The Single-Cell and Spatial Transcriptomics Atlas of Epithelial-Fibroblast Interactions in Colorectal Cancer.

BackgroundLung adenocarcinoma (LUAD) exhibits significant cellular heterogeneity, yet the precise interactions between epithelial and stromal cells remain unclear. This study integrates single-cell and spatial transcriptomics to delineate tumor microenvironment dynamics, aiming to uncover key cellular subpopulations and their roles in LUAD progression.MethodsWe analyzed single-cell RNA sequencing (scRNA-seq) data from 21 LUAD patients and performed spatial transcriptomic deconvolution. Epithelial and fibroblast subpopulations were identified using Seurat and Harmony. Cell-cell communication was inferred via CellChat, while metabolic interactions were assessed using MEBOCOST. Copy number variation (CNV) analysis distinguished malignant cells, and trajectory inference mapped differentiation states. Spatial colocalization was examined via CellTrek. Prognostic signatures were derived from Cox regression, and a six-gene MCI score was validated using survival analysis.ResultsWe identified eight epithelial (e.g., MUC21 + Epi, ASCL1 + Epi) and nine fibroblast subpopulations (e.g., Fb_IGFBP4, Fb_COL11A1), with tumor-enriched subsets showing elevated CNVs and metabolic crosstalk. Fb_IGFBP4 correlated with poor prognosis, while MUC21 + Epi exhibited amplified COL1A1/SDC4-mediated interactions with fibroblasts. Pathway analysis highlighted tumor-specific MK and collagen signaling between fibroblasts and epithelial cells, suggesting stromal-epithelial synergy drives progression. Spatial analysis revealed colocalization of epithelial and fibroblast subclusters in tumors, contrasting with normal tissue. The MCI score, derived from six genes (e.g., ADAM10, MARVELD1), independently predicted survival and stratified high-risk patients (AUC > 0.6).ConclusionThis study identifies key stromal-epithelial subset interactions in LUAD, proposing prognostic biomarkers and therapeutic targets.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00432-025-06250-6.

Colorectal cancer (CRC) progression is heavily influenced by the tumor microenvironment (TME), where cancer-associated fibroblasts (CAFs) are key players. However, the heterogeneity, plasticity, and functional roles of CAFs in CRC remain poorly understood. We integrated single-cell RNA sequencing (scRNA-seq) data from four public CRC datasets and spatial transcriptomics data. Using computational approaches such as Harmony, Monocle2, and CellChat algorithms, we analyzed cellular landscapes, CAF subtype identification, developmental trajectories, transcription factor networks, and cell-cell communications to reveal CAF heterogeneity and their crosstalk with other cell subtypes in CRC. We identified eight distinct CAF subtypes with unique gene expression profiles and developmental plasticity. The CTHRC1+ CAF subtype was significantly associated with T cell exclusion and upregulated expression of immune checkpoint genes. We uncovered a specific communication axis between CTHRC1+ CAFs and MMP7+ malignant epithelial (Malig-Epi) cells mediated by the thrombospondin (THBS)2-SDC4 ligand-receptor signaling. High infiltration of both cell types synergistically correlates with worse prognosis and unfavorable response to immunotherapy. Our study delineates CAF heterogeneity in CRC and highlights the CTHRC1+ CAF subtype as a critical organizer of an immunosuppressive niche. The THBS2-SDC4 signaling pathway between CTHRC1+ CAFs and MMP7+ epithelial cells acts as a potential therapeutic target to disrupt protumorigenic crosstalk and improve clinical outcomes for CRC patients.

