Loss Of 10q Research Articles

Angiomyolipomatous Lesions of the Nasal Cavity (Sinonasal Angioleiomyoma with Adipocytic Differentiation): A Multi-Institutional Immunohistochemical and Molecular Study.

Mesenchymal neoplasms composed of vascular, smooth muscle, and adipocytic components are uncommon in the nasal cavity. While angioleiomyoma (AL) is a smooth muscle tumor in the Head & Neck WHO classification, it is considered of pericytic origin in the Skin as well as Soft Tissue and Bone classifications. For nasal AL with an adipocytic component, the terms AL with adipocytic differentiation and angiomyolipoma (AML) have been applied, among others. AML is a type of perivascular epithelioid cell tumor (PEComa), most often arising in the kidney, sometimes associated with the tuberous sclerosis complex (TSC). It is uncertain whether nasal cavity AML and AL are best considered hamartomas or neoplasms, as their genetics are largely unexplored. We performed a multi-institutional retrospective study of nasal cavity mesenchymal lesions. Patient demographics, clinical histories, and histologic and immunohistochemical findings were collected. DNA and RNA were extracted from formalin-fixed, paraffin-embedded tissue and analyzed by SNP-based chromosomal microarray, targeted RNA fusion sequencing, and whole-exome sequencing. Fifteen lesions (3-42mm) were identified, predominantly in male (87%) patients with a median age of 60. Patients typically presented with obstructive symptoms, and none had a history of TSC. One AL was a recurrence from six years prior; 11 cases showed no recurrence (median 4.7 years, range: 0.88-12.4). Morphologically, 11 AML contained 30-80% smooth muscle, 10-25% vasculature, and 2-60% adipose tissue, while four AL contained 70-80% smooth muscle and 20-30% vasculature. Other histologic observations included ulceration, thrombosis, inflammation, myxoid change, senescent nuclei, and extramedullary hematopoiesis; no well-developed epithelioid cell morphology was identified. Immunohistochemically, all cases were positive for smooth muscle markers (actin, desmin, and/or caldesmon) and negative for melanocytic markers. Molecular analysis revealed loss of 3p and 11q in a single AML. No other known pathogenic copy number or molecular alterations were seen, including in TSC1/2, TFE3, or NOTCH2. Nasal cavity AML lacks morphologic, immunophenotypic, and genetic features of PEComa family AML. The significant histologic overlap between nasal AML and AL without distinguishing molecular features in either entity suggests "sinonasal angioleiomyoma with adipocytic differentiation" may be the most appropriate terminology for hybrid vascular and smooth muscle lesions containing adipocytic components.

Angiomyolipomatous Lesions of the Nasal Cavity (Sinonasal Angioleiomyoma with Adipocytic Differentiation): A Multi-Institutional Immunohistochemical and Molecular Study.

Mesenchymal neoplasms composed of vascular, smooth muscle, and adipocytic components are uncommon in the nasal cavity. While angioleiomyoma (AL) is a smooth muscle tumor in the Head & Neck WHO classification, it is considered of pericytic origin in the Skin as well as Soft Tissue and Bone classifications. For nasal AL with an adipocytic component, the terms AL with adipocytic differentiation and angiomyolipoma (AML) have been applied, among others. AML is a type of perivascular epithelioid cell tumor (PEComa), most often arising in the kidney, sometimes associated with the tuberous sclerosis complex (TSC). It is uncertain whether nasal cavity AML and AL are best considered hamartomas or neoplasms, as their genetics are largely unexplored. We performed a multi-institutional retrospective study of nasal cavity mesenchymal lesions. Patient demographics, clinical histories, and histologic and immunohistochemical findings were collected. DNA and RNA were extracted from formalin-fixed, paraffin-embedded tissue and analyzed by SNP-based chromosomal microarray, targeted RNA fusion sequencing, and whole-exome sequencing. Fifteen lesions (3 to 42 mm) were identified predominantly in male (87%) patients with a median age of 60. Patients typically presented with obstructive symptoms, and none had a history of TSC. One AL was a recurrence from six years prior; 11 cases showed no recurrence (median 4.7 years, range: 0.88-12.4). Morphologically, 11 AMLs contained 30-80% smooth muscle, 10-25% vasculature, and 2-60% adipose tissue, while four ALs contained 70-80% smooth muscle and 20-30% vasculature. Other histologic observations included surface ulceration, vascular thrombosis, chronic inflammation, and myxoid change; no well-developed epithelioid cell morphology was identified. Immunohistochemically, all cases were positive for smooth muscle markers (actin and/or desmin) and negative for melanocytic markers. Molecular analysis revealed loss of 3p and 11q in a single AML. No other known pathogenic copy number or molecular alterations were seen, including in TSC1/2, TFE3, or NOTCH2. Nasal cavity AML lacks morphologic, immunophenotypic, and genetic features of PEComa family AMLs. The significant histologic overlap between nasal AML and AL without distinguishing molecular features in either entity suggests "sinonasal angioleiomyoma with adipocytic differentiation" may be the most appropriate terminology for hybrid vascular and smooth muscle lesions containing adipocytic components.

Long-term follow-up of patients with intermediate-risk neuroblastoma treated with response- and biology-based therapy: A report from the Children's Oncology Group study ANBL0531.

