Search

Purpose:Regorafenib (REG) is approved for the treatment of metastatic colorectal cancer, but has modest survival benefit and associated toxicities. Robust predictive/early response biomarkers to aid patient stratification are outstanding. We have exploited biological pathway analyses in a patient-derived xenograft (PDX) trial to study REG response mechanisms and elucidate putative biomarkers.Experimental Design:Molecularly subtyped PDXs were annotated for REG response. Subtyping was based on gene expression (CMS, consensus molecular subtype) and copy-number alteration (CNA). Baseline tumor vascularization, apoptosis, and proliferation signatures were studied to identify predictive biomarkers within subtypes. Phospho-proteomic analysis was used to identify novel classifiers. Supervised RNA sequencing analysis was performed on PDXs that progressed, or did not progress, following REG treatment.Results:Improved REG response was observed in CMS4, although intra-subtype response was variable. Tumor vascularity did not correlate with outcome. In CMS4 tumors, reduced proliferation and higher sensitivity to apoptosis at baseline correlated with response. Reverse phase protein array (RPPA) analysis revealed 4 phospho-proteomic clusters, one of which was enriched with non-progressor models. A classification decision tree trained on RPPA- and CMS-based assignments discriminated non-progressors from progressors with 92% overall accuracy (97% sensitivity, 67% specificity). Supervised RNA sequencing revealed that higher basal EPHA2 expression is associated with REG resistance.Conclusions:Subtype classification systems represent canonical “termini a quo” (starting points) to support REG biomarker identification, and provide a platform to identify resistance mechanisms and novel contexts of vulnerability. Incorporating functional characterization of biological systems may optimize the biomarker identification process for multitargeted kinase inhibitors.

Supplementary Figures S1 - S8 and Table S1. Fig S1: Analysis of copy number burden in a PDX population trial of REG treated mCRC PDXs. Fig S2: Effect of REG on microvessel density and proliferation according to CNA cluster. Fig S3 - S6: BCL-2 family profiling of mCRC PDX models via quantitative Western blotting. Fig S7: RPPA protein scores indicating proteins' association with the PDX models' response to REG. Fig S8: Western blot validation of differential EPHA2 protein expression between REG post- and pre-treatment progressor and non-progressor PDX models. Table S1: List of antibodies and dilution factors used against desired targets in RPPA analysis.

Introduction: Colorectal cancer (CRC) is a disease of older patients, but evidence-based guidelines for chemotherapy in older patients are scarce. Geriatric assessment (GA) evaluates a patient’s functional status (FS) and helps in decision-making when choosing chemotherapy for older patients. However, the change of FS during chemotherapy is rarely studied as GA is mostly performed once instead of sequentially. Methods: We performed a subgroup analysis of a prospective, multicenter study EpiReal 75. Patients aged ≥75 years with gastrointestinal malignancy prior to initiation of chemotherapy or receiving palliative chemotherapy were screened. We defined geriatric core assessments including the Eastern Cooperative Oncology Group score, Barthel’s activities of daily living (ADL) scale, Lawton’s instrumental activities of daily living (IADL) scale, and G-8 questionnaire, which were performed at baseline and repeated every 3 months. Quality of life (QoL) assessed by QLQ-C30 questionnaire was also re-evaluated every 3 months. We defined any deterioration in any of the geriatric parameters as unstable in the corresponding function. Results: 28 patients with CRC were enrolled between April 2014 and December 2018. 20 patients were evaluable for statistical analysis with a mean age of 78.5 years (range, 75–88). Most patients received chemotherapy in palliative setting. During 3 months of chemotherapy, 25% of patients became more dependent as measured by ADL or IADL. During a median follow-up of 15 months, patients with unstable ADL or IADL had a significantly shorter overall survival (OS) than those with stable ADL or IADL (plogrank = 0.0055 and 0.0253, respectively), without a significant difference in progression-free survival (PFS). Also, unstable IADL correlated with a deterioration in aspects of QoL such as role functioning and emotional functioning (p = 0.0189 and 0.0239, respectively). 20% of patients experienced treatment-related grade 3 adverse events (AEs), no grade 4–5 AEs occurred. Conclusion: Sequential GA revealed changes in FS in older patients with CRC receiving chemotherapy. A deterioration of FS during chemotherapy did not influence PFS but had a negative impact on OS and QoL. It is therefore important to maintain FS in older patients with cancer, and regular performance of geriatric core assessments should be encouraged in the clinical practice.

