Independent Tumors Research Articles

Abstract C011: Targeting STAT3 overcomes PDAC resistance to EGFR/RAF1 inhibition leading to complete and durable tumor regression

Abstract Pancreatic ductal adenocarcinoma (PDAC) belongs to the tumors with worst prognosis due to the lack of effective treatment. It was previously demonstrated that combined elimination of EGFR and RAF1, two main mediators of the KRAS signaling, induces complete regression of a limited subset of small tumors in a genetically engineered mouse model (Blasco et al., 2019). In the current study, we show that resistance of PDACs with big sizes to the elimination of RAF1 and EGFR is mediated by STAT3. Indeed, combined genetic ablation of EGFR, RAF1 and STAT3 both in vitro and in vivo, results in complete and irreversible regression of more than 40 independent mouse PDAC tumors driven by Kras/Trp53 mutations. Importantly, all PDACs were characterized by increased initial tumor size and highly heterogeneous molecular profile similar to that of Basal human tumors. Moreover, we have validated the efficacy of this novel therapeutic strategy using pharmacological agents, such as EGFR inhibitors approved for clinical use and the STAT3 PROTAC degrader SD-36. Finally, inhibition of RAF1, EGFR and STAT3 expression effectively blocked in vitro and in vivo tumor progression in ten independent patient-derived organoid (PDO) and patient-derived xenograft (PDX) cultures carrying KRAS and TP53 mutations. These results open the door to the development of novel targeted therapies for PDAC patients. Citation Format: Vasiliki Liaki, Mariano Barbacid, Carmen Guerra. Targeting STAT3 overcomes PDAC resistance to EGFR/RAF1 inhibition leading to complete and durable tumor regression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C011.

Genomic and immune heterogeneity of multiple synchronous lung adenocarcinoma at different developmental stages

Multiple synchronous lung cancers (MSLCs) constitute a unique subtype of lung cancer. To explore the genomic and immune heterogeneity across different pathological stages of MSLCs, we analyse 16 MSLCs from 8 patients using single-cell RNA-seq, single-cell TCR sequencing, and bulk whole-exome sequencing. Our investigation indicates clonally independent tumours with convergent evolution driven by shared driver mutations. However, tumours from the same individual exhibit few shared mutations, indicating independent origins. During the transition from pre-invasive to invasive adenocarcinoma, we observe a shift in T cell phenotypes characterized by increased Treg cells and exhausted CD8+ T cells, accompanied by diminished cytotoxicity. Additionally, invasive adenocarcinomas exhibit greater neoantigen abundance and a more diverse TCR repertoire, indicating heightened heterogeneity. In summary, despite having a common genetic background and environmental exposure, our study emphasizes the individuality of MSLCs at different stages, highlighting their unique genomic and immune characteristics.

Treatment of patients with multiple primary malignant neoplasms (solid tumors and malignant diseases of the hematopoietic tissue) using systems for independent prolonged infusion therapy. Clinical observations

Study of the problem of multiple primary malignant neoplasms (MPMNs) becomes more important and significant in modern clinical oncology. MPMN is a varied group of independent tumors of varying origin located in one or several organs. Due to introduction of single-use elastomeric microinfusion pumps into the clinical practice, continuous cyclical drug outpatient and in-hospital treatment of oncological patients with MPMNs can be performed. Significant successes of specific therapy led to increased lifespan of patients with MPMNs. Prolonged administration of drugs using infusion pump with set velocity allows to deliver cytostatics into the patient’s body in strictly controlled amounts. The article presents 2 clinical cases of effective antitumor treatment of patients with MPMNs using infusion pum

