutation subtypes and MET amplification were quantified. Immunogenic signatures and potential pathways of invasion were characterized using single-sample gene set enrichment analysis and mRNA gene signatures. ResultsA total of 533tumors (2.47%) with METex14 were identified. The most common alterations were point mutations (49.5%) at donor splice sites. Most alterations translated to increased MET expression, with MET co-amplification resulting in synergistic increase in expression (q < 0.05). Common coalterations were amplifications of MDM2 (19.0% versus 1.8% wild-type [WT]), HMGA2 (13.2% versus 0.98% WT), and CDK4 (10.0% versus 1.5% WT) (q < 0.05). High programmed death-ligand 1 > 50% (52.5% versus 27.3% WT, q < 0.0001) and lower proportion of high tumor mutational burden (>10 mutations per megabase, 8.3% versus 36.7% WT, p < 0.0001) were associated with METex14, which were also enriched in both immunogenic signatures and immunosuppressive checkpoints. Pathways associated with METex14 included angiogenesis and apical junction pathways (q < 0.05). ConclusionsMETex14 splicing alterations and MET co-amplification translated to higher and synergistic MET expression at the transcriptomic level. High frequencies of MDM2 and CDK4 co-amplifications and association with multiple immunosuppressive checkpoints and angiogenic pathways provide insight into potential actionable targets for combination strategies in METex14 NSCLC.

Amplification of CDK4 and MDM2: a detailed study of a high-risk neuroblastoma subgroup

In neuroblastoma, MYCN amplification and 11q-deletion are important, although incomplete, markers of high-risk disease. It is therefore relevant to characterize additional alterations that can function as prognostic and/or predictive markers. Using SNP-microarrays, a group of neuroblastoma patients showing amplification of one or multiple 12q loci was identified. Two loci containing CDK4 and MDM2 were commonly co-amplified, although amplification of either locus in the absence of the other was observed. Pharmacological inhibition of CDK4/6 with ribociclib or abemaciclib decreased proliferation in a broad set of neuroblastoma cell lines, including CDK4/MDM2-amplified, whereas MDM2 inhibition by Nutlin-3a was only effective in p53wild-type cells. Combined CDK4/MDM2 targeting had an additive effect in p53wild-type cell lines, while no or negative additive effect was observed in p53mutated cells. Most 12q-amplified primary tumors were of abdominal origin, including those of intrarenal origin initially suspected of being Wilms’ tumor. An atypical metastatic pattern was also observed with low degree of bone marrow involvement, favoring other sites such as the lungs. Here we present detailed biological data of an aggressive neuroblastoma subgroup hallmarked by 12q amplification and atypical clinical presentation for which our in vitro studies indicate that CDK4 and/or MDM2 inhibition also could be beneficial.

Genomic features of IDH-mutant adult diffuse astrocytoma.