Lung carcinoids are neuroendocrine tumors representing 1 to 2% of lung cancers. This study outlines the case of a patient with a metastatic lung atypical carcinoid who presented with a pleural effusion and progression of liver metastases after developing resistance to conventional treatments. Personalized functional profiling (PFP), i.e. drug screening, was performed in ex-vivo spheroids obtained from the patient’s liver metastasis to identify potential therapeutic options. The drug screening results revealed cediranib, an antiangiogenic drug, as a hit drug for this patient, from a library of 66 Food and Drug Administration (FDA)-approved and investigational drugs. Based on the PFP results and the reported evidence of clinical efficacy of bevacizumab and capecitabine combination in gastro-intestinal neuroendocrine tumors, this combination was given to the patient. Four months later, the pleural effusion and pleura carcinosis regressed and the liver metastasis did not progress. The patient experienced 2 years of a stable disease under the PFP-guided personalized treatment.

Colorectal cancer (CRC) is a high‐incidence malignancy worldwide which still needs better therapy options. Therefore, the aim of the present study was to investigate the responses of normal or malignant human intestinal epithelium to bone morphogenetic protein (BMP)‐9 and to find out whether the application of BMP‐9 to patients with CRC or the enhancement of its synthesis in the liver could be useful strategies for new therapy approaches. In silico analyses of CRC patient cohorts (TCGA database) revealed that high expression of the BMP‐target gene ID1, especially in combination with low expression of the BMP‐inhibitor noggin, is significantly associated with better patient survival. Organoid lines were generated from human biopsies of colon cancer (T‐Orgs) and corresponding non‐malignant areas (N‐Orgs) of three patients. The N‐Orgs represented tumours belonging to three different consensus molecular subtypes (CMS) of CRC. Overall, BMP‐9 stimulation of organoids promoted an enrichment of tumour‐suppressive gene expression signatures, whereas the stimulation with noggin had the opposite effects. Furthermore, treatment of organoids with BMP‐9 induced ID1 expression (independently of high noggin levels), while treatment with noggin reduced ID1. In summary, our data identify the ratio between ID1 and noggin as a new prognostic value for CRC patient outcome. We further show that by inducing ID1, BMP‐9 enhances this ratio, even in the presence of noggin. Thus, BMP‐9 is identified as a novel target for the development of improved anti‐cancer therapies of patients with CRC.

Patient derived organoids closely resemble the biology of tissues and tumors. They are enabling ex vivo modeling of human diseases and dissecting key features of tumor biology like anatomical diversity or inter- and intra-tumoral heterogeneity. In the last years, organoids were established as models for drug discovery and explored to guide clinical decision making. In this review, we discuss the recent developments in organoid based research, elaborating on the developments in colorectal cancer as a prime example. We focus our review on the role of organoids to decode cancer cell dynamics and tumor microenvironmental complexity with the underlying bi-directional crosstalk. Additionally, advancements in the development of living biobanks, screening approaches, organoid based precision medicine and challenges of co-clinical trials are highlighted. We discuss ongoing efforts to overcome challenges that the field faces and indicate potential future directions.