Abstract 3988: Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics

Abstract The most well-described mechanism of immune evasion in PDAC is exclusion of immune cells by a desmoplastic stroma, but such immune exclusion is not uniform. Through single-nucleus RNA-seq and whole-transcriptome digital spatial profiling of patient tumors, we previously reported marked heterogeneity in the immune composition of PDAC. Here, we developed a novel multi-omics workflow integrating single cell spatial proteomics (96-plex, Akoya Phenocycler®-Fusion) and transcriptomics (990-plex, Nanostring CosMx) in primary patient PDAC tumors to systematically study spatial relationships and functional states of malignant, stromal, and immune cells. Consecutive sections from four independent tumors enabled whole slide proteomic assessment of >3.5M cells, and transcriptomic assessment of >200K cells. Strikingly, we observed an abundance of immune cell aggregates in all tissue sections, up to 57 within a 1 cm2 tumor section. Aggregates were diverse in composition with representation of myeloid cells, CD8+ T cells, and stromal cells, including fibroblasts and nerves. B/T lymphoid aggregates were well vascularized with co-localized CD31+CD34+ endothelial cells and appeared at various phases of maturation towards putative tertiary lymphoid structures (TLSs) containing CD38+ B cells, CD4+ T cells, and CD21+ follicular DCs. These tissues were enriched for interferon stimulated genes in T cells and type 1 interferon response signatures in malignant cells and cancer associated fibroblasts (CAFs) forming inflammatory multicellular hubs that have been associated with improved response to immune checkpoint blockade. Surprisingly, B cells, in addition to macrophages and CD8+ T cells, were major producers of interferon gamma (B cells: 13.7%, macrophages: 36.6%, CD8+ T cells: 12.1% of IFNG+ cells). Finally, to gain insight into the necessary factors for TLS formation in PDAC, we quantified the expression of chemokines and adhesion molecules associated with TLS initiation. Activated dendritic cells expressed CCL19 and CCL21, endothelial cells expressed CCL21, and in concert with inflammatory CAFs (iCAFs), CXCL12. Malignant cells expressed CXCL13 and CCL20 and displayed high levels of cell adhesion molecules ICAM1, VCAM1, and ICAM2. Together, our data suggests that the functions of lymphoid tissue organizer cells can be substituted by endothelial cells, dendritic cells, iCAFs, and, surprisingly, malignant cells. Our spatial immunophenotyping study of pancreatic cancer thus highlights the marked immune heterogeneity of the disease and suggests a central role for malignant and stromal cells in mediating diverse mechanisms of immune evasion and engagement. Functional studies will be conducted to investigate these mechanisms of tumor-immune crosstalk and identify novel immunotherapeutic vulnerabilities for this deadly disease. Citation Format: Dennis Gong, Jingyi Cao, Peter Wang, Niyati Jhaveri, Yue Hou, Bassem B. Cheikh, HaYeun Ji, Mark Gregory, Jason Reeves, Michael Patrick, Carina Shiau, Jennifer Su, Jimmy A. Guo, Joseph M. Beechem, Martin Hemberg, William L. Hwang, Ryan Park. Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics [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 3988.

Abstract 5659: Origin of synchronous or metachronous multiple pancreatic cancers

Abstract [Introduction] The majority of patients with pancreatic cancer are inoperable at the time of diagnosis and even those cases with successful tumor resection for early-stage tumors frequently had second tumors within the remaining pancreas shortly after the surgery, contributing to a poor clinical outcome. Additionally, synchronous multiple pancreatic cancers have been frequently documented. One of the long-standing issues about such synchronous and/or metachronous multiple pancreatic cancers is their clonal origins. This study is designed to elucidate the origin of multiple pancreatic cancers through an unbiased detection of somatic mutations in primary and metachronous cancers, along with adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 20 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and precancerous lesions, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. The investigation into the shared and private mutations across different samples aimed to unveil the clonal evolution history of these lesions. [Results] We analyzed 20 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 16 metachronous, and 2 synchronous tumors. In metachronous cases, all patients exhibited margin-negative pathology for the primary cancer, with a median interval of 38.9 months (ranging from 7 to 85 months) between the initial and second surgery. In addition, a total of 20 pancreatic intraepithelial neoplasia (PanIN) from 5 of these cases were analyzed. We identified a median of 66 (range: 31-232) and 20 (14-42) somatic mutations in cancer and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In most cases (N=19), sharing many somatic mutations, multiple tumors had a common clonal origin. In another synchronous case, the synchronous tumors shared only one mutation, suggesting that they were clonally independent tumors. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor and cancer lesions. While multiple independent clones were observed in precancerous lesions, most of multiple cancers originated from a common ancestor. Moreover, negative pathology of the margins at the initial surgery suggested that those multiple lesions arose from distant dissemination or metastasis, rather than contiguous, intraductal invasion. [Conclusions] In conclusion, our study suggests that even in early pancreatic cancer, dissemination within the pancreas may occur, contributing to the development of synchronous and metachronous cancers. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Takayuki Anazawa, Kazuyuki Nagai, Toshihiko Masui, Sachiko Minamiguchi, Hironori Haga, Takeshi Tanaka, Atsuhiro Masuda, Yuzo Kodama, Norimitsu Uza, Hiroshi Seno, Satoru Miyano, Hiroko Tanaka, Seishi Ogawa. Origin of synchronous or metachronous multiple pancreatic cancers [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 5659.