e14032 Background: The fifth edition of the WHO Classification of Tumors of the Central Nervous System (CNS), published in 2021, which indicated that all IDH mutant diffuse astrocytic tumors are considered a single type (Astrocytoma, IDH-mutant) and are then graded as CNS WHO grade 2, 3, or 4. Besides, the guideline eliminates the term “Glioblastoma (GBM), IDH-mutant”. Moreover, grading is no longer entirely histological, since the presence of CDKN2A/B homozygous deletion results in a CNS WHO grade of 4, even in the absence of microvascular proliferation or necrosis. Therefore, more clinical and genomic characteristics of IDH-mutant astrocytoma under molecular typing need to be further studied. Methods: Adults pathological diagnosed with diffuse astrocytoma and glioblastoma in our Chinese dataset were identified retrospectively. All patients included in the analysis were tested for 131 CNS tumor-related genes (including IDH1/2), chromosomes 1p/19q and chromosomes 7/10. Results: 63 initial diagnosed IDH-mutant diffuse astrocytomas including 46 patients with grade 2-3 and 17 patients with uncertain grade. While, 5.2% of GBM (9/174) harbored IDH1/2 mutation which should be redefined as diffuse astrocytoma, IDH-mutant, WHO 4 grade. And these patients had a high frequency of CDKN2A/B homozygous deletion (88.9%, 8/9). In patients with 2-3 grade and unclear grade, CDKN2A/B homozygous deletion accounted for 18.7% (9/48) and 27.8% (5/18), respectively, which meant the grade of these diffuse astrocytomas should be revised to WHO 4 grade according to guideline. While mutation frequency of crucial prognosis factors in glioma, including chromosomes 7 loss, chromosomes 10 gain, EGFR amplification, PIK3CA/R1, PDGFRA amplification, CDK4 amplification, were 9.7%, 2.8%, 5.6%, 13.9%, 6.9% and 8.3% in all 72 astrocytomas, respectively. In addition, we found 83.3% (5/6) KDR copy number variant were occurred in which initially pathological diagnosed as IDH-mutant GBM. Conclusions: we conducted comprehensive analysis of IDH mutant diffuse astrocytoma. CDKN2A/B homozygous deletion as a specific grade-classified molecular marker could better re-distinguish about 20%-30% 2-3 grade and uncertain grade astrocytoma. KDR copy number variant may be a marker in IDH-mutant astrocytoma to identify the tumor which pathology is closer to GBM.

Increased phosphorylation of RB and protein expression of Cyclin E confers resistance to CDK4/6 inhibitors in dedifferentiated liposarcoma.

e23525 Background: Dedifferentiated liposarcomas (DDLPS) are malignant adipocytic cancers characterized by an amplification of the cell cycle regulatory gene, CDK4. Palbociclib, a CDK4/6-inhibitor, is a standard of care treatment; however, innate and acquired resistance to the drug limits efficacy in patients with DDLPS. Molecular mechanisms and biomarkers of palbociclib resistance have been posited in non-mesenchymal tumors but are poorly understood in DDLPS. To this end, we developed palbociclib-resistant DDLPS cell lines derived from several DDLPS patient samples to better characterize these phenotypic and genomic differences. Methods: Three human CDK4 amplified DDLPS cell lines (224A, 246, 863) and one control liposarcoma control cell line without CDK4 amplification and RB loss (LiSa2) were brought into culture. Each cell line was intermittently treated with palbociclib at their respective IC25 for 6 months to develop resistance in long-term treated cell lines. Surviving cells were allowed to recover for at least 2 weeks before retreatment. Parental lines were grown as controls in tandem with the developing resistant lines to compare molecular changes in response to palbociclib. Cell viability was measured by XTT, cell cycle analysis was measured with Flow cytometry using a PI stain, and protein expression was measured using whole cell lysates for Western blotting. Results: The four cell lines had IC50s to palbociclib ranging from 15.0, 14.8, 12.0, and 11.4 µM for LiSa-2, 224A, 246, and 863 cell lines respectively. Long-term treated strains demonstrated consistent reductions in the measured IC50s (8.99, 7.32, 8.27, and 7.08 µM respectively). After treatment with 10 µM of palbociclib for 48 hours, two parental cell lines showed a greater increase in cells in the G1 fraction than long-term treated cells: LiSa-2 (-2.4% increase in parental vs -16.5% increase in long-term treated) and 863 (16.2% vs 11.7%). 224A and 246 demonstrated the opposite where parental cell lines had fewer cells in G1 after treatment compared to long-term treated cell: 224A (12.6% vs. 20.5%) and 246 (12.6% vs. 21.6%). Western blots indicated that the initial response to palbociclib in parental cell lines decreased pRb protein levels and increased Cyclin E and CDK6. After 6 months of treatment for all cell lines, a significant increase in pRb and Cyclin E were observed in the long-term treated cell lines and no changes were seen in CDK4, Cyclin D, nor p16. Conclusions: Here we present the development of two novel, palbociclib resistant, DDLPS cell lines and two long-term treated cell lines. Our resistant cell lines indicated that the ability to bypass the G1/S-checkpoint in the presence of CDK4/6-inhibitors may result from increased phosphorylation of Rb and increased total of Cyclin E. Next generation sequencing of RNA expression in parental and resistant lines are underway.