BackgroundGastric cancer is a highly aggressive malignancy characterized by a complex tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs), which are a key component of the TME, exhibit significant heterogeneity and play crucial roles in tumor progression. Therefore, a comprehensive understanding of CAFs is essential for developing novel therapeutic strategies for gastric cancer.MethodsThis study investigates the characteristics and functional information of CAF subtypes and explores the intercellular communication between CAFs and malignant epithelial cells (ECs) in gastric cancer by analyzing single-cell sequencing data from 24 gastric cancer samples. CellChat was employed to map intercellular communication, and Seurat was used to integrate single-cell sequencing data with spatial transcriptome data to reconstruct a comprehensive single-cell spatial map. The spatial relationship between apCAFs and cancer cells was analyzed using multicolor immunohistochemistry.ResultsCells were categorized into nine distinct categories, revealing a positive correlation between the proportions of epithelial cells (ECs) and fibroblasts. Furthermore, six fibroblast subpopulations were identified: inflammatory (iCAFs), pericytes, matrix (mCAFs), antigen-presenting (apCAFs), smooth muscle cells (SMCs), and proliferative CAFs (pCAFs). Each of these subpopulations was linked to various biological processes and immune responses. Malignant ECs exhibited heightened intercellular communication, particularly with CAF subpopulations, through specific ligand-receptor interactions. High-density regions of CAF subpopulations displayed spatial exclusivity, with pericytes serving as a source for iCAFs, mCAFs, and apCAFs. Notably, malignant ECs and apCAFs showed increased interactions, with certain ligand-receptor pairs potentially impacting the prognosis of gastric cancer. Multiplex immunohistochemistry (mIHC) confirmed the close spatial proximity of apCAFs to cancer cells in gastric cancer.ConclusionOur study provided a comprehensive characterization of CAF heterogeneity in gastric cancer and revealed the intricate intercellular networks within the TME. The identified CAF subpopulations and their interactions with malignant cells could serve as potential therapeutic targets.

Heterogeneity in colorectal cancer (CRC) manifests in the diversity and interactions among tumor cells, support stroma, and immune components, critically impacting patient prognosis and therapeutic efficacy. In this study, spatial transcriptomics (ST, 10x Genomics Visium Spatial Gene Expression) was performed on 40 retrospectively collected CRC samples, along with detailed clinicopathological parameters and long-term clinical outcomes, with the goal of unraveling the molecular topography of the tumor and surrounding stromal compartments, and associating their spatial interactions to clinical outcomes. Five primary RNA groups were identified through unsupervised clustering on ST data, correlating with tissue classes detected in H&E-stained images via a pre-trained deep learning model. Markers and pathways specific to these tissues were identified. Furthermore, immune cell infiltration in these regions was deconvoluted using reference single-cell RNA sequencing (scRNA-seq) data. Sub-clustering within the tumor groups revealed five major tumor subtypes, each characterized by a distinct set of differentially expressed marker genes. Enrichment analysis across these subtypes highlighted distinct activation and suppression in pathways including KRAS, p53, MAPK, and Integrin. Tumors exhibited intra- and inter-tumoral heterogeneity. Location-specific tumor subtypes were observed, with those in right-sided CRC displaying increased immune cell infiltration, linked to enriched pathways such as Type II Interferon Signaling and Cytokine Signaling. Conversely, left-sided CRC subtypes had reduced immune cell presence, and were characterized by enhanced pathways related to vascular development, Hypoxia, and p53 Signaling. Inferred clonal lineages from copy number variation (CNV) using ST suggested a spatially structured phylogenetic organization corresponding to transcriptional clusters. Additionally, the ST data unveiled distinct spatial distribution patterns across various cancer-associated fibroblast (CAF) phenotypes, such as matrix, inflammatory, vessel-associated, tumor-promoting, and antigen-presenting CAFs. Transcriptional patterns uncovered through scRNA-seq, including epithelial-mesenchymal transition (EMT) signatures and consensus molecular subtypes (CMS), displayed unique spatial layouts within the tumor microenvironment. To our knowledge, this is the most comprehensive study to date on spatial transcriptomic heterogeneity within CRC patients, revealing distinct tumor, stroma and immune compositions and spatial structures. This spatially detailed map provides novel insights into CRC heterogeneity, critical for advancing personalized treatment. Citation Format: Jie Zhou, Todd B. Sheridan, Sergii Domanskyi, Stephanie L. Cowles, Sunghee Park, Ilham Putra, Olga Anczukow, Jeffrey H. Chuang, Jill C. Rubinstein. Unveiling the spatial landscape of tumor and stroma heterogeneity in colorectal cancer with spatial 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 1150.