Gradual and synergistic correlation of tumor thickness and histological grade in penile invasive carcinomas

Histological grade and depth of invasion are among the best outcome pathological predictors in penile cancer. The TNM system is based on a combination of both for some stages. It is assumed that high-grade and deep tumors carry the worst prognosis, and the opposite occurs with superficial and low-grade neoplasms. However, there is no systematic evaluation of the phenomenon. We studied 147 patients from the Hospital de Oncologia - Instituto Mexicano del Seguro Social (period 2000 to 2013). They were treated by total or partial penectomies. Lymph node involvement was evaluated by bilateral inguinal node dissection (126 cases) or ultrasonography (21 cases). Tumor thickness was measured in mm from tumor surface to deepest invasion point, using a cut-point for superficial (≤10 mm) vs deep (>10 mm) tumors. Histological grade was from 1 to 3 according to WHO and AFIP criteria and considering G1 and G2 as low-grade and G3 as high-grade. Average age was 62 (26–98) years old. Tumor thickness mean was 15 mm (2–30 mm). G1, G2 and G3 tumors corresponded to 19 (13 %), 48 (33 %), and 80 (54 %) cases, respectively. Follow-up ranged from 10 to 82 months (median: 57 months). Fifty-three (36 %) patients died of disease. There was an overall correlation of tumor thickness and grade in most of the cases. Low-grade tumors were encountered in 92 % (12/13 cases) of superficial tumors. Deep tumors showed high-grade in 75 % of cases (73/97 cases). Superficial tumors with low histological grade had negative inguinal nodes and no mortality whereas deep tumors showing high histological grade were associated with high metastatic risk to lymph nodes (62/73 cases) and mortality (52/73 cases). Out of 24 deep tumors with low histological grade, seven had nodal spread (29 %) but only one died of disease. No outcome difference was found in HPV associated vs HPV independent tumors. Tumor thickness and grade are important synergistic and predictive pathological factors in relation to prognosis.

Syndromic MEN1 parathyroid adenomas consist of both subclonal nodules and clonally independent tumors

Primary hyperparathyroidism with parathyroid tumors is a typical manifestation of Multiple Endocrine Neoplasia Type 1 (MEN1) and is historically termed “primary hyperplasia”. Whether these tumors represent a multi-glandular clonal disease or hyperplasia has not been robustly proven so far. Loss of Menin protein expression is associated with inactivation of both alleles and a good surrogate for a MEN1 gene mutation. The cyclin-dependent kinase inhibitor 1B (CDKN1B) gene is mutated in MEN4 and encodes for protein p27 whose expression is poorly studied in the syndromic MEN1 setting.Here, we analyzed histomorphology and protein expression of Menin and p27 in parathyroid adenomas of 25 patients of two independent, well-characterized MEN1 cohorts. The pattern of loss of heterozygosity (LOH) was assessed by fluorescence in situ hybridization (FISH) in one MEN1-associated parathyroid adenoma. Further, next-generation sequencing (NGS) was performed on eleven nodules of four MEN1 patients.Morphologically, the majority of MEN1 adenomas consisted of multiple distinct nodules, in which Menin expression was mostly lost and p27 protein expression reduced. FISH analysis revealed that most nodules exhibited MEN1 loss, with or without the loss of centromere 11. NGS demonstrated both subclonal evolution and the existence of clonally unrelated tumors.Syndromic MEN1 parathyroid adenomas therefore consist of multiple clones with subclones, which supports the current concept of the novel WHO classification of parathyroid tumors (2022). p27 expression was lost in a large fraction of MEN1 parathyroids and must therefore be used with caution in suggesting MEN4.

Rare giant ovarian metastasis arising from a small primary lung adenocarcinoma: a case report.