Characterization of MET exon 14 skipping alterations (METex14) in non–small cell lung cancer (NSCLC) using whole transcriptome sequencing (WTS).

9122 Background: Multiple DNA alterations in exon 14 splice sites have been identified in NSCLC and result in skipping of the juxtamembrane domain Cbl-E3 ubiquitin ligase binding region, leading to increased MET stability and oncogenesis. The effects of these alterations on transcriptome-level have not been fully characterized. We present the largest cohort study of METex14 using WTS and identify key cellular pathways associated with invasion and metastases in METex14. Methods: 17,666 NSCLC tumor samples underwent genomic profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq, whole exome sequencing, NovaSeq) and RNA (NovaSeq, WTS). METex14 was captured via WTS. ssGSEA analysis was used to evaluate pathway enrichment. Wilcoxon, Fisher’s exact were used for statistical significance (p without and q values with multiple comparison correction). Results: 440 patients (2.5%) with METex14 were identified. METex14 patients were of older age, female gender, and enriched in sarcomatoid histology (Table 1). The most common alterations were point mutations (51.5%) and deletions (17.3%) at donor splice sites. Splice site alterations except point mutations at splice acceptor site translated to increased mRNA expression compared to wild-type MET (WT). MET amplification translated to higher mRNA expression compared to METex14 and WT with synergistic expression when co-altered with METex14 (q&lt;0.05). The most common co-alterations were amplifications of MDM2 (18.5% vs. 1.8% WT), HMGA2 (13.7% vs 0.9% WT), and CDK4 (10.4% vs 1.4% WT) (q &lt; 0.05). METex14 were mutually exclusive to mutations in KRAS and EGFR. High PD-L1 (22c3) &gt; 50% (53% vs. 27.6% WT, q&lt;0.001) and lower TMB (4 mut/Mb vs. 7 mut/Mb WT, p&lt;0.001) were observed with METex14 and pathways associated with skipping variants included IFNγ signaling, angiogenesis, and apical junction pathways on univariate analysis (q&lt;0.05). Conclusions: We present the largest WTS analysis of METex14. Splicing alterations and MET co-amplification translated to higher and synergistic MET expression at transcriptome level, respectively. Association with upregulated angiogenic and apical junction pathways support preclinical observation of vascular and cytoskeletal remodeling as potential mechanisms of invasion and metastases in MET ex14 NSCLC.[Table: see text]

Phase II trial of palbociclib in advanced sarcoma overexpressing CDK4 gene excluding dedifferentiated liposarcoma (DD LPS): A study from the Spanish Group for Research on Sarcoma (GEIS).