The progression of colorectal cancer (CRC) is profoundly influenced by the tumor microenvironment (TME). However, the spatial functional specialization of cancer-associated fibroblasts (CAFs) in driving malignancy and immune evasion remains poorly defined. This study aims to delineate the spatial and functional heterogeneity of specific CAF subpopulations in CRC. We performed single-cell RNA sequencing (scRNA-seq) on CRC tissues and integrated public datasets to map the stromal landscape. Key cellular interactions inferred from scRNA-seq were validated using high-resolution Xenium in situ transcriptomics and further corroborated by public spatial transcriptomics (ST) data and immunohistochemistry (IHC). ScRNA-seq identified two distinct, tumor-enriched CAF subpopulations: INHBA+ SULF1+ CAFs, associated with an epithelial-mesenchymal transition (EMT) signature, and INHBA+ BNIP3+ CAFs, which exhibited an immunoregulatory phenotype. Xenium spatial mapping localized these transcriptional identities to spatially segregated populations of INHBA+ myofibroblastic CAFs (myCAFs) and INHBA+ inflammatory CAFs (iCAFs), respectively. Both subsets localized to 'Invasive Tumor-Enriched Regions' alongside EMT+ malignant epithelial cells. Spatial analysis further revealed that INHBA+ myCAFs frequently clustered adjacent to SPP1+ macrophages, whereas INHBA+ iCAFs resided within specialized immunosuppressive niches. Orthogonal validation using ST and IHC confirmed that INHBA+ SULF1+ CAFs directly colocalized with the Malignant C2 (characterized by an EMT signature), while INHBA+ BNIP3+ CAFs were closely associated with CD4+ regulatory T cells (Tregs). These findings suggest that two spatially and functionally distinct INHBA+ CAF lineages coexist: one orchestrating EMT-driven invasion and the other fostering local immunosuppression via Treg recruitment. Our study reveals a spatial division of labor among CAFs in CRC. The conserved expression of INHBA across both pro-tumorigenic lineages positions it as a central stromal regulator and a promising therapeutic target for simultaneously disrupting tumor invasion and immune evasion. These findings highlight the potential of stroma-targeted strategies in advancing CRC treatment.

Colorectal cancer (CRC) is a heterogeneous malignancy shaped by genetic alterations and immune microenvironmental interactions. While single-cell RNA sequencing (scRNA-seq) has uncovered diverse epithelial and T cell subsets, the spatial architecture of their crosstalk remains poorly understood. We integrated scRNA-seq and spatial transcriptomics data from 36 CRC patients to comprehensively characterize epithelial and T cell heterogeneity, differentiation dynamics, and intercellular communication networks. Data preprocessing and integration were conducted using Seurat and Harmony. Cell trajectory inference was performed via CytoTRACE and Slingshot. Ligand-receptor signaling was assessed using CellChat, while spatial mapping was achieved using CellTrek. Nine epithelial and eight T cell subpopulations were identified, each exhibiting distinct transcriptional states, CNV burdens, and pseudotime trajectories. CD8⁺ T_GZMK cells displayed tumor-specific activation signatures and engaged in enriched communication with epithelial subsets through CEACAM, APP, and MIF signaling pathways. Spatial transcriptomics confirmed the in situ colocalization of CD8⁺ T_GZMK cells with PTP4A3⁺ epithelial cells in tumor regions, revealing spatially organized epithelial-immune niches. These interactions suggest potential mechanisms of immune modulation and tumor progression. This study provides an integrative single-cell and spatial atlas of CRC, revealing structured epithelial-T cell communication and spatial architecture within the tumor microenvironment. Our findings offer novel insights into immune-epithelial crosstalk and identify signaling pathways that may serve as therapeutic targets or biomarkers for CRC precision treatment.

Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are known to play supportive roles in tumor development and progression, but their interactions in colorectal cancer (CRC) remain unclear. Here, we investigated the effects of colon-cancer-derived CAFs on TAM differentiation, migration, and tumor immunity, both in vitro and in vivo. When co-cultured with monocytes, CAFs attracted monocytes and induced their differentiation into M2 macrophages. Immunohistology of surgically resected human CRC specimens and orthotopically transplanted mouse tumors revealed a correlation between numbers of CAFs and numbers of M2 macrophages. In a mouse model of CRC orthotopic transplantation, treatment with an inhibitor of the colony-stimulating factor-1 receptor (PLX3397) depleted M2 macrophages and increased CD8-positive T cells infiltrating the tumor nest. While this treatment had a minor effect on tumor growth, combining PLX3397 with anti-PD-1 antibody significantly reduced tumor growth. RNA-seq following combination therapy showed activation of tumor immunity. In summary, CAFs are involved in the induction and mobilization of M2 macrophage differentiation in the CRC tumor immune microenvironment, and the combination of cancer immunotherapy and PLX3397 may represent a novel therapeutic option for CRC.