This intricate case report details an exceptionally rare incidence of ovarian metastasis originating from a primary lung adenocarcinoma (LUAD). The relative rarity of this metastatic pathway in medical literature indicates significant diagnostic challenges. This patient was initially found to have both the ovarian tumor and lung nodule and they were originally considered independent primary tumors, derived from radiological interpretations and biomarker profiling. Nevertheless, subsequent postoperative histopathological and immunohistochemical staining evaluations identified ovarian tumors as invasive adenocarcinoma metastasized from lung. The lung and ovary tumor both showed marked anaplastic lymphoma kinase gene (ALK) protein expression by immunohistochemistry. The molecular pathologic genetic testing for lung tumor also revealed ALK rearrangement positive. The complexity of this case underscores the essentiality of maintaining a high degree of diagnostic vigilance, particularly when confronting synchronous tumors. In addition, immunohistochemical staining plays an important role in diagnosing the ovarian neoplasm's metastatic nature and determining the primary site of metastatic adenocarcinoma. For lung cancer with ovary metastasis patients, the adopting an adaptable treatment approach responsive to evolving diagnostic evidence can improve the accuracy of diagnosis and avoid excessive treatment of patients.

BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

Fusion transcriptome profiling defines the monoclonal origin of multifocal epithelioid haemangioma of bone.

Epithelioid haemangioma (EH) of bone remains a highly controversial entity. Indeed, the WHO classifies EHs of soft tissues as benign tumours, whereas bone EHs are considered intermediate-locally aggressive tumours due to common multifocal presentation and local destructive growth. To gain insights into theclinical behaviour and biology of EH of bone we retrospectively analysed 42 patients treated in a single institution from 1978 to 2021. Multifocal presentation was detected in 17 of 42 patients (40%) primarily as synchronous lesions. Patients were treated with curettage (57%), resection (29%) or biopsy, followed by radiotherapy or embolisation (14%). Follow-up (minimum 24 months) was available for 38 patients, with only five local recurrences (13%) and no death of disease. To clarify whether the synchronous bone lesions in multifocal EH represent multicentric disease or clonal dissemination, four cases were profiled by RNA-sequencing. Separate lesions from the same patient, which showed a similar transcriptional profile, expressed the same fusion transcript (involving FOS or FOSB) with identical gene breakpoints. These results indicate that, in EH of bone, multifocal lesions are clonally related and therefore represent the spread of a same neoplastic clone rather than simultaneous independent tumours. This finding is in apparent contradiction with the benign clinical course of the disease, and suggests that tumour dissemination in bone EH probably reflects a phenomenon of passive spreading, with tumour cells colonising distal sites while maintaining their benign biological nature.

Clinical and molecular characteristics of multiple primaries identified by whole-exome sequencing-based genetic divergence in non-small-cell lung cancer.

e20558 Background: With the recommendation of high-resolution chest computed tomography scan in lung cancer screening, multiple primary lung cancers (MPLC) is gaining more attention. Identifying MPLC from intrapulmonary metastases (IPM) is crucial to the clinical management. The current guidelines for the identification are always based on expert consensus rather than clinical evidence. We aim to develop a reliable identification method and further figure out the clinical and genetic features of MPLC. Methods: We retrospectively screened 305 lung lesions from 131 patients who underwent radical resection for multiple lung carcinomas. To minimize the effect of therapeutic intervention on prognostic outcomes, patients with each single tumor size smaller than 4cm, free of nodal or systemic metastases, and had high-quality whole-exon sequencing (WES) data were included. We selected 11 patients with confirmed intrapulmonary metastases for training. All patients’ follow-up information was collected until disease recurrence or metastases. A WES genetic divergence method for identifying MPLC from IPM was developed. The disease-free survival (DFS) was used as a validation tool for identification reliability. Results: A total of 61 eligible multi-lesion patients with 128 resected non-small cell lung cancer (NSCLC) lesions were finally analyzed. Based on WES genetic divergence identification, 51 MPLC patients were identified, whose DFS was significantly prolonged (HR, 0.27; 95% CI, 0.08 to 0.91; P = 0.02), while other recommended methods for identifying MPLC patients failed to show a DFS advantage. Among MPLC patients, 43.1% (22/51) had a first-degree relatives cancer family history, of whom 54.5% (12/22) had a lung cancer family history. EGFR was still the most common mutation gene (72.5%, 37/51); however, TP53 mutations was less frequent in MPLC (21% vs. 48%, P < 0.05), and tumor mutation burden (TMB) was lower (median, 0.73 muts/Mb vs. 1.15 muts/Mb, P < 0.01). Notably, despite the lack of shared alterations, unique germline variants in cancer-predisposing genes, particularly nonsynonymous SNV in solute carrier gene (SLC) family, were observed in most MPLC patients, rarely overlapping with IPM patients. Conclusions: WES-based genetic divergence clearly distinguishes independent primary tumors from IPM in NSCLC patients. MPLC patients represent a unique population which deserves further study.