We previously reported excellent three-year overall survival (OS) for patients with newly diagnosed intermediate-risk neuroblastoma treated with a biology- and response-based algorithm on the Children's Oncology Group study ANBL0531. We now present the long-term follow-up results. All patients who met the age, stage, and tumor biology criteria for intermediate-risk neuroblastoma were eligible. Treatment was based on prognostic biomarkers and overall response. Event-free survival (EFS) and OS were estimated by the Kaplan-Meier method. The 10-year EFS and OS for the entire study cohort (n=404) were 82.0% (95% confidence interval (CI), 77.2%-86.9%) and 94.7% (95% CI, 91.8%-97.5%), respectively. International Neuroblastoma Staging System stage 4 patients (n=133) had inferior OS compared with non-stage 4 patients (n=271; 10-year OS: 90.8% [95% CI, 84.5%-97.0%] vs 96.6% [95% CI, 93.9%-99.4%], p=.02). Infants with stage 4 tumors with ≥1 unfavorable biological feature (n=47) had inferior EFS compared with those with favorable biology (n=61; 10-year EFS: 66.8% [95% CI, 50.4%-83.3%] vs 86.9% [95% CI, 76.0%-97.8%], p=.02); OS did not differ (10-year OS: 84.4% [95% CI, 71.8%-97.0%] vs 95.0% [95% CI, 87.7%-100.0%], p=.08). Inferior EFS but not OS was observed among patients with tumors with (n=26) versus without (n=314) 11q loss of heterozygosity (10-year EFS: 68.4% [95% CI, 44.5%-92.2%] vs 83.9% [95% CI, 78.7%-89.2%], p=.03; 10-year OS: 88.0% [95% CI, 72.0%-100.0%] vs 95.7% [95% CI, 92.8%-98.6%], p=.09). The ANBL0531 trial treatment algorithm resulted in excellent long-term survival. More effective treatments are needed for subsets of patients with unfavorable biology tumors.

Revised Diagnosis From Histiocytic Neoplasm to Optic Chiasm Glioblastoma After Genetic Analysis.

A 46-year-old man presented with left eye blurring. Automated visual field testing showed an incongruous right hemianopia, with sparing of the lower temporal quadrant in the right eye. MRI revealed foci of gadolinium enhancement in the optic chiasm and optic tracts. Serologic testing (including myelin oligodendrocyte glycoprotein and neuromyelitis optica antibodies) and cerebrospinal fluid analysis were negative. Whole-body PET/CT scan found no malignancy. Biopsy of the optic chiasm revealed a moderately cellular neoplasm composed of atypical, discohesive cells with enlarged nuclei, prominent eosinophilic nucleoli, and abundant vacuolated cytoplasm. Immunohistochemical stains for CD68 and S100 were positive, whereas those for GFAP, OLIG2, SOX10, and multiple others were negative, supporting a diagnosis of histiocytic neoplasm. Five weeks later, results became available from next-generation sequencing targeting the coding regions of hundreds of malignancy-associated genes and select introns. Alterations associated with histiocytic neoplasms (i.e. BRAF and MAP2K1 mutations) were absent. However, there was a nonsense mutation in the PTEN gene, a hotspot mutation in the TERT gene promotor, and focal amplifications of the CDK4 and MDM2 genes. Additionally, there was chromosome 6q loss, 7 gain, and 10q loss. Based on these findings, the diagnosis was revised to glioblastoma, IDH-wildtype, CNS WHO grade 4. The patient began treatment with temozolomide while continuing radiation therapy. This case illustrates how next-generation sequencing can at times provide more accurate diagnostic information than standard tissue histopathology.

EPEN-08. MULTI-OMICS PROFILES REVEAL UNIQUE DEFINING MOLECULAR FEATURES OF POSTERIOR FOSSA EPENDYMOMA SUBTYPE A FROM INFANTS

Abstract BACKGROUND Ependymoma is a heterogeneous brain cancer with multiple subgroups based on locations and molecular profiles. Tumours of the methylation class, posterior fossa group A (PF-EPN-A) represent the most common and aggressive neoplasms and have a poor outcome. PF-EPN-A tumours in infants (under three years of age) are understudied; this project aimed to characterise these infant tumours. METHODS We collected DNA methylation (n=570) and gene expression (n=56) profiles of PF-EPN-A tumours, and single-cell RNA sequencing data of human fetal cerebellum from published studies. RESULTS Analysis of copy number profiles derived from DNA methylation arrays showed that infants with PF-EPN-A have relatively quiescent genomes when compared to children aged four to eighteen years (mean fraction of changes in infants (n=300) = 0.018 and children (n=270) = 0.055; p-value < 2.2 × 10-16). No recurrent focal amplifications or losses were detected, however, copy number loss involving chromosome arms, 12q (12%), 7q (9.7%), 5p (8.3%), and 16p (6%) were relatively common in infants, while 1q gain, 6q loss, and 22q loss were more frequent in children. When we stratified PF-EPN-A based on copy number alterations, infants with altered genomes were associated with poor outcomes compared to infants with stable genomes (fraction of changes = 0), which in turn showed poor overall survival compared to children with stable genomes. Gene set enrichment analysis using data from microarrays showed that infant tumours were specifically enriched with genes involved in the extracellular matrix organization, interferon gamma signaling, neuronal system, autophagy, and signaling by Interleukins, VEGF and PDGF. Integration with single-cell data from the human fetal cerebellum revealed that infants in PF-EPN-A had significantly higher proportions of glial progenitor cells and decreased granule cell progenitor subpopulations. CONCLUSIONS Together, our study identifies distinct genetic and transcriptional programs among infants in PF-EPN-A tumours, representing opportunities to refine future treatment.

MDB-63. AN INTERNATIONAL META-ANALYSIS OF SHH MEDULLOBLASTOMA SUBTYPES DEFINES A CLINICALLY SIGNIFICANT HIGH-RISK VARIANT OF SHH-SUBTYPE 3