BackgroundRecurrent bladder cancer is the most common type of urinary tract malignancy; nevertheless, the mechanistic basis for its recurrence is uncertain. Innovative technologies such as single‐cell transcriptomics and spatial transcriptomics (ST) offer new avenues for studying recurrent tumour progression at the single‐cell level while preserving spatial data.MethodThis study integrated single‐cell RNA (scRNA) sequencing and ST profiling to examine the tumour microenvironment (TME) of six bladder cancer tissues (three from primary tumours and three from recurrent tumours).FindingsscRNA data‐based ST deconvolution analysis revealed a much higher tumour heterogeneity along with TME in recurrent tumours than in primary tumours. High‐resolution ST analysis further identified that while the overall natural killer/T cell and malignant cell count or the ratio of total cells was similar or even lower in the recurrent tumours, a higher interaction between epithelial and immune cells was detected. Moreover, the analysis of spatial communication reveals a marked increase in activity between cancer‐associated fibroblasts (CAFs) and malignant cells, as well as other immune cells in recurrent tumours.InterpretationWe observed an enhanced interplay between CAFs and malignant cells in bladder recurrent tumours. These findings were first observed at the spatial level.

Colorectal cancer (CRC) represents the second leading cause of cancer-related death worldwide. One of the main reasons for this high mortality rate is because CRC develops into invasive lesions with high metastasis potential. The cancer-associated fibroblasts (CAFs) present in the tumour reactive stroma promote epithelial cancer cell motility and aggressiveness. However, whereas the role of tumour-stroma crosstalks in cancer progression is increasingly documented, how “normal” resident fibroblasts are converted into pro-invasive CAFs and how CAFs participate to the epithelial-stroma dialogue remains an open question. Precursor of the hormone gastrin, progastrin, now established as a growth factor for colon cells, is detected in colorectal polyps and tumours while it is absent in healthy colon. Using immunohistochemical and biochemical approaches we found that progastrin induces the expression of fibroblast activation markers (alphaSMA, FAPalpha) in the normal colon mucosa of progastrin-overexpressing mice as well as in the human colon fibroblast cell line CCD18Co. To investigate the role of progastrin-induced fibroblast activation, we compared the migration of colon epithelial cancer cells expressing or not the hormone in presence or absence of fibroblasts in Boyden chamber. Progastrin-expressing epithelial cells have increased migration capacities in presence of fibroblasts while not difference between the two epithelial cell lines was observed in absence of fibroblasts. Moreover, we identified the implication of different chemokines in this process. Thus, here we demonstrate that progastrin, secreted by tumour epithelial cells, contributes to the activation of the fibroblasts present in the cancer-associated stroma and participates to the epithelial-stroma dialogue. Citation Format: Nicolas Fénié, Claudine Bertrand, Aurélien Lacombe, Serge Roche, Corinne Bousquet, Valérie Gouazé-Andersson, Christine Toulas, Elizabeth Moyal, Audrey Ferrand. Progastrin activates colon fibroblasts and participates to the dialogue between tumor epithelial cells and stromal fibroblasts in colorectal cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B16. doi:10.1158/1538-7445.CHTME14-B16