11511 Background: CDK4/6 inhibitors showed a favorable progression-free survival (PFS) in DD LPS, a sarcoma bearing 12q 13-15 amplicon that implies CDK4 amplification. The median PFS was 4 and 7 months (m) for palbociclib and abemaciclib, respectively. Preclinical experiments in 10 sarcoma cell lines and 6 PDX models, including only one DD LPS, showed higher efficacy of anti-CDK4 in cases with high expression of CDK4 and low expression of p16. This rationale supported the design of a phase II trial exploring palbociclib in a wide range of sarcomas, excluding DD LPS. Methods: Progressing pretreated advanced soft tissue sarcoma, excluding DD LPS, or osteosarcoma adult patients (pts), whose tumors overexpressed CDK4 and underexpressed CDKN2A mRNA in a baseline mandatory biopsy, were enrolled. CDK4 and CDKN2A expression were assessed by qRT-PCR, using an external control as reference (Universal human reference RNA; Agilent Technologies). The primary endpoint was 6-m PFS rate. Minimax Simon’s two-stage with type 1 and 2 errors of 10%, and null and alternative hypothesis of H0 15%, H1 40%, 6-month PFS rates were specified. The study will warrant further investigation if 6 or more pts had a PFS &gt; 6 m from 21 evaluable pts. Palbociclib was administered orally at 125 mg/ day 21 out of 28 days. Pre-screening intended to increase the probability of positive profile in the baseline biopsy. Results: A total of 214 pts with 236 CDK4/ CDKN2A determinations were assessed for enrolment; 141 for prescreening, in archive tumor sample, and 95 for screening, in a baseline biopsy. There were 38/141 (27%) and 28/95 (29%) pts with favorable mRNA profile from pre and screening, respectively. Twenty-two pts were enrolled with a median of previous systemic lines of 3 (1-5). There were 9 different sarcoma subtypes, including 2 osteosarcomas. With a median FU of 10 m (0.4-23.3), the median PFS was 4.2 m (95% CI 0.9-7.4), while the 6- and 12-m PFS rates were 30% (95% CI 9-51) and 18% (95% CI 12-48) respectively. From 19 evaluable pts (1 early death by COVID, 1 withdrew consent and for 1 it was too early to be assessed) 11 had stable disease (58%) and 8 progressed (42%) as the best response. Patients with CDK4 expression above the median value had significantly longer mPFS in the univariate analysis: 5.9 m (95% CI 1.4-10.4) vs 1.9 m (95% CI 0.6-3.2), p = 0.046; and longer OS: 15.5 m (95% CI 6.8-24.3) vs 10.6 m (95% CI 0-23.2), p = 0.047, respectively. The probability to find a positive profile in the screening was 29%, but this proportion increased up to 41% if in pre-screening had been positive. Conclusions: Palbociclib showed to be effective in a wide variety of sarcoma subtypes, other than DD LPS, selected by CDK4/CDKN2A biomarkers. Clinical trial information: NCT03242382.

Characterization of MET exon 14 skipping in pancancer.

e20530 Background: MET exon 14 skipping ( METex14) is characterized oncogenic that possesses susceptibility to targeted inhibitors. We retrospectively investigated a Chinese Pan-cancer cohort to characterize METex14 alterations and co-mutation. Methods: We screened tumor tissues or body fluids of a pan-cancer patients by DNA-based next-generation sequencing assay (Genetron Health) from 2017 to 2020. And distribution of co-mutation with METex14 was analyzed. Results: 233 samples from 187 patients (190 tissues and 43 circulating tumor DNA) were identified with METex14. Average age of these METex14 patients was 67.84 ± 12.19 years old, indicating they were characterized by advanced age. The cancer distribution involved METex14 was 95.72% of lung cancer (179 cases), 3.21% of brain tumors (4 glioma blastoma, 1 anaplastic astrocytoma and 1 ependymoma), 0.535% of gastrointestinal stromal tumor (1 case) and 0.535% of sarcoma (1 case). Referring to different transcripts (NM_001127500.1/2 or NM_000245.2), a total of 68 distinct sequence variants of METex14 were identified spanning functional regions: 31 forms in splice donor site (SD, 148 cases, including 1 case of D1010H), 5 in splice acceptor site (SA, 5 cases), 13 in poly-pyrimidine tract (PPT, 15 cases), 19 cover PPT and SA (19 cases). In non-lung cancers, 5 of 8 cases were detected of METex14 variation in SD site. Among the driver coexisting cases of lung cancer, METex14 samples mainly coexisted with MET amplification, EGFR driver mutations and functional fusions. Co-mutation cases with MET amplification (13) and SEMA3D-MET fusion (1) may benefit from MET inhibitors, which may be the same as the samples of METex14 alone. 13 of METex14 cases occurred with MDM2 and 9 of CDK4 amplification, respectively. There were 2 of METex14 cases coexisted with EGFR 19del or L858R, which may benefit from combination or sequential therapies of MET inhibitor and EGFR TKIs. In addition, 2 of METex14 cases were detected with functional fusions (1 ALK-EML4, 1 KIFI5B-RET), resulting in potential benefit from combination of corresponding targeted agents. Among non-lung cancer cases, 2 of 8 case had coexistence of METex14 and MET amplification, 6 of 8 patients had coexistence of METex14 and TP53, similar phenomenon was also observed in lung cancer samples. Conclusions: Here we reported diverse METex14 alterations in a Chinese pan-cancer cohort that contribute to oncogenic transformation. METex14 mutations of pan-cancer mainly occurred in SD site. TP53 was its main co-mutant gene among pan-cancers, and MET amplification was also observed.