Abstract BACKGROUND The distinction of MBSHH into four methylation-dependent subgroups was recognized by the WHO in 2021. However, these subgroups have not previously been defined by international experimental consensus and their clinico-molecular features and behavior have not formally been investigated in large cohorts. We aimed to robustly identify SHH subgroups through analysis of MBSHH with DNA-methylation profiling. METHODS A cohort of 683 MBSHH, confidently classified using the Heidelberg classifier v12.5, was assembled for analysis from multiple international studies. To define subgroups, we applied consensus sampling-based clustering approaches to tumor methylomes, including assessment of confidence in class-definition and inter-technique concordance. The clinico-molecular features of consensus subgroups were investigated. RESULTS Lowest complexity analysis supported the division of MBSHH into the 4 WHO-subtypes. SHH-1 and SHH-2 were associated with lower age and infrequent mutations of PTCH1/SUFU; SHH-2 was distinguished from SHH-1 by enriched MBEN histology, absence of chromosome 2 gain, less frequent metastasis and more favorable overall-survival. SHH-4 presented in older children and adults (3-57; median 24 years), and was primarily defined by mutations in U1-snRNA (67/72). Consensus analyses supported the division of SHH-subgroup-3 into three. Importantly, the SHH-3C subtype was associated with a coalescence of high-risk features (LCA histology, TP53mut, MYCNamp/GLI2amp) alongside 3p, 10q and 17p loss, and had dismal survival. The remaining SHH-3A/B subtypes were characterized by 9q loss, frequent focal amplifications of TERT and PPM1D, and equivalent better survival. CONCLUSIONS This study affirms the distinction of MBSHH into 4 major subgroups. For infant disease, SHH-2 is a favorable-risk marker. For childhood-MBSHH, the SHH-3C subtype provides a molecular definition of high-risk that subsumes other previously-defined high-risk disease markers (LCA, MYCNamp, TP53mut) into a unified high-risk disease group. The other SHH-3 subtypes behave similarly and are favorable-risk. These subtypes have potential to enhance molecularly-guided risk-stratification in routine diagnostics, to improve patient outcomes and quality-of-life.

EPEN-21. EXPLORATION OF TUMOR SUPPRESSOR LATS1 LOSS IN POSTERIOR FOSSA GROUP A EPENDYMOMA WITH LOSS OF CHROMOSOME 6Q

Abstract INTRODUCTION Chromosome 6q loss (6q-) has recently been identified as an ultra-high-risk factor in posterior fossa group A ependymoma (PFA). This study seeks to pinpoint the biological mechanism underlying the aggressive biology of PFA 6q-. We hypothesize that loss of chromosome 6q gene large tumor suppressor kinase 1 (LATS1) in PFA 6q- results in increased Hippo pathway activity that drives malignant tumor progression. LATS1 is a key negative regulator of the Hippo pathway which may represent a novel therapeutic vulnerability in 6q- PFA. METHODS We are testing this hypothesis using genomic and transcriptomic analysis of PFA patient samples, and by gene knock-in and pharmaceutical interventions of LATS1 and Hippo pathway components in PFA 6q- models. RESULTS Tumor suppressor LATS1 shows reduced expression in PFA 6q- patient samples. Although complete loss of the entire 6q arm is common in PFA 6q-, partial loss of chromosome 6q is also observed, and in all cases this partial loss encompasses the LATS1 locus on chromosome 6q. We used lentiviral transduction to knock-in LATS1 in PFA 6q- cell lines to determine the effect on tumor growth. This demonstrated loss of viability that was specific for 6q- PFA cell lines versus 6q WT control cell lines. DISCUSSION Data point to LATS1 loss being a key driver of the aggressive phenotype of PFA6q-, implicating the Hippo pathway in PFA 6q- and potential for novel therapeutic strategies.

EPEN-14. MOLECULAR MARKERS AND PREDICTORS OF OUTCOME FOR PAEDIATRIC INTRACRANIAL EPENDYMOMA IN THE PROSPECTIVE, MULTICENTRE E-HIT2000 TRIAL AND SUBSEQUENT HIT-REGISTRIES: A POOLED ANALYSIS OF 244 PATIENTS

Abstract BACKGROUND Throughout the past decade, molecular tools have revolutionised ependymoma classification, yet their application has been limited in clinical trial cohorts. METHODS We present the molecular analysis of paediatric ependymoma patients from the E-HIT2000 trial (n = 165), or subsequent HIT2000-interim and I-HIT-MED registries (n = 79) based on DNA-methylation profiling with multi-level classification using the Heidelberg Brain Tumor Classifier v12.5 and the analysis of chromosome 1q, 6q, and CDKN2A copy number status. Prior to further analysis, cases without ependymoma group prediction were excluded (n = 16/244). RESULTS 5-year PFS and OS rates varied by group: EPN-PFA (n=146/228) 45±4% and 76±4%; EPN-PFB (n=19/228) 90±7% and 100%; EPN-ZFTA (n=59/228) 63±7% and 87±5%; and EPN-YAP1 (n=4/228) 50±25% and 100%. EPN-PFA was subclassified into nine subtypes. Poor median PFS was observed for PFA1c (n=22, 5-yr-PFS 32±10%/5-yr-OS 77±9%), PFA1d (n=13, 35±14%/72±14%), PFA1e (n=15, 47±13%/59±13%), and PFA2a (n=16, 27±12%/50±14%). Presence of 1q gain was associated with inferior survival (n = 39/146, 29±8%/65±8%). 6q loss impacted PFS, but not OS (n=8, 25±14%/100%). Among 59 EPN-ZFTA cases, 46 clustered with EPN ZFTA-RELA fused, and 12 clustered within recently described subgroups of EPN-ZFTA (EPN-ZFTA alt., Cluster 1: 1/12, Cluster 2: 11/12). Strikingly, 6/11 EPN ZFTA-alt. (Cluster 2) cases showed infratentorial (4/6) or supratentorial midline location (2/6). Homozygous deletion of CDKN2A (CDKN2A-/-; n=8) was only observed in EPN ZFTA-RELA. Presence of EPN ZFTA-alt. (cluster 2) (5-yr-PFS: 36±15%; p<0.001), and CDKN2A-/- (19±16%; p<0.0001) was associated with inferior PFS compared to EPN ZFTA-RELA CDKN2A-wt (81±6%). OS was only impacted for patients with EPN ZFTA-RELA CDKN2A-/- (5-yr-OS: 38±20%, p>0.0001). CONCLUSIONS We present strong evidence supporting the inclusion of molecular criteria into the next generation of clinical trials for children with ependymoma.