Promoter methylation and enhanced SKP2 are associated with the downregulation of CDKN1C in cervical squamous cell carcinoma

PurposeCervical Squamous Cell Carcinoma (CSCC) is one of the significant causes of cancer deaths among women. Distinct genetic and epigenetic-altered loci, including chromosomal 11p15.5–15.4, have been identified. CDKN1C (Cyclin-Dependent Kinase Inhibitor 1C, p57KIP2), a member of the CIP/KIP family of cyclin-dependent kinase inhibitors (CDKIs), located at 11p15.4, is a putative tumor suppressor. Apart from transcriptional control, S-Phase Kinase Associated Protein 2 (SKP2), an oncogenic E3 ubiquitin ligase, regulates the protein turnover of CDKN1C. But the molecular status of CDKN1C in CSCC and the underlying mechanistic underpinnings have yet to be explored. MethodsTCGA and other publicly available datasets were analyzed to evaluate the expression of CDKN1C and SKP2. The expression (transcript/protein) was validated in independent CSCC tumors (n = 155). Copy number alteration and promoter methylation were correlated with the expression. Finally, in vitro functional validation was performed. ResultsCDKN1C was down-regulated, and SKP2 was up-regulated at the transcript and protein levels in CSCC tumors and the SiHa cell line. Notably, promoter methylation (50%) was associated with the downregulation of the CDKN1C transcript. However, high expression of SKP2 was found to be associated with the decreased expression of CDKN1C protein. Independent treatments with 5-aza-dC, MG132, and SKP2i (SKPin C1) in SiHa cells led to an enhanced expression of CDKN1C protein, validating the mechanism of down-regulation in CSCC. ConclusionCollectively, CDKN1C was down-regulated due to the synergistic effect of promoter hyper-methylation and SKP2 over-expression in CSCC tumors, paving the way for further studies of its role in the pathogenesis of the disease.

Efficacy of concurrent chemoradiotherapy with retrograde super selective intra-arterial infusion combined with cetuximab for synchronous multifocal oral squamous cell carcinomas

BackgroundThe incidence of multicentric oral cancer is increasing. However, treatment encounters difficulty if each tumor needs to be treated simultaneously. The objective of this clinical case report is to highlight the effect of concurrent chemoradiotherapy with retrograde superselective intra-arterial infusion combined with systemic administration of cetuximab on synchronous multifocal oral squamous cell carcinomas.Case presentationA 70-year-old man presented to the hospital with multiple tumors and oral pain. Three independent tumors were found in the right dorsal tongue, left edge of the tongue, and left lower lip. Based on the characteristic appearance of the lesions and further evaluation, clinical diagnoses of right tongue cancer “T3”, left tongue cancer “T2” and lower left lip cancer “T1”, N2cM0 were made. Treatment was initiated with systemic administration of cetuximab, followed by intra-arterial chemoradiotherapy. Treatment results were complete response on all three local lesions, and left neck dissection was performed following the initial treatment. The patient showed no evidence of recurrence during the 4 years follow-up period.ConclusionsThis novel combination treatment seems to be a promising strategy for patients with synchronous multifocal oral squamous cell carcinoma.