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

Ovarian cancer is one of the most lethal gynecologic malignancies, primarily due to its late-stage diagnosis and intrinsic tumor heterogeneity. Histologically, epithelial ovarian cancer (EOC) accounts for approximately 70% of cases and exhibits distinct molecular and clinical characteristics. High-grade serous ovarian cancer (HGSOC), the most aggressive form of EOC, stands out for its widespread genomic instability and poor prognosis. Copy number variations (CNVs) play a critical role in tumor heterogeneity, treatment resistance, and disease progression. While CNVs have been extensively studied using bulk sequencing approaches, their spatial heterogeneity within tumors remains underexplored. To investigate this, we integrated spatial transcriptomics (10x Genomics Visium Spatial) with whole-exome sequencing (WES) in 24 fresh-frozen HGSOC samples. This dual approach enabled us to validate CNV patterns inferred from spatial data while leveraging WES for a genome-wide view of CNVs. First, spatial transcriptomics data were processed using the Space Ranger pipeline (10x Genomics) to generate spatially resolved gene expression matrices. Tumor purity scores were estimated using the R package ESTIMATE, and were then used to inform CNV inference with the tool InferCNV. This analysis revealed varied CNVs across spatial domains, indicating intratumoral heterogeneity and the existence of subclones with distinct CNV profiles and spatial niches. We further resolved loss of heterozygosity (LOH) in these samples using the R package tLOH. Clonal LOH of chr 17p, encompassing the TP53 locus, was detected in several samples. Pseudotime and spatial trajectory analysis confirmed that chr17 LOH is an early event in the trajectory of CNV development. To validate the CNV and LOH patterns inferred from spatial transcriptomics, we performed WES on paired tumor and normal tissues from the same cohort. WES data were first aligned to the human reference genome GRCh38 using BWA, followed by CNV profiling with GATK CNV. Recurrent CNV patterns, including gains on chromosomes 1q, 3q, 6p, 8q, and 20q, and losses on chromosomes 4, 5q, 6q, 9p, 13q, 14q, and 17, were observed, consistent with previous reports. A comparison between spatially inferred CNV clusters and GATK CNV results showed strong concordance, further validating the robustness of spatial CNV inference. Interestingly, germline BRCA-mutated samples exhibited a higher frequency of CNVs than BRCA wild-type samples, suggesting a link between BRCA status and genomic instability. By placing CNVs in tissue context, our findings highlight the genetic and spatial heterogeneity of HGSOC, and enhance our understanding of the driving role of CNVs in tumor evolution and growth. This abstract was developed with the assistance of generative AI to revise and clarify the text. Citation Format: Yuxin Jin, Jing Qian, Michelle G. Webb, David W. Craig, Lynda Roman, John D. Carpten, Rania Bassiouni. Multi-omics characterization of copy number variation in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5359.

Cancer Associated Fibroblasts in Multiple Myeloma: The Urokinase Receptor System in Tumor Growth Regulation

3543 Background: Cancer associated fibroblast (CAF) in tumor microenvironment is associated with poor prognosis and chemo-resistance in multiple solid tumors, however, there is lack of universal measure of CAF in colorectal cancer (CRC). The aim of this study was to assess fibroblast-signature for predicting outcome and analyze relevant mechanism. Methods: A dataset including 316 CRC patients without adjuvant chemotherapy was used as the discovery cohort for the identification of prognostic fibroblast-related genes (FRGs). A total of 1,352 CRC patients were then divided into one training cohort (n = 461) and two validation cohorts (n = 338, n = 553, respectively) for the construction of fibroblast-related gene signature (FRGS) and the verification of its prognostic value in stage II/III CRC patients. Functional annotation and analysis were performed to reveal the relevant mechanism. Results: A 11-gene signature was derived, which was prognostic for stage II/III CRC patients in two validation cohort (Validation-1 cohort: HR = 1.90, 95%CI = 1.16-3.12, P< 0.01; Validation-2 cohort: HR = 1.95, 95%CI = 1.39-2.73, P< 0.001). High CAF risk was correlated with worse prognosis in CRC patients without adjuvant chemotherapy (HR = 3.63, 95%CI = 2.24-5.88, P< 0.001), but not in patients who received adjuvant chemotherapy ( P= 0.154). Similar trends were found in Validation-1 cohort. After integrated with clinical characteristics, FRGS was confirmed as an independent prognostic factor after adjusted for TNM stage of tumor in multivariate analysis (Training cohort: HR = 3.19, 95%CI = 1.88-5.41, P< 0.001; Validation-1 cohort: HR = 5.00, 95%CI = 1.58-15.85, P= 0.007; Validation-2 cohort: HR = 2.99, 95%CI = 1.44-6.21, P= 0.003). Furthermore, enrichment analysis found that anti-tumor immune response was suppressed in the high CAF risk group. Conclusions: The 11-gene FRGS had independent prognostic value for CRC patients, as well as in prediction of benefit from chemotherapy. CAF in tumor microenvironment might impact on the prognosis of CRC patients via inhibiting immune response.