Abstract PO2-20-09: Hiding in plain sight: A heterogeneous response in a patient with metastatic HR+/HER2- breast cancer

Abstract INTRODUCTION: Metastatic breast cancer is an infamously heterogeneous disease—characterized by a range of clinical presentations and treatment responses. In light of this heterogeneity, ASCO guidelines recommend patients with new, accessible metastatic tumors undergo a biopsy to inform treatment decisions. Here, we present a patient with HR+/HER2- disease with a long history of unusually heterogeneous responses—and a repeat metastatic biopsy that provided an answer. CASE: A 55-year-old woman presented for consultation of treatment-refractory metastatic breast cancer. She was originally diagnosed with a right-sided Stage III HR+/HER2- invasive ductal carcinoma (IDC) at the age of 33. Upon staging, she was incidentally found to have a right lower-lobe lung carcinoid tumor that was removed by lobectomy. She underwent a partial mastectomy, axillary lymph node dissection, followed by adjuvant chemo- and radiation therapy. She tolerated one week of tamoxifen which was discontinued due to adverse effects. Fifteen years later, screening mammography revealed a suspicious lesion in the right breast—biopsy consistent with a new IDC. She underwent bilateral mastectomies which showed a pT1cNxM0 G2 IDC with associated DCIS (ER+/PR+/HER2 IHC 2+, FISH-negative, Oncotype RS 12). Genetic testing was negative. The patient trialed adjuvant tamoxifen, which was discontinued due to repeat adverse effects. Two years later, rising tumor markers prompted restaging scans that demonstrated an enlarging liver lesion. Liver biopsy was consistent with metastatic adenocarcinoma of the breast (ER+/PR+/HER2 IHC 0), GATA3 positive. Patient was started on letrozole with lupron and palbociclib with disease control for two years until progression in the bone, prompting a switch of letrozole to fulvestrant with continued palbociclib. Her disease was stable for two additional years until progression of multiple liver lesions. She was then started on single-agent paclitaxel. At this point, she was seen in our office for consultation and next-line therapy options. Repeat staging scans showed heterogeneous response in the liver with overall progression. ctDNA testing revealed a mutation in MEN1 (p.Q354*, 2.8% VAF) without other actionable alterations. Patient opted to enroll into a clinical trial of an experimental ER-antagonist but experienced progression of her liver lesions on first staging scans. A second liver biopsy was performed on a progressive lesion which showed ER-/PR-/HER2- disease. She opted to enroll into a Phase 1b/2 trial with sacituzumab govitecan-hziy in combination with a PARPi. Staging scans again showed a mixed response in the liver. Given a history of mixed responses and discordant receptor staining, the differential diagnosis included loss of hormone receptor expression in the breast carcinoma versus metastases from an occult neoplasm. Additional immunohistochemical studies were performed on a subsequent liver biopsy showing positivity for chromogranin, synaptophysin, and INSM1, and negative results for ER, PR, GATA3, and TRPS1. Next-generation sequencing of the progressive lesion revealed a MEN1 SNV (p.Q354*, VAF 82%) as well as loss of 11q (MEN1 locus) suggestive of biallelic loss of MEN1—providing molecular evidence of a metastatic carcinoid tumor. A history of mixed response in the liver was concluded to be two coexisting neoplasms—a metastatic HR+/HER2- breast cancer as well as a late recurrence of a right-lower lobe carcinoid tumor from 22 years prior. DISCUSSION: The unanticipated discovery of an occult, coexisting neoplasm in the setting of a metastatic HR+/HER2- breast cancer illustrates the value of meticulous pathological review, repeat molecular testing and the use of next-generation diagnostic tools such as tumor sequencing in the advanced setting—particularly when a clinical story defies conventional expectations—to personalize care and optimize oncologic management in the face of complex biology. Citation Format: Nolan Priedigkeit, Jane Brock, Olivia Cunningham, Molly Skeffington, Melissa Hughes, Nancy Lin, Susan Lester, Heather Parsons. Hiding in plain sight: A heterogeneous response in a patient with metastatic HR+/HER2- breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-20-09.

SALL3 mediates the loss of neuroectodermal differentiation potential in human embryonic stem cells with chromosome 18q loss

Human pluripotent stem cell (hPSC) cultures are prone to genetic drift, because cells that have acquired specific genetic abnormalities experience a selective advantage invitro. These abnormalities are highly recurrent in hPSC lines worldwide, but their functional consequences in differentiating cells are scarcely described. In this work, we show that the loss of chromosome 18q impairs neuroectoderm commitment and that downregulation of SALL3, a gene located in the common 18q loss region, is responsible for this failed neuroectodermal differentiation. Knockdown of SALL3 in control lines impaired differentiation in a manner similar to the loss of 18q, and transgenic overexpression of SALL3 in hESCs with 18q loss rescued the differentiation capacity of the cells. Finally, we show that loss of 18q and downregulation of SALL3 leads to changes in the expression of genes involved in pathways regulating pluripotency and differentiation, suggesting that these cells are in an altered state of pluripotency.

Abstract 6944: Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer

Abstract Intratumor heterogeneity (ITH) is a substrate for tumor evolution and confers amenability to stressors and therapies. Sources of ITH include subclonal mutations, microenvironmental factors, and plasticity whereby a cell has the ability to adopt different cell states. Clear cell kidney cancer (ccRCC) has a well-characterized genetic initiating event - VHL inactivation - resulting in activation of HIF-1 and HIF-2 transcription factors. Although VHL inactivation is consistently clonal in nature, ccRCC nonetheless exhibits extensive genetic and morphological ITH. This makes ccRCC an excellent model in which to study ITH. We performed single cell RNA-seq on multiregional samples from kidney tumors and macroscopically normal kidney tissue. We obtained 55 samples from 16 tumors and used the 10x Genomics platform to obtain gene expression data from ~150,000 single cells, including ~80,000 cancer and normal epithelial cells. We identified three cancer cell states, co-existing in all clear cell kidney cancer tumors analyzed and marked by different gene expression programs: 1) differentiated-like cells marked by proximal tubule cell genes characteristic of the cancer cell of origin; 2) epithelial-mesenchymal transition (EMT)-like cells and 3) injured-like cells. Differentiated markers and EMT markers were associated with good and poor prognosis, respectively, indicating that these cell subpopulations are clinically important. Notably, these cell states are also present in the normal proximal tubule although in different proportions to the tumor samples, which contain more cells in the EMT-like and injured-like states. This suggests that the core features of ITH are inherent to the cell of origin of the cancer, but that the cell state trajectories are dysregulated in the tumor. Furthermore, in tumors these states exist as a continuum, whereby transitional cells in between states appear to exist, suggesting that cancer cells are continually and dynamically transitioning between states. Lastly, we used chromosomal copy number variation (CNV) patterns to study genetic ITH. We examined the effect of 14q loss, which is associated with ccRCC metastasis, on gene expression. We found that 14q loss is associated with downregulation of HIF-1 transcriptional activity together with a reciprocal increase in HIF-2 activity, MYC activity and oxidative phosphorylation, highlighting potential mechanisms by which 14q loss may driver ccRCC metastasis. Citation Format: Olivia Lombardi, Ran Li, Lisa Browning, Faiz Jabbar, Hannah Evans, Peter J. Ratcliffe, David R. Mole. Transcriptional programs underpinning reoccurring cancer cell states, plasticity and tumour phylogenies in clear cell kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6944.