A novel risk prediction nomogram for early death in patients with resected synchronous multiple primary colorectal cancer based on the SEER database

BackgroundSynchronous multiple primary colorectal cancer (SMPCC) involves the simultaneous occurrence of 2 or more independent primary malignant tumors in the colon or rectum. Although SMPCC is rare, it results in a higher incidence of postoperative complications and mortality compared to patients with single primary colorectal cancer (SPCRC).MethodsThe clinical factors and survival outcomes of SMPCC patients registered on the Surveillance, Epidemiology, and End Results (SEER) database between 2000 and 2017 were extracted. The patients were divided into the training and validation cohorts using a ratio of 7:3. Univariate and multivariate logistic regression analyses were used to identify the independent risk factors for early death. The performance of the nomogram was evaluated using the concordance index (C-index), calibration curves, and the area under the curve (AUC) of a receiver operating characteristics curve (ROC). A decision curve analysis (DCA) was used to evaluate the clinical utility of the nomogram and standard TNM system.ResultsA total of 4386 SMPCC patients were enrolled in the study and randomly assigned to the training (n = 3070) and validation (n = 1316) cohorts. The multivariate logistic analysis identified age, chemotherapy, radiotherapy, T stage, N stage, and M stage as independent risk factors for all-cause and cancer-specific early death. The marital status was associated with all-cause early death, and the tumor grade was associated with cancer-specific early death. In the training cohort, the nomogram achieved a C-index of 0.808 (95% CI, 0.784–0.832) and 0.843 (95% CI, 0.816–0.870) for all-cause and cancer-specific early death, respectively. Following validation, the C-index was 0.797 (95% CI, 0.758–0.837) for all-cause early death and 0.832 (95% CI, 0.789–0.875) for cancer-specific early death. The ROC and calibration curves indicated that the model had good stability and reliability. The DCA showed that the nomogram had a better clinical net value than the TNM staging system.ConclusionOur nomogram can provide a simple and accurate tool for clinicians to predict the risk of early death in SMPCC patients undergoing surgery and could be used to optimize the treatment according to the patient's needs.

Abstract 3168: DE-meta: Revealing tumor gene expression by meta-analysis of RNASeq and proteomics datasets

Abstract Detecting differentially expressed genes under different biological conditions is crucial to characterize mechanisms of cancer development and identifying determinants of patient outcome. High throughput technologies such as RNA sequencing and mass spectrometry-based proteomics have been widely used to identify differentially expressed genes (DEG), on a transcript and on a peptide basis, respectively. However, both transcription and translation of genes provide information about gene expression. Thus, leveraging both RNA and protein expression data could potentially produce more accurate results. Various statistical tools have been developed to tackle the differential expression problem for a single platform, such as edgeR, DESeq2, etc. However, a tool that integrates both transcriptome and proteomics data for differential expression analysis has not yet been developed. Meta-analysis can potentially increase statistical power and generate a more consensus conclusion by combining multiple datasets. Here we present DE-Meta, a new tool developed using R, which performs combined meta-analysis of RNA-seq and MS-based proteomics on matched tumor specimens. To characterize the performance of our method using real world data, we utilized a published lung cancer dataset and set a truth standard of DEG between known two expression subtypes: terminal respiratory unit (TRU) and proximal proliferative (PP). Briefly, we restricted to 7,458 common genes detected by RNA-seq and MS-based proteomics. We then, randomly selected tumors as the truth sample set, n=20 TRU and n=20 PP tumors. With this truth sample set, DEGs were calculated separately by RNA or by protein, using DESeq2 and regression models. The union of these DEGs defined the truth DEG set. Using independent tumors (n=10 TRU and n=10 PP), we applied DE-meta to detect DEGs by RNA, by protein or joint RNA and protein data. Through receiver operating characteristic curve analysis, we show that the DE-meta joint model performed better than the single platform models (joint AUC=0.651, RNA AUC=0.610, protein AUC=0.623). Focusing genes relevant to tumor biology, we found that the DE-meta joint model called an 169 cancer-relevant DEGs, such as MSH2, CD4, WDR3, PIK3R1, and RPS6KA3. which were not detected by the RNA or protein models, at the same false discovery rate threshold of 0.05 (Benjamini-Hochberg procedure). In summary, our new DE-meta tool, by joint analysis of RNA-seq and MS-based proteomics datasets, yields more accurate and biologically-relevant results. The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, Department of Defense, USUHS, HJF, or U.S. Government. Citation Format: Xijun Zhang, Clifton L. Dalgard, Matthew D. Wilkerson. DE-meta: Revealing tumor gene expression by meta-analysis of RNASeq and proteomics datasets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3168.