Establishment and characterization of a novel patient-derived cell line of dedifferentiated liposarcoma, NCC-DDLPS6-C1

Dedifferentiated liposarcoma (DDLPS) is morphologically characterized by well-differentiated liposarcomas associated with high-grade non-lipogenic sarcoma and molecularly characterized by the coamplification of MDM2 and CDK4(12q14-15). DDLPS is highly aggressive, and effective systemic chemotherapy has not been developed yet. In this study, we established a novel DDLPS cell line, NCC-DDLPS6-C1, as a potential tool for the development of novel therapies. NCC-DDLPS6-C1 cells were established from surgically resected tumor tissues of a patient with DDLPS. Amplification and overexpression of MDM2 and CDK4 were observed in NCC-DDLPS6-C1 cells. NCC-DDLPS6-C1 cells proliferated rapidly, invaded aggressively, and formed spheroids. Moreover, NCC-DDLPS6-C1 cells formed tumors in mice. These observations suggested that the malignant potentials that may reflect the original features of DDLPS were retained in the NCC-DDLPS6-C1. Anticancer drugs that significantly reduced the proliferation of NCC-DDLPS6-C1 cells were identified by drug library screening. Thus, NCC-DDLPS6-C1 may recapitulate the original genotypes and phenotypes, and we conclude that the NCC-DDLPS6-C1 cell line is a useful resource for the study of DDLPS.

Abstract IA013: Genetic heterogeneity and tumor microenvironment in glioblastoma

Abstract Glioblastoma (GBM), the most aggressive brain tumor, remains an unmet medical need. Despite aggressive chemotherapy, radiation and surgery, only 5.6% of patients survive beyond 5 years post-diagnosis. Targeted approaches have not been successful in this tumor type due to the extreme heterogeneity of GBM. Mosaic amplification of oncogenes suggests that multiple genetically distinct clones are present in each tumor. However, little is known about how different subpopulations of GBM cells interact with each other or with the surrounding tumor microenvironment (TME). To address this, we employed spatial protein profiling coupled with single-cell spatial maps of fluorescence in situ hybridization (FISH) for key oncogenes frequently amplified in GBM. Our analysis spanned 3-4 areas from 17 formalin-fixed paraffin-embedded (FFPE) GBM cases, 96 sub-regions of interest and total of 35,843 single nuclei. Digital spatial profiling for 79 protein markers associated with GBM biology, TME and neurobiology revealed high intra-tumor variability of vasculature, between distinct regions of the same tumor biopsy. The single-cell FISH signal quantification allowed us to classify tumors based on the relative frequency of cells harboring co-occurring of EGFR and CDK4 amplifications. Tumors with low odds ratio for EGFR and CDK4 co-amplification in a cell had a significantly higher EGFR copy number in cells harboring this amplification. Cell-to-cell variation in EGFR copy number was also much higher in these tumors, suggesting a possibility for extrachromosomal origin of this amplification. Interestingly, the tumors with high frequency of cells harboring co-amplification of EGFR and CDK4 exhibit higher infiltration by CD163+ immunosuppressive macrophages. Our results suggest that high throughput assessment of genomic alterations at the single cell level could provide a measure for predicting the immune state of GBM. Citation Format: Kacper A. Walentynowicz, Dalit Engelhardt, Shreya Yadav, Ugoma Onubogu, Roberto Salatino, Cristina Vincentelli, Thomas O. McDonald, Franziska Michor, Michalina Janiszewska. Genetic heterogeneity and tumor microenvironment in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr IA013.