Abstract 7564: DNA methylation profiling identifies two distinct groups of oligosarcoma, IDH-mutant with distinct genetic alterations and clinical outcomes

Abstract DNA methylation has been extensively adopted for the classification of brain tumors and is endorsed by current neuropathology guidelines. Recently, oligosarcoma, IDH-mutant has been characterized as a distinct group of IDH mutant gliomas by DNA methylation classification. However, emerging studies revealed the variety in this group. Here, we employed DNA methylation-based t-SNE analysis to characterize oligosarcomas identified by the dkfz brain tumor classifier (v12.5). Thereby, we uncovered that oligosarcomas were segregated into two distinct groups, namely, subgroup A and subgroup B, which were further validated by genetic alterations and clinical outcomes. Next-generation Sequencing revealed subtype A tumors enriched with chr 1p/19q co-deletion, chr 6q loss, CDKN2A/B homogeneous deletion, TERT promoter mutation, and NF1 mutation, while subtype B tumors showed chr7 gaining and TP53 mutation. RNA sequencing and gene set enrichment analysis (GSEA) revealed that neuronal-tumor interaction and muscular progression pathways were more enriched in subtype A tumors. In contrast, subtype B tumors showed enrichment for angiogenesis and mTOR pathways. Additionally, inter-chromosomal fusion genes were more frequently found in subtype A tumors. Clinically, patients with subgroup A oligosarcomas manifested poorer survival and distinct imaging features in magnetic resonance imaging (MRI) compared to those with subgroup B. Collectively, we provided more cases of oligosarcoma, IDH-mutant, confirming the existence of this newly identified group, and revealed the subgroups harboring different genetic alterations and suffering different prognoses, providing new evidence for the molecular pathology in diffuse glioma. Citation Format: Kenan Zhang, Lingyu Liu, Xing Liu, Tao Jiang. DNA methylation profiling identifies two distinct groups of oligosarcoma, IDH-mutant with distinct genetic alterations and clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7564.

Chondrolipoma of the Breast: A Myofibroblastoma Variant or a Distinct Lesion?

The entity commonly referred to as chondrolipoma is a rare and enigmatic breast lesion with unclear histogenesis and a complete lack of molecular characterization. It is uncertain whether it represents a hamartoma, choristoma, or a distinct neoplasm, including possibly a variant of mammary-type myofibroblastoma. We report two additional chondrolipomatous lesions of the breast. The lesions had varying histologic and immunohistochemical features similar to myofibroblastoma, including the loss of retinoblastoma (Rb) protein expression in one lesion. Molecular analysis by chromosomal microarray analysis performed on a second lesion did not demonstrate a loss of 13q14 or 16q typical of myofibroblastoma. Our findings further support the concept that at least a subset of breast lesions that historically have been classified as chondrolipoma are related to myofibroblastoma. However, the lack of myofibroblastoma-specific molecular alterations in one lesion suggests chondrolipomas may also have varying origins.

Proteogenomic characterization of small cell lung cancer identifies biological insights and subtype-specific therapeutic strategies

We performed comprehensive proteogenomic characterization of small cell lung cancer (SCLC) using paired tumors and adjacent lung tissues from 112 treatment-naive patients who underwent surgical resection. Integrated multi-omics analysis illustrated cancer biology downstream of genetic aberrations and highlighted oncogenic roles of FAT1 mutation, RB1 deletion, and chromosome 5q loss. Two prognostic biomarkers, HMGB3 and CASP10, were identified. Overexpression of HMGB3 promoted SCLC cell migration via transcriptional regulation of cell junction-related genes. Immune landscape characterization revealed an association between ZFHX3 mutation and high immune infiltration and underscored a potential immunosuppressive role of elevated DNA damage response activity via inhibition of the cGAS-STING pathway. Multi-omics clustering identified four subtypes with subtype-specific therapeutic vulnerabilities. Cell line and patient-derived xenograft-based drug tests validated the specific therapeutic responses predicted by multi-omics subtyping. This study provides a valuable resource as well as insights to better understand SCLC biology and improve clinical practice.

Genomic and epigenomic integrative subtypes of renal cell carcinoma in a Japanese cohort

Renal cell carcinoma (RCC) comprises several histological types characterised by different genomic and epigenomic aberrations; however, the molecular pathogenesis of each type still requires further exploration. We perform whole-genome sequencing of 128 Japanese RCC cases of different histology to elucidate the significant somatic alterations and mutagenesis processes. We also perform transcriptomic and epigenomic sequencing to identify distinguishing features, including assay for transposase-accessible chromatin sequencing (ATAC-seq) and methyl sequencing. Genomic analysis reveals that the mutational signature differs among the histological types, suggesting that different carcinogenic factors drive each histology. From the ATAC-seq results, master transcription factors are identified for each histology. Furthermore, clear cell RCC is classified into three epi-subtypes, one of which expresses highly immune checkpoint molecules with frequent loss of chromosome 14q. These genomic and epigenomic features may lead to the development of effective therapeutic strategies for RCC.