Abstract 1511: Origin of synchronous or metachronous multiple pancreatic cancers

Abstract Introduction: The majority of patients with pancreatic cancer are inoperable at the time of diagnosis and even those cases with successful tumor resection for early-stage tumors frequently had second tumors within the remaining pancreas shortly after the surgery, contributing to a poor clinical outcome. Moreover, synchronous multiple pancreatic cancers were often reported. One of the long-standing issues about such synchronous and/or metachronous multiple pancreatic cancers is their clonal origins. In this study, we aimed to reveal the origin of multiple pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. Methods: Serially obtained formalin-fixed paraffin-embedded surgical specimens from 18 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and precancerous lesions, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. Results: We analyzed 18 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 14 metachronous, and 2 synchronous tumors. In metachronous cases, pathology for primary cancer were margin-negative in all patients, and the median interval between the initial and second surgery was 38.9 months (7 - 85 months). In addition, a total of 20 pancreatic intraepithelial neoplasia (PanIN) from 5 of these cases were analyzed. We identified a median of 66 (range: 31-232) and 20 (14-42) somatic mutations in cancer and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In most cases (N=17), sharing many somatic mutations, multiple tumors had a common clonal origin. In another synchronous case, the synchronous tumors shared only one mutation, suggesting that they were clonally independent tumors. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor and cancer lesions. While multiple independent clones were observed in precancerous lesions, most of multiple cancers originated from a common ancestor. Moreover, negative pathology of the margins at the initial surgery suggested that those multiple lesions arose from distant dissemination or metastasis, rather than contiguous, intraductal invasion. Conclusions: In conclusion, our study suggests that even early pancreatic cancer might be disseminated within the pancreas and contribute to synchronous and metachronous cancers. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Tomomi Nishimura, Toshihiko Masui, Kazuyuki Nagai, Takayuki Anazawa, Sachiko Minamigushi, Hironori Haga, Norimitsu Uza, Hiroshi Seno, Yuzo Kodama, Atsuhiro Masuda, Takeshi Tanaka, Seishi Ogawa, Hiroko Tanaka, Satoru Miyano. Origin of synchronous or metachronous multiple pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1511.

Abstract 133: Rewinding the tape of life: somatic profiling in Von Hippel-Lindau (VHL) disease reveals evolutionary contingency and constraints

Abstract Whether cancer evolution follows a deterministic route prescribed by host factors (germline genetics and/or environment) or proceeds along multiple alternative trajectories due to the stochastic nature of evolution is unknown. Patients with VHL disease have a pathogenic germline mutation in VHL (3p25) and tumorigenesis occurs when the wild-type copy of VHL is lost. Patients develop multiple clonally independent tumors including clear cell renal cell carcinoma (ccRCC), pheochromocytoma or paraganglioma (PPGL) and pancreatic neuroendocrine tumors (pNET), offering an opportunity to test repeatability of evolutionary trajectories between and within patients. We performed deep targeted exome sequencing, in 1520 tumors (993 solid ccRCC; 156 cystic ccRCC; 135 mixed solid/cystic ccRCC; 236 extra-renal) from 130 patients with VHL disease to evaluate somatic variants and copy number alterations, ploidy and chromosomal complexity. Tumor phenotype was evaluated from pathology reports and longitudinal growth kinetics from diagnostic MRI data. Analysis of 889 tumor samples is complete and profiling of the full cohort will be presented at the 2023 AACR meeting. Within clonally independent tumors, we performed two tests to assess for evidence of convergence between tumors from the same patient. First, we tested for parallel events in canonical ccRCC drivers. We found significant enrichment of somatic VHL variants occurring in one patient with a germline whole VHL gene deletion encompassing neighboring BRK1. Contiguous BRK1 loss is associated with less aggressive ccRCC phenotype and our findings suggest a specific constraint in this patient. We next assessed for evidence of convergence by measuring the genetic distance of copy number and mutational profiles between and within patients with resampling and permutation. Similar somatic profiles between tumors were only detected in 1/76 patients whose tumors were characterized by infrequent somatic alterations beyond 3p loss indicating little evidence of convergence. Cystic ccRCCs represent an indolent phenotype not associated with metastasis that may progress to solid tumors over time; we found mixed tumors closely resemble somatic profiles of solid tumors whereas cysts were characterized by infrequent copy number gains and low chromosomal complexity. We also identified cysts with specific losses (chr16, chr17, chr19p) mutually exclusive with 3p LOH suggestive of VHL-loss independent cyst formation. In solid ccRCCs, we found chromosomal complexity and loss of specific chromosomal loci (9p and/or 14q) are associated with diameter potentially reconciling the observation linking risk of metastasis to tumor diameter in VHL-related ccRCC. In summary, within the constrained setting of VHL-related ccRCC, evolution is dominated by chance although specific patient and phenotype specific constraints are observed. Citation Format: Scott T. Shepherd, Alex Coulton, Cathy D. Vocke, Chris J. Ricketts, Mark Ball, Fiona Byrne, Yuliia Dovga, Annika Fendler, Barber Taja, Olga O'Neill, Lina Gerontogianni, Gavin Kelly, Laura S. Schmidt, Daniel R. Crooks, Kirstin Choo, Rabindra Gautam, Zoe Blake, Krista Reynolds, Kevin Litchfield, Maria Merino, Marston W. Linehan, Samra Turajlic. Rewinding the tape of life: somatic profiling in Von Hippel-Lindau (VHL) disease reveals evolutionary contingency and constraints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 133.