Genomic and Transcriptomic Analysis of Neuroendocrine Transformation in ALK-Rearranged Lung Adenocarcinoma After Treatments With Sequential ALK Inhibitors: A Brief Report

IntroductionNeuroendocrine (NE) transformation has been reported in patients with ALK-rearranged NSCLC after ALK inhibition, but unlike EGFR-mutant NSCLC, the exact mechanism of NE transformation in ALK-rearranged NSCLC is poorly studied. MethodsWe collected the matched pre- and post-transformation samples from a patient with ALK-rearranged lung adenocarcinoma (LUAD) and performed targeted panel sequencing, whole exome sequencing, and bulk RNA sequencing. ResultsMultiple mutations were shared between the pretransformation and post-transformation samples. Neither RB1 nor TP53 mutation was detected, but CDKN2A deletion and CDK4 amplification were found instead. Mismatch repair-associated mutational signature was significantly enriched after transformation. Genes associated with Notch signaling and PI3K/AKT pathway were significantly up-regulated, whereas genes related to lymphocyte activation and NF-kB signaling were down-regulated. Signatures relating to homologous recombination, mismatch repair, and Notch signaling pathways were enriched, which were further validated in The Cancer Genome Atlas cohorts. Macrophages M2 were found to have prominently higher abundance in the tumor immune microenvironment after NE transformation. ConclusionsThe mechanism of NE transformation in ALK-rearranged LUAD may be different from that in EGFR-mutant LUAD.

MDM2 Gene Amplification and Expression of MDM2 and CDK4 are Rare in Ossifying Fibroma of Craniofacial Bones.

Ossifying fibroma of the craniofacial bones is a fibro-osseous lesion characterized by varied patterns of bone formation in a fibroblastic stroma. Ossifying fibroma is a putatively benign lesion with no reports of malignant transformation or metastasis. Differentiation from other fibro-osseous lesions can be challenging necessitating synthesis of clinical, radiological and pathological findings. The molecular pathogenesis of ossifying fibroma is poorly understood but recent studies have reported MDM2 gene amplification and chromosomal copy number changes in a subset of ossifying fibromas. MDM2 amplification in ossifying fibroma, if true, presents a diagnostic problem because this genetic event, at least among craniofacial fibro-osseous lesions, was previously considered specific for low-grade osteosarcoma. In the present study, we investigated the utility of MDM2 and CDK4 immunohistochemistry, and fluorescence in situ hybridization for MDM2 gene amplification, in the diagnosis of 44 craniofacial bone ossifying fibromas. Focal MDM2 and CDK4 nuclear immunoreactivity was found in 11 and 1 ossifying fibromas, respectively, but none demonstrated MDM2 amplification by fluorescence in situ hybridization. A single tumor displayed MDM2 amplification without nuclear immunoreactivity to either MDM2 or CDK4. Our data suggest that while focal MDM2 and CDK4 nuclear expression may be detected in a minority of ossifying fibromas, this expression does not correlate with MDM2 amplification. In addition, MDM2 amplification is extremely rare in ossifying fibroma so the detection of this genetic abnormality should continue to raise concern for osteosarcoma.

Endometrial Stromal Sarcomas With BCOR Internal Tandem Duplication and Variant BCOR/BCORL1 Rearrangements Resemble High-grade Endometrial Stromal Sarcomas With Recurrent CDK4 Pathway Alterations and MDM2 Amplifications.