The vasoactive peptide endothelin-1 (ET-1) acts via two endothelin receptor subtypes, ETA (ETAR) and ETB (ETBR). ET-1 and ETAR are overexpressed in colorectal cancer tissues. In vitro, ET-1 acting via ETAR, is a mitogen for colorectal cancer cells. To identify other potential stimulatory loops, we investigated the distribution and cell-specific localization of both ETAR and ETBR in tissue sections from patients with colorectal cancer. Frozen sections from specimens of colorectal cancer (n=9) and normal colon (n=9) were cut and subjected to either (i) autoradiography or (ii) a combination of cell type-specific immunohistochemistry, using antibodies against fibroblasts (AS02), endothelial cells (CD31) or nerve fibres (NF200) and in-vitro receptor microautoradiography, using ETAR-specific and ETBR-specific radioligands. ETARs were upregulated in all cell types, apart from nerve, in cancer compared with normal colon (1:1.59 normal to cancer). Specifically, ETAR binding was highest in cancer-associated blood vessels and fibroblasts and to a lesser extent in epithelial cancer cells. In contrast, ETBRs were the predominant receptors in normal colon (1:0.59 normal to cancer) and were markedly down-regulated in cancer-associated blood vessels, fibroblasts and to a lesser extent in epithelial cells. Nerve colocalization was demonstrated, but remained unchanged for all tissues. The shift in ET receptor binding observed in epithelial cancer cells and cancer-associated fibroblasts and endothelial cells may favour ET-1 signals contributing to colorectal cancer growth and neovascularization via ETAR. This may provide the basis for therapeutic use of specific ETAR antagonists as adjuvant treatment of colorectal cancer.

Targeted deep massive parallel sequencing (MPS) has been implemented in routine molecular diagnostics for high-throughput genetic profiling of formalin-fixed paraffin-embedded cancer samples. This approach is now widely used to interrogate simple somatic mutations but experience with the analysis of copy number variations (CNV) is still limited. Here, we retrospectively analyzed CNVs in 822 cancer cases (n = 135 melanoma, n = 468 non-small cell lung cancers (NSCLC), n = 219 colorectal cancers (CRC)) that were sent to our institution for routine molecular profiling using a semiconductor based sequencing platform. Amplifications and deletions inferred by MPS coverage data were independently validated by a qPCR assay. We observed a decreasing frequency of CNV in clinically actionable genes from melanoma to NSCLC to colorectal cancer. Of 56 melanomas with genetic aberrations in BRAF, 31 showed co-occurring CNV in other genes, mainly affecting CDKN2A. Some tumors (5 cases each) revealed clustered deletions affecting either ABL1, NOTCH1, and RET or STK11, GNA11, and JAK3. 8.1% of the cases had amplifications in clinically actionable genes. In the group of NRAS mutant tumors (n = 39), 26 showed co-occurring CNVs in other genes, such as CDKN2A and FGFR3, and 9 NRAS mutant cases were additionally mutated in BRAF. 19.1% had amplifications in clinically actionable genes. In contrast to BRAF mutant tumors, we did not see any specific CNV clusters. In the group of BRAF/NRASwt tumors (n = 11), we observed 5 cases with co-amplification of KDR, KIT, PDGFRA and another 6 cases with KIT mutations. While co-amplified cases had many gene deletions, KIT mutated tumors harbored only very few genetic aberrations in other genes. Across both NSCLC data sets, we identified 14 cases with amplified EGFR (10 of them harboring co-occurring EGFR mutations) and detected 8 NSCLC with KRAS amplifications (of which 7 had co-occurring mutations of KRAS). KRAS mutated tumors displayed frequent amplifications in MYC (n = 10) and MDM2 (n = 5). Of the 22 BRAF mutant tumors, two harbored mutated KRAS. In contrast to melanoma, we observed no clustering of CNVs in BRAFmut NSCLCs. Within the group of KRAS/EGFR/BRAFwt tumors, we identified 15 cases harboring genetic aberrations in MET (n = 8 mutations, n = 7 amplifications). Compared to melanoma and NSCLC, the number of CNV in CRC was rather low. IGF2 amplifications were most prevalent (n = 13) followed by MYC (n = 9). Two cases showed amplified wild-type alleles of KRAS. Two KRAS mutant tumors showed concomitant amplification of NRAS and three cases harbored amplified EGFR. In conclusion we demonstrate that i) detection of CNVs by targeted MPS data obtained from FFPE material is feasible and ii) could be validated independently, iii) this approach enables detection of known CNV patterns, and iv) uncovers new CNV patterns in clinically actionable targets across cancers. Citation Format: Albrecht Stenzinger, Roland Penzel, Frederick Klauschen, Arne Warth, Regine Brandt, Daniel Heim, Peter Schirmacher, Wilko Weichert, Volker Endris, Nicole Pfarr. CNV patterns in 822 routine diagnostic cases of NSCLC, melanoma, and colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3167.