Purpose: Cell-free DNA (cfDNA) analysis in plasma is an emerging technique with numerous applications in oncology. We employed cfDNA to determine chromosomal instability (CIN), nucleosome footprints (NF) and methylation profiles in metastatic colorectal cancer (mCRC) patients and to predict outcome to bevacizumab (BVZ) combination therapy. Experimental Design: Low coverage whole-genome sequencing (LC-WGS) was performed on matched tumor and plasma samples from 74 mCRC patients participating in the CTRIAL-IE 12-16 AC-ANGIOPREDICT Phase II clinical trial (NCT01822444), prior to receiving BVZ, and analyzed for CIN (3 subclusters with low, intermediate and high CIN, respectively) and nucleosomes. For 61/74 mCRC patients, plasma samples before and after BVZ treatment were available and used for targeted methylation sequencing. A validation cohort of mCRC plasma samples (n= 24) from the University Medical Center Mannheim (UMM) was similarly profiled. Results: Based on cfDNA CIN profiles, we subtyped mCRC patients with 92.3% accuracy of sample distinction into low and high CIN clusters (cluster 1 against cluster 2 and 3), demonstrating concordance between matched plasma and tumor samples. Improved survival outcome was observed in CIN high patients. Plasma-based CIN clustering and improved survival for CIN high patients was also confirmed in the UMM cohort. NF and methylation profiles differed between CIN clusters and healthy individuals, and could reliably separate cluster 1 from cluster 2 and 3 samples (AUC=0.72 and 0.85 respectively). A large methylation score decrease after BVZ treatment was associated with improved overall survival (p = 0.013). Conclusions: cfDNA can be analyzed to predict outcome to BVZ in mCRC patients. Detection of CNAs, NFs and methylation profiles facilitated stratification of samples into CIN clusters and informed patient response to treatment. Citation Format: Ian S. Miller, Valentina Thomas, Tom Venken, Ingrid Arijs, Ana Barat, Johannes Betge, Tianzuo Zhan, Timo Gaiser, Matthias P. Ebert, Jochen Prehn, Rut Klinger, Darran P. O’Connor, Brian Brian Moulton, Verena Murphy, Ray McDermott, Brian Bird, Gregory Leonard, Liam Grogan, Anne Horgan, Nadine Schulte, Markus Moehler, Nicole Prannikar, Judith Franz-Werner, Hans-Peter Feustel, Diether Lambrechts, Annette T. Byrne. Analysis of cell free DNA to predict outcome to Bevacizumab combination therapy in metastatic colorectal cancer patients. [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 5584.

Epigenetic dysregulation is an important feature of colorectal cancer (CRC). Combining epigenetic drugs with other antineoplastic agents is a promising treatment strategy for advanced cancers. Here, we exploited the concept of synthetic lethality to identify epigenetic targets that act synergistically with histone deacetylase (HDAC) inhibitors to reduce the growth of CRC. We applied a pooled CRISPR-Cas9 screen using a custom sgRNA library directed against 614 epigenetic regulators and discovered that knockout of the euchromatic histone-lysine N-methyltransferases 1 and 2 (EHMT1/2) strongly enhanced the antiproliferative effect of clinically used HDAC inhibitors. Using tissue microarrays from 1066 CRC samples with different tumor stages, we showed that low EHMT2 protein expression is predominantly found in advanced CRC and associated with poor clinical outcome. Cotargeting of HDAC and EHMT1/2 with specific small molecule inhibitors synergistically reduced proliferation of CRC cell lines. Mechanistically, we used a high-throughput Western blot assay to demonstrate that both inhibitors elicited distinct cellular mechanisms to reduce tumor growth, including cell cycle arrest and modulation of autophagy. On the epigenetic level, the compounds increased H3K9 acetylation and reduced H3K9 dimethylation. Finally, we used a panel of patient-derived CRC organoids to show that HDAC and EHMT1/2 inhibition synergistically reduced tumor viability in advanced models of CRC.

Lung carcinoids are neuroendocrine tumors representing 1 to 2% of lung cancers. This study outlines the case of a patient with a metastatic lung atypical carcinoid who presented with a pleural effusion and progression of liver metastases after developing resistance to conventional treatments. Personalized functional profiling (PFP), i.e. drug screening, was performed in

Patient-derived organoids resemble the biology of tissues and tumors, enabling ex vivo modeling of human diseases. They have heterogeneous morphologies with unclear biological causes and relationship to treatment response. Here, we use high-throughput, image-based profiling to quantify phenotypes of over 5 million individual colorectal cancer organoids after treatment with >500 small molecules. Integration of data using multi-omics modeling identifies axes of morphological variation across organoids: Organoid size is linked to IGF1 receptor signaling, and cystic vs. solid organoid architecture is associated with LGR5 + stemness. Treatment-induced organoid morphology reflects organoid viability, drug mechanism of action, and is biologically interpretable. Inhibition of MEK leads to cystic reorganization of organoids and increases expression ofLGR5, while inhibition of mTOR induces IGF1 receptor signaling. In conclusion, we identify shared axes of variation for colorectal cancer organoid morphology, their underlying biological mechanisms, and pharmacological interventions with the ability to move organoids along them.