The distinction between low-grade and high-grade endometrial stromal sarcomas (LGESS, HGESS) is increasingly defined by genetics. Recently, variant genomic alterations involving BCOR or BCORL1 have been reported in endometrial stromal sarcoma (ESS), although it remains unclear whether these justify a diagnosis of LGESS or HGESS. In this study, we describe clinicopathologic and molecular features of ESS with such alterations to help clarify their classification in the spectrum of ESS. We collected a cohort of 13 ESS harboring variant alteration involving BCOR (6 with internal tandem duplication, 1 with EP300::BCOR fusion, 1 with BCOR::LPP fusion) and BCORL1 ( 4 with JAZF1::BCORL1 fusion, 1 with EPC1::BCORL1 fusion). The median patient age at primary diagnosis was 51 years (range: 18 to 70 y). Median tumor size at primary diagnosis was 9.3 cm (range: 4.5 to 21 cm), and extrauterine disease spread (stage IIIB-C) was present in 27%. The tumors were composed of round to spindled cells with cellularity and cytologic atypia ranging from mild to marked and a median mitotic count of 18/10 HPFs (range: 2 to 85/10 HPFs). At least focally myopermeative growth was noted in 8/8 assessable cases. Of 12 patients with follow-up data (median: 25 mo), 4 patients died of disease and 3 were alive with recurrent disease. Unsupervised hierarchical clustering of DNA methylation data together with a large cohort of uterine mesenchymal tumors that included YWHAE::NUTM2 and Z C3H7B::BCOR HGESS and molecularly confirmed LGESS revealed a common methylation signature for all ESS with variant BCOR and BCORL1 alterations and HGESS with YWHAE::NUTM2 and ZC3H7B::BCOR gene fusion. Copy number analysis revealed amplifications of CDK4 and MDM2 , as well as homozygous deletions of CDKN2A/B and NF1 in a subset of tumors. Our results indicate that ESS with BCOR internal tandem duplication and variant BCOR and BCORL1 rearrangements clinically and molecularly resemble conventional HGESS.

Molecular Stratification of Medulloblastoma: Clinical Outcomes and Therapeutic Interventions

Medulloblastoma (MB) is the most common malignant pediatric posterior fossa tumor. Recent genetic, epigenetic, and transcriptomic analyses have classified MB into three subgroups, Wingless Type (WNT), Sonic Hedgehog (SHH), and non-WNT/non-SHH (originally termed Group 3 and Group 4), with discrete patient profiles and prognoses. WNT is the least common subgroup with the best prognosis, characterized by nuclear β-catenin expression, mutations in Catenin beta-1 (CTNNB1), and chromosome 6 monosomy. SHH tumors contain mutations and alterations in GLI1, GLI2, SUFU, and PTCH1 genes, which constitutively activate the SHH pathway. Originally, the presence of TP53 gene alterations and/or MYC amplifications was considered the most reliable prognostic factor. However, recent molecular analyses have subdivided SHH MB into several subtypes with distinct characteristics such as age, TP53 mutation, MYC amplification, presence of metastases, TERT promoter alterations, PTEN loss, and other chromosomal alterations as well as SHH pathway-related gene mutations. The third non-WNT/non-SHH MB (Group3/4) subgroup is genetically highly heterogeneous and displays several molecular patterns, including MYC and OTX2 amplification, GFI1B activation, KBTBD4 mutation, GFI1 rearrangement, PRDM6 enhancer hijacking, KDM6A mutation, LCA histology, chromosome 10 loss, isochromosome 17q, SNCAIP duplication, and CDK6 amplification. However, based on molecular profiling and methylation patterns, additional non-WNT/non-SHH MB subtypes have been described. Recent WHO (2021) guidelines stratified MB into four molecular subgroups with four and eight further subgroups for SHH and non-WNT/non-SHH MB, respectively. In this review, we discuss advancements in genetics, epigenetics, and transcriptomics for better characterization, prognostication, and treatment of MB using precision medicine.