Prognostic Value of Copy Number Aberrations and Copy Neutral Loss of Heterozygosity in Acute Myeloid Leukemia

Background: Chromosomal abnormalities are key to the diagnosis and prognosis of patients with acute myeloid leukemia (AML). Chromosomal genomic array testing (CGAT) can identify copy neutral loss of heterozygosity (cnLOH) and genomic copy number alterations (CNAs) that are undetected by conventional karyotype or fluorescence in situ hybridization (FISH). Previous studies have shown that cnLOH is correlated with a shorter duration of complete remission (CR) and worse overall survival (OS) in AML (Grosneth et. al, Cancer, 2015). The primary objective of this exploratory study is to examine the predictive power of CGAT in a cohort of AML patients with majority normal karyotype. Methods: CGAT analyses were collected from 187 subjects with AML or other high grade myeloid neoplasm (myeloid blasts >10% in peripheral blood or bone marrow) between 2012 and 2022 at Fred Hutchinson Cancer Center. CGAT utilized CytoScan™ HD Array (ThermoFisher, MA), targeting genome-wide region with 2.4 million markers for copy number and approximately 750,000 genotype-able SNPs. The data were analyzed using the Nexus Copy Number software with manual curation. Based on the assay validation in our CLIA-certified diagnostic lab, the cutoff is 100 Kb for copy number aberrations and 10 Mb for cnLOH. Survival predictive power analysis (SPP), a log-rank based test using Nexus, was applied to identify aberrations significant for event free survival (EFS) and overall survival (OS). SPP analysis tabulated genomic segments based on p-values with the alpha value cutoff at 0.05. Using frequency as the filtering criteria (present in >5 patients), 132 SPP for EFS, and 73 SPP for OS were identified. SPP were then manually curated to (1) remove those representing constitutional absence of heterozygosity (AOH) and those overlapping with benign copy number variance and (2) connect adjacent SPP into contiguous regions. The presence and absence of the identified SPP were then tabulated, cleaned up, and used to generate Kaplan-Meier curves for EFS and OS. This process identified 5 SPP that are significant for EFS and 2 SPP for OS. A multivariate analysis was performed to determine the hazard ratio (HR) of these 5 genetic aberrations for EFS and OS, adjusted for patient age, secondary/treatment related AML, ELN 2022 risk, and receipt of allogeneic hematopoietic cell transplantation (HCT) as a time-varying covariate. Results: In our 187 subject cohort, the median age was 63 (range 20-87). 76 (41%) had secondary or treatment related disease. 97 (52%) went on to receive a hematopoietic stem cell transplant. 3 had favorable risk disease, 14 had adverse risk, and 170 had intermediate risk by karyotype and FISH using ELN 2022 criteria. 120 (64%) had normal karyotype. 5 subjects had 4q cnLOH, 6 had 5q ( EGR1) loss, 7 had 9p cnLOH, 10 had mixed-lineage leukemia gene partial tandem duplication ( MLL-PTD), and 4 had 7q deletion. Univariate analysis identified five SPP that are significant for EFS: 4q cnLOH (p=0.002), 5q ( EGR1) loss (p<0.001), 9p cnLOH (p<0.001), MLL-PTD (p<0.001), and 7q deletion (p<0.001). 2 SPP were significant for OS: 9p cnLOH (p=0.001) and MLL-PTD (p=0.036). Multivariate analysis for EFS adjusted for age, secondary/treatment related disease, ELN 2022 risk, and receipt of HCT as a time-varying covariate demonstrated a hazard ratio (HR) of 3.5 for 4q cnLOH (p=0.008), 2.3 for 5q ( EGR1) loss (p=0.16), 4.5 for 7q deletion (p=0.007), 5.7 for 9p cnLOH (p<0.001), and 2.9 for MLL-PTD (p=0.002). Multivariate analysis for OS adjusted for age, secondary/treatment related disease, ELN 2022 risk, and receipt of HCT as a time-varying covariate demonstrated a hazard ratio (HR) of 2.0 for 4q cnLOH (p=0.23), 0.5 for 5q ( EGR1) loss (p=0.36), 1.3 for 7q deletion (p=0.66), 4.9 for 9p cnLOH (p=0.001), and 2.3 for MLL-PTD (p=0.04). Discussion: This study has found 5 key genetic changes identifiable via CGAT that are significant for shorter EFS and/or OS in AML patients. Our next step is to validate these findings in a large independent cohort of AML patients. CGAT is a powerful adjunct to conventional karyotype and FISH for the detection of cnLOH and can be used to identify CNAs below the level of detection of karyotype. For these reasons, CGAT may be most useful in intermediate risk patients. Further research is warranted to fully understand the prognostic power of CGAT in AML.

Multiplex Detection of Copy Number Alterations in Multiple Myeloma Including Emerging Therapeutic Targets By Digitalmlpa