Abstract F1-2: Clonal evolution of DCIS to invasion

Abstract F1-2: Clonal evolution of DCIS to invasion

A novel cancer risk prediction score for the natural course of FA patients with biallelic BRCA2/FANCD1 mutations.

Biallelic germline mutations in BRCA2 occur in the Fanconi anemia (FA)-D1 subtype of the rare pediatric disorder, FA, characterized clinically by severe congenital abnormalities and a very high propensity to develop malignancies early in life. Clinical and genetic data from 96 FA-D1 patients with biallelic BRCA2 mutations were collected and used to develop a new cancer risk prediction score system based on the specific mutations in BRCA2. This score takes into account the location of frameshift/stop and missense mutations relative to exon 11 of BRCA2, which encodes the major sites for interaction with the RAD51 recombinase, and uses the MaxEnt and HBond splicing scores to analyze potential splice site perturbations. Among 75 FA-D1 patients with ascertained BRCA2 mutations, 66 patients developed 102 malignancies, ranging from one to three independent tumors per individual. The median age at the clinical presentation of peripheral embryonal tumors was 1.0, at the onset of hematologic malignancies 1.8 and at the manifestation of CNS tumors 2.7years, respectively. Patients who received treatment lived longer than those without. Using our novel scoring system, we could distinguish three distinct cancer risk groups among FA-D1 patients: in the first, patients developed their initial malignancy at a median age of 1.3years (n = 36, 95% CI = 0.9-1.8), in the second group at 2.3years (n = 17, 95% CI = 1.4-4.4) and in the third group at 23.0years (n = 22, 95% CI = 4.3-n/a). Therefore, this scoring system allows, for the first time, to predict the cancer manifestation of FA-D1 patients simply based on the type and position of the mutations in BRCA2.

A human adenovirus encoding IFN-γ can transduce Tasmanian devil facial tumour cells and upregulate MHC-I.

The devil facial tumour disease (DFTD) has led to a massive decline in the wild Tasmanian devil (Sarcophilus harrisii) population. The disease is caused by two independent devil facial tumours (DFT1 and DFT2). These transmissible cancers have a mortality rate of nearly 100 %. An adenoviral vector-based vaccine has been proposed as a conservation strategy for the Tasmanian devil. This study aimed to determine if a human adenovirus serotype 5 could express functional transgenes in devil cells. As DFT1 cells do not constitutively express major histocompatibility complex class I (MHC-I), we developed a replication-deficient adenoviral vector that encodes devil interferon gamma (IFN-γ) fused to a fluorescent protein reporter. Our results show that adenoviral-expressed IFN-γ was able to stimulate upregulation of beta-2 microglobulin, a component of MHC-I, on DFT1, DFT2 and devil fibroblast cell lines. This work suggests that human adenoviruses can serve as a vaccine platform for devils and potentially other marsupials.