Purpose: Regorafenib (REG) is approved for the treatment of metastatic colorectal cancer, but has modest survival benefit and associated toxicities. Robust predictive/early response biomarkers to aid patient stratification are outstanding. We have exploited biological pathway analyses in a patient-derived xenograft (PDX) trial to study REG response mechanisms and elucidate putative biomarkers. Experimental Design: Molecularly subtyped PDXs were annotated for REG response. Subtyping was based on gene expression (CMS, consensus molecular subtype) and copy-number alteration (CNA). Baseline tumor vascularization, apoptosis, and proliferation signatures were studied to identify predictive biomarkers within subtypes. Phospho-proteomic analysis was used to identify novel classifiers. Supervised RNA sequencing analysis was performed on PDXs that progressed, or did not progress, following REG treatment. Results: Improved REG response was observed in CMS4, although intra-subtype response was variable. Tumor vascularity did not correlate with outcome. In CMS4 tumors, reduced proliferation and higher sensitivity to apoptosis at baseline correlated with response. Reverse phase protein array (RPPA) analysis revealed 4 phospho-proteomic clusters, one of which was enriched with non-progressor models. A classification decision tree trained on RPPA- and CMS-based assignments discriminated non-progressors from progressors with 92% overall accuracy (97% sensitivity, 67% specificity). Supervised RNA sequencing revealed that higher basal EPHA2 expression is associated with REG resistance. Conclusions: Subtype classification systems represent canonical “termini a quo” (starting points) to support REG biomarker identification, and provide a platform to identify resistance mechanisms and novel contexts of vulnerability. Incorporating functional characterization of biological systems may optimize the biomarker identification process for multitargeted kinase inhibitors.

303 Background: Overall survival of patients with advanced and refractory esophageal squamous cell carcinoma (ESCC) is poor. Most patients are 65 years or older, present with advanced and metastatic disease and suffer from extensive co-morbidity and decreased functionality. While approved therapies beyond first-line therapy have not been available for decades, just recently treatment with PD-1 antibodies has shown to improve progression-free and overall survival in this patient cohort. Thus, we assessed the combination of nivolumab and ipilimumab in this vulnerable and older patient population. Methods: In this multi-center, open-label phase II trial older patients with ESCC with progression or recurrence of disease following first-line therapy were treated with nivolumab and ipilimumab. Patients had to pass a brief geriatric assessment using the G8 screening tool in combination with the Deficit Accumulation Frailty Index (DAFI). A safety run–in phase was initiated with nivolumab (240mg fixed dose Q2W). Following a safety assessment, patients then went on to receive the combination therapy of nivolumab/ipilimumab (nivolumab 240 mg fixed dose Q2W; ipilimumab 1 mg/kg Q6W), in case safety was critical patients were allowed to continue with nivolumab monotherapy. The primary outcome was overall survival. Progression-free survival, quality of life and adverse events were also assessed. Results: In total 66 evaluable patients (16 female, 50 male) with ESCC were enrolled in this trial after successful geriatric assessment, median age was 70.5 years (range 55-84 years). 44 patients were treated with the combination therapy of nivolumab and ipilimumab, 22 patients with nivolumab only. The primary endpoint was met with a median OS of 7.2 months (95% CI, 5.7 to 12.4 months) (p < 0.006; versus historical control treated with standard chemotherapy). Median PFS was 2.7 months (95% CI, 2.5 to 2.9 months). ORR was 18.2% (95% CI, 9.8 to 29.6), all cases were partial responses. Grade 3 or more treatment related adverse events were observed in ̃25% of patients. Conclusions: The combination therapy of nivolumab and ipilimumab demonstrates improved overall survival and sustained confirmed responses in the second line therapy of older European patients with ESCC. Geriatric assessment is feasible in the setting of a prospective immune therapy trial. Overall, the RAMONA trial confirmed efficacy and safety of combination checkpoint inhibitor therapy in G8 prescreened older patients with ESCC in Europe beyond first line therapy. Clinical trial information: NCT03416244.