Recent advances in immunotherapy have highlighted that in multiple myeloma (MM), besides risk-determining copy number alterations (CNAs) there is a need for investigation of CNAs in CAR-T cell therapy-related targets (e.g. BCMA, GPRC5D, FcRH5). DigitalMLPA technology has been successfully used for characterization of recurrent CNAs on chromosome arms 1p, 1q, 13q and 17p in newly diagnosed and relapsed MM cases, as well as in cancer cell lines of MM origin. Adding probes aimed at CAR-T cell therapy-related target genes complements the already included predictive target probes ( IKZF1/3, IRF4, MYC, RPL5, SLAMF7 and BRAF V600E point mutation) in SALSA® digitalMLPA™ Probemix D006 Multiple Myeloma. In addition, due to a large overlap of affected genomic regions between hematologic neoplasms, we aimed to demonstrate usefulness of this multiplex assay not only on cell lines of MM origin, but also of myeloid and lymphoid lineages. Methods. D006 Multiple Myeloma digitalMLPA probemix contains (i) probes targeting chromosomal arms with recurrent CNAs, including 1p (33 probes), 1q (29 probes), 13q (23 probes), 17p (20 probes); (ii) probes for promising predictive targets such as BCMA, CRBN, GPRC5D, FcRH5, IKZF1/3, IRF4, MYC, RPL5, SLAMF7 - 2-4 probes per gene, and BRAF V600E mutation-specific probe; (iii) 278 probes for subtelomeric, pericentromeric and middle regions of chromosomal arms for all chromosomes; and (iv) control probes for quality control and troubleshooting. DigitalMLPA reactions were performed with 20-40 ng DNA from 29 cancer cell lines of lymphoid and myeloid lineage, 16 of which were of MM origin. Commercial blood-derived genomic DNA samples from healthy individuals were used as references for data normalization using data analysis software Coffalyser digitalMLPA™. Results. Losses of 1p were among the most frequent CNAs in MM cell lines (n=13) and were mainly local (1p21.1-p22.1), including homozygous deletion of CDKN2C in MM cell lines co-occurring with TENT5C subclonal loss in 2 of those. 13q loss (encompassing predominantly the whole arm) was detected in 13 MM cell lines. 1q and 17p losses were present in 12 MM cell lines. One MM cell line showed loss of BCMA (LOPRA-1) and one of CRBN (EJM), 6 had loss of GPRC5D, 9 had MYC gain. None of the MM cell lines carried the BRAF V600E mutation. In the EOL-1 myeloid lineage cell line, trisomy of chromosomes 4, 6, 8 and 19 were correctly identified by increased ratios for all probes covering these chromosomes: 24, 27, 20 and 11 probes respectively; and in the HG-3 lymphoid lineage cell line 11q25 gain and 13q loss was detected. Examples of CNAs detected by D006 Multiple Myeloma probemix are described in Table 1. Copy number ratios of predictive target genes in MM cell lines are shown in Table 2. Ratios between 0.7-1.3 (green cells) indicate normal copy number, ratios below 0.7-1.3 range indicate copy number loss (red cells) and higher ratios indicate gains (blue cells). CNAs detected by digitalMLPA were highly concordant with those reported in public databases. Conclusions. digitalMLPA technology is well suited for multiplex CNA detection of routinely analyzed genomic regions in MM and provides a unique opportunity to research molecular genetic markers of emerging significance in MM in the same reaction. In addition, this MM assay also gives the possibility to analyze gross CNAs in other hematopoietic neoplasms with overlapping affected regions. Input DNA requirements that are a fraction of current WGS, simple protocol and possibility of combining of digitalMLPA libraries with other NGS libraries make D006 Multiple Myeloma digitalMLPA probemix a reliable, cost-effective and robust method to detect well-established and emerging CNAs.

Beyond the Primary: Unveiling the Prognostic Value of Secondary Cytogenetic Abnormalities in Multiple Myeloma

Introduction Fluorescent In-Situ Hybridization (FISH) has been an indispensable tool to characterize the cytogenetic landscape of multiple myeloma (MM), mainly for identifying primary abnormalities such as heavy chain gene (IgH) translocations and odd-number chromosome trisomies. These aberrations play a crucial role in risk stratifying the disease given their correlation with disease aggressiveness, treatment response and overall prognosis. High-risk (HR) cytogenetics are identified through the presence of t(14;16), t(14;20) or loss of the p53 gene locus. Less is known about the prognostic implication of secondary abnormalities, in particular gain of 1q21. We aimed to evaluate the association between primary and secondary cytogenetic abnormalities and their impact on overall survival (OS) in MM patients. Methods We performed a retrospective analysis on a FISH records database from three Mayo Clinic campuses. Cohort included patients >18 years of age, diagnosed with multiple myeloma between 2005 and 2022, with FISH performed within two years of diagnosis. Demographics including age, date of diagnoses and OS data were retrieved from electronic medical records. Statistical analyses were done using BlueSky Statistics software. Study was conducted in accordance with the principles of the Declaration of Helsinki; informed consent was obtained from all participants. Results We analyzed a total of 2221 patients, among which 59.8% were male, with a median age of diagnosis 65 years. Hyperdiploidy was the most prevalent primary cytogenetic abnormality observed in 44.3% of patients, followed by t(11;14) as the most common translocation (23%), and t(4;14) (8.2%). Gain(1q21) was assessed in 925 patients, among which 64%, 30.3% and 5.7% had 2, 3 and ≥4 copies respectively. Furthermore, 11.4% (n=253) exhibited either mutated TP53 or del17p, and 37.8% (n=839) presented with 13q loss or monosomy 13. The median follow-up for the overall population was 102 months (8.5 years), with a median OS of 75.5 months. Patients with high-risk translocations had significantly shorter median OS of 40.01 months compared to 80.65 months for those with standard risk translocations (p <0.001). The presence of ≥4 1q copies was associated with significantly reduced overall survival (2 copies: not reached, 3 copies: 69.4 months, ≥4 copies: 40.1774; p <0.001). This was consistent even when considering the presence of high-risk translocations (Figure 1). To confirm the independent significance of these findings, we performed multivariate Cox regression models, incorporating age at diagnosis, year of diagnosis, gender, presence of hyperdiploid disease, and presence of high-risk translocations. Given that overall survival outcomes were analyzed irrespective of treatment received, we incorporated year of diagnosis as a variable to control for potential impact of evolving treatment regimens over the years. The presence of ≥4 1q copies was associated with reduced OS, with a hazard ratio (HR) of 2.12 (CI: 1.38-3.26; p <0.001). The presence of 3 copies also showed prognostic significance (HR: 1.43; p: 0.012). Similarly, both the presence of TP53mut/mon17 (HR: 1.96, CI: 1.66-2.31; p <0.001) and 13qdel/mon13 (HR: 1.21, CI: 1.08-1.38) were associated with decreased OS. On the other hand, 1p deletion did not have any significant prognostic implications (HR: 1.84, CI: 0.38-1.59; p 0.113). When incorporating all cytogenetic events into a multivariate model, variables associated with reduced OS included age at diagnosis, the presence of both of 3 and ≥4 1q copies, TP53mut/mon17, t(4;14), t(14;16) and t(14;20). However, 13qDel/mon13 lost significance when incorporated with other abnormalities (Table 1). Conclusion Our investigation sheds light on the less explored realm of secondary cytogenetic abnormalities and confirm a strong association between increasing number of 1q copies and reduced overall survival, independent of other cytogenetic abnormalities and treatment regimens. These results underscore the utility of FISH-based comprehensive cytogenetic profiling at baseline, which enables reliable identification of high-risk patients. Future prospective trials should further elucidate the pathogenic mechanism for these findings. Our analysis used OS as primary endpoint and included over 2000 MM patients, reinforcing the clinical relevance of our findings.