Selected Articles from This Issue

Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are both characterized by amplification of MDM2 and CDK4. Although inhibitors of both MDM2 and CDK4 have shown clinical promise as monotherapy, preclinical studies have revealed synergy between MDM2 and CDK4 inhibitors. To assess this combination treatment strategy, Abdul Razak and colleagues conducted a clinical trial combining siremadlin, a p53–MDM2 inhibitor, with ribociclib, a CDK4/6 inhibitor, in patients with locally advanced/metastatic WDLPS or DDLPS. The toxicity profile of this combination was manageable. Furthermore, preliminary signs of clinical activity were observed, with 3 patients achieving a partial response and 38 achieving stable disease. These results suggest that the combination of siremadlin and ribociclib should be studied in future clinical trials.The process of autophagy has been shown to lead to resistance to BRAF/MEK inhibition in BRAFV600-mutant melanoma. To investigate whether this resistance mechanism can be circumvented, Mehnert and colleagues conducted a phase I/II clinical trial assessing the combination of hydroxychloroquine (HCQ), an autophagy inhibitor, with dabrafenib and trametinib, in patients with BRAFV600-mutant melanoma. This combination was well tolerated but did not meet a previously determined criteria for success. That said, evidence of clinical activity was observed: a complete response rate of 41% was observed across the entire cohort, while a response rate of 88% was observed in patients with elevated LDH. A follow-up clinical trial is underway to further study this combination in patients with LDH-elevated, BRAFV600-mutant melanoma.Ionizing radiation has been proposed to enhance the efficacy of checkpoint inhibitors. This combinatorial strategy has been shown to be effective in non-small cell lung cancer and other advanced cancer subtypes. To assess the efficacy of this strategy in patients locally advanced rectal cancer (LARC), Bando and colleagues conducted a phase I trial assessing chemotherapy followed by nivolumab and surgery. A pathological complete response (pCR) was observed in 30% and 60% of patients with MSS and MSI-H LARC, respectively. The authors observed elevated PD-L1 levels and CD8+/effector T-reg cell ratio in patients with pCR. Further clinical trials are needed to confirm these results and further establish prognostic biomarkers for this strategy.Tislelizumab is an anti-PD-1 monoclonal antibody specifically designed to minimize binding to Fcγ receptors (FcγR) and subsequent antibody-dependent phagocytosis by macrophages. Song and colleagues conducted an extended three-year follow-up of a phase II clinical trial of tislelizumab in patients with relapsed/refractor classical Hodgkin lymphoma. Tislelizumab remained generally well-tolerated over this extended period. The overall response rate was 87.1%, and the complete response rate was 67.1%, with 3-year progression-free and overall survival rates of 40.8% and 84.8, respectively. Interestingly, FcγRI-positive macrophages did not correlate with CR or PFS. These results are encouraging, warranting further clinical investigation of tislelizumab.

Primary cardiac undifferentiated pleomorphic sarcoma is associated with TP53 mutation during lack of MDM2 amplification, and targeted sequencing analysis reveals potentially actionable targets

Cardiac undifferentiated pleomorphic sarcoma (UPS) is a rare malignancy. Several studies have revealed frequent MDM2, CDK4, PDFGRA, and KIT amplifications and CDKN2A and CDKN2B deletions. Cases lacking the above copy number alterations may harbor alternative driver mutations; however, little is known about such occurrences. This study was conducted to gain further insights into the molecular features of cardiac UPS using targeted sequencing of 560 cancer-related genes, and fluorescence in situ hybridization and immunohistochemistry of MDM2, CDK4, CDKN2A, TP53, and RB1 in 9 cardiac UPS cases. TP53 mutation or CDKN2A deletion was found in cases lacking MDM2 amplification. Further, p53 overexpression was detected in the case with TP53 mutation, while p16 expression was completely lost in the case with CDKN2A homozygous deletion. p16 overexpression was found in cases with MDM2 and CDK4 amplification but without CDKN2A deletion. Immunohistochemistry of MDM2, CDK4, p53, and p16 is expected to be preliminarily used for gene status analysis. As cardiac UPS and intimal sarcomas are merging into a single spectrum, mutation data for 3 cardiac UPS and 9 intimal sarcomas from the literature, as well as data for 5 cardiac UPS in our study were evaluated, and known recurrently mutated cancer driver genes, including PDGFRB, TP53, ALK, PTCH1, RET, ERBB4, JAK3, GATA1, PIK3CG, and RARA, were identified. Several new potentially actionable mutations, including those in RARA, ALK, PTCH1, RET, ROS1, ABL1, and MET, were also found. These findings improve the molecular understanding of this rare malignancy and are expected to provide a basis for developing precision therapeutics for cardiac UPS and intimal sarcomas.