Incidence of RSPO3 rearrangements in advanced colorectal cancer patients detected by NGS and their relationship with disease characteristics

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Incidence of RSPO3 rearrangements in advanced colorectal cancer patients detected by NGS and their relationship with disease characteristics

https://doi.org/10.21203/rs.3.rs-4945373/v1

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Colorectal cancer (CRC) is the second most frequent cause of cancer-related deaths. Mortality is largely due to limited treatment options for patients who present with advanced disease. Genomic abnormalities that are potentially characteristic of such advanced stages of the disease are complex and are not yet fully understood. In fact, only 30% of cases with advanced disease benefit from targeted treatments. The molecular heterogeneity of primary advanced CRC (aCRC) tumor samples was evaluated by next-generation sequencing (NGS) in 53 consecutive patients (pT4a-b). The genetic abnormalities found in primary tumors were most frequently mutations in TP53 (57% of cases), KRAS (45%), PI3KCA (27%), BRAF (15%) and RET (10%), as well as RSP03 fusions (8%). Alterations in the TP53 and NRAS genes were more commonly observed in the left colon, while BRAF mutations and RSPO3 fusions were more frequently detected in the right or transverse colon. We also show a strong association between the presence of RSPO3 rearrangements and patients with small, low histological grade tumors, with normal CEA levels, preferentially located on the right side of the colon. Furthermore, aCRC patients with PTPRK::RSPO3 fusions showed a higher frequency of deaths, and RSPO3 gene expression levels had an adverse impact on overall survival in two large, independent series of patients with CRC. We show that RSPO3 rearrangements occur very frequently in advanced disease, making it a promising therapeutic target for this subtype of patient.

Biological sciences/Cancer/Cancer genomics

Biological sciences/Cancer

Biological sciences/Molecular biology

Advanced colorectal cancer

NGS

PTPRK:RSPO3 fusion

Sporadic colorectal cancer (sCRC) is the third most frequently diagnosed cancer worldwide and is the second most common cause of cancer-related deaths. Mortality is largely due to limited treatment options for patients who present with advanced disease ¹. In fact, the five-year survival rate for advanced colorectal cancer (aCRC) is less than 10% ². Up to 25% of sCRC cases present with stage IV disease and approximately 25–50% who initially present with early-stage sCRC go on to develop metastases over the course of the disease ². This urgent clinical need has meant that the research currently being carried out by various research groups is focused on the development of safe, effective, and tolerable therapies for advanced disease. Since 2004, cetuximab and panitumumab have been introduced as monoclonal anti-EGFR antibody therapeutic drugs to treat aCRC ³. Mutations in any of the genes that are part of the EGFR signaling pathway (KRAS and NRAS genes) determine the response to this therapy, for which reason they are used as predictive biomarkers ⁴. However, data on the prevalence of mutations in these genes, in different locations of aCRC tumors, are limited. More recently, aCRC patients with deficient mismatch repair (dMMR) (15%) have shown an excellent response to treatment with immune checkpoint inhibitors ⁵. Additional therapeutic targets, such as HER2 (which has a 3–5% amplification rate in CRC), have also led to new molecularly based treatment options, including those with monoclonal antibodies (trastuzumab and pertuzumab) and tyrosine kinase inhibitors (tucatinib) ³. Likewise, 0.5%-1% of CRC tumors present structural rearrangements in the neurotrophic receptor tyrosine kinase (NTRK) genes and generally exhibit a high tumor mutational load and high degree of microsatellite instability (MSI-H), which is associated with an adverse prognosis ⁶; however, there are two FDA-approved NTRK inhibitor molecules, larotrectinib and entrectinib, that have demonstrated excellent results for patients whose disease has progressed under standard therapy or for those without treatment options ⁷. Similarly, selpercatinib received FDA tumor-agnostic approval for refractory advanced solid tumors with a RET gene fusion, based on the results from the LIBRETTO-001 trial ⁸, reported in < 1% of patients with sCRC ⁹. Finally, the FDA approved the combination of encorafenib with cetuximab for patients who received prior treatment for aCRC with BRAF V600E mutations (8–10% of cases) ¹⁰. This clearly indicates that, as our understanding of the genomic landscape of tumors deepens, the therapeutic paradigm for aCRC expands. In fact, in the present study we found that of the 53 patients with aCRC analyzed by NGS techniques, 4 (8% of cases) showed RSPO3 fusions. Several studies have reported that RSPO3 fusions are mutually exclusive with APC mutations and also contribute to the activation of WNT signaling and the development of sCRC in this subgroup of patients ^11–13. Interestingly, blocks of secretion of WNT ligand and RSPO3 neutralizing antibodies have shown an antitumoral effect in in vitro and in vivo models of CRC expressing the RSPO3 fusion ^14–18. However, despite abundant evidence that hyperactivation of the WNT pathway drives CRC and that therapies targeting this pathway have been effective in preclinical models of CRC, targeted WNT therapies have not progressed in clinical settings ¹⁹. The arrival of effective therapies in the clinical setting that target the Wnt signaling pathway imply that, as part of the daily routine, it would be necessary to evaluate whether the components of this pathway are altered in CRC tumors.

In the present study, we focused on the search for new biomarkers using NGS techniques in patients with aCRC to find new targets for the treatment of patients with advanced disease. For this purpose, we analyzed the genetic abnormalities (mutations, copy number and fusions) of 50 genes in 53 patients with aCRC, with a median follow-up of 53 months. We find that RSPO3 fusions occur at a high frequency in advanced disease. We also demonstrate that elevated levels of RSPO3 transcripts have an adverse influence on recurrence-free survival (RFS) and overall survival (OS) in two independent series, making them important markers for future therapeutic decision-making.

2.1. Patients and samples

Fifty-three consecutive advanced colorectal cancer (aCRC) patients who had undergone surgical resection of primary tumor tissues between January 2013 and December 2018 in the Surgical Department of Hospital Nuestra Señora de Sonsoles (Ávila, Spain) were included in the study. Informed consent was given by all patients before they entered the study, in accordance with the Declaration of Helsinki. The study was approved by the local ethics committee of the Hospital Nuestra Señora de Sonsoles. Formalin-fixed, paraffin-embedded (FFPE) tissue samples were obtained from surgical tumor resection in all patients (n = 53). The tumors were diagnosed and classified according to the AJCC (American Joint Committee on Cancer) criteria ²⁰ by a pathologist from the Pathology Department of the Hospital Nuestra Señora de Sonsoles. Primary tumors were localized in the rectum (n = 4), or in the transverse (n = 3), right (caecum, ascending; n = 20) or left (descending and sigmoid; n = 26) colon. The median size of the primary tumors was 5.4 ± 2.2 cm. Most patients exhibited tumors on the surface of the visceral peritoneum (T4a; 77% of cases; Table 1).

2.2. Next-generation sequencing study

Next-generation sequencing (NGS) was performed in the University Hospital of Salamanca (Anatomical Pathology Service, Salamanca, Spain) as follows. From 1 to 6 5-µm sections were obtained from each of the FFPE samples. These samples were deparaffinized in 1