Chromoanagenesis in Haematological Malignancy: Review of Samples from Patients with Acute Leukemia and MDS

Background: Complex chromosome rearrangements (CCRs) include a variety of structural aberrations grouped as chromothripsis, chromoanasynthesis and chromoplexy. Although the underlying mechanisms for these phenomena are unknown and likely to be different, they appear to originate from a single event. While chromothripsis and chromoanasynthesis affect limited genome sites their role and genome associations in haematological malignancy remain to be elucidated. Aims: This study aimed to provide clarity on the location, incidence and association with TP53 gene profile of chromothripsis (cth) and chromoanasynthesis (cha) events in MDS/AML. Methods: We analysed data from routine investigations carried out by haematological malignancy diagnostic service (HMDS) at University College London Hospital (UCLH) on 2,506 samples of acute myeloid leukaemia (AML) and myelodysplasia (MDS), including G-banding, FISH (Cytocell), chromosome microarray analysis (CMA, Agilent) and target NGS(Illumina). Analysis of 87 samples with complex bone marrow karyotypes were reported following the ISCN 2020 as: (i) cth(alternating disomy and heterozygous loss along a chromosome or chromosomal segment) or (ii) cha (deletions and one or two copy number gains in a single chromosome or chromosome region including copy number variants without the clustered breakpoints of chromothripsis). Statistical analysis used RStudio (v 1.4.1106) to carry Pearson's chi-squared (χ²) and Fisher's exact (2-sided) tests. Results: Complex karyotypes harbouring cth and cha type aberrations were detected in 65 out of 727 AML and 22 out of 1779 MDS samples. The cth events were seen alongside cha in 67% of cases, rarely presenting as a sole abnormality (2%), while cha alone was found in 31% of samples. Most frequently cth was mapped at chromosome 21 (17%), chromosome 7 (15%), chromosome 17 and 19 (13%) and chromosome 5 (11%) (Fig.1a). In this cohort of 87cases, chromothripsis was found to be associated with TP53 deletions ( TP53Del/WT) in 7 (7%) and with TP53 mutations ( TP53Dpl/mut) in 22 (25%), while concurrent TP53 deletions and mutations ( TP53Del/mut) were detected in 29 (33%) ( p = 0.0005). Samples with TP53Del/mut harboured cth in chromosomes 7 (7%), 17 (6%), 8, 9, 19 and 21 (5%) whilst cases with TP53Dpl/mut had cth in chromosomes 21 (9%), 19 (8%), 5 (7%) and 7 (5%). (Fig 1 b). The cha aberrations generally followed the genome location of cth events with some exception. Firstly, chromosomes X,13 and 21 appear to have only cth and is seen at slightly higher level on chromosome 19 (cth 13% vs cha 9%). Secondly, cha was most frequently seen on 5q (61 %), chromosome 17 (40%) and 7q (34%). However, the same chromosomes also had the greatest number of cases presenting with concurrent cth and cha, seen in 63/87 (72%) for chromosome 5, 46/87 (53%) for chromosome 17 and 43/87 (49%) for chromosome 7. Importantly, only cases harbouring 5q deletions showed a statistically significant association with TP53 status ( p = 0.0011) in this cohort irrespective of cth and cha presence. Notably, 24/29 cases (83%) with TP53Del/mut were cth positive and had 5q31 deletion. This finding is an important observation but not in keeping with reports of exclusive association of TP53 mutations with concurrent 5q and 7q loss (1, 2 and 3). The concurrent recording of cth and cha performed by this study could explain the discrepancy, most likely due to high frequency of cha events in 5q. Summary This study highlights issues pertinent to risk stratification in routine diagnostic settings: (i) MDS/AML cases with complex genome harbouring either or both cth and cha share common TP53 aberrations (del/mut) and (ii) deletion of 5q31 is positively associated with TP53 aberrations and presence of cth events. (iii) CMA offers a comprehensive, sensitive, fast and financially superior way to detect cth, cha events along with genome complexity compared to G banding complemented by FISH. Overall, combining CMA and TP53 mutation screening offers a fast, reliable way to detect high risk disease in MDS/AML. Keywords: Complex genome, CMA, chromothripsis, TP53 mutations

Genetic and Epigenetic Characteristics in Isolated Pancreatic Metastases of Clear-Cell Renal Cell Carcinoma.

Isolated pancreatic metastases of renal cell carcinoma (IsPMRCC) are a rare manifestation of metastatic, clear-cell renal cell carcinoma (RCC) in which distant metastases occur exclusively in the pancreas. In addition to the main symptom of the isolated occurrence of pancreatic metastases, the entity surprises with additional clinical peculiarities: (a) the unusually long interval of about 9 years between the primary RCC and the onset of pancreatic metastases; (b) multiple pancreatic metastases occurring in 36% of cases; (c) favourable treatment outcomes with a 75% 5-year survival rate; and (d) volume and growth-rate dependent risk factors generally accepted to be relevant for overall survival in metastatic surgery are insignificant in isPMRCC. The genetic and epigenetic causes of exclusive pancreatic involvement have not yet been investigated and are currently unknown. Conversely, according to the few available data in the literature, the following genetic and epigenetic peculiarities can already be identified as the cause of the protracted course: 1. high genetic stability of the tumour cell clones in both the primary tumour and the pancreatic metastases; 2. a low frequency of copy number variants associated with aggressiveness, such as 9p, 14q and 4q loss; 3. in the chromatin-modifying genes, a decreased rate of PAB1 (3%) and an increased rate of PBRM1 (77%) defects are seen, a profile associated with a favourable course; 4. an increased incidence of KDM5C mutations, which, in common with increased PBRM1 alterations, is also associated with a favourable outcome; and 5. angiogenetic biomarkers are increased in tumour tissue, while inflammatory biomarkers are decreased, which explains the good response to TKI therapy and lack of sensitivity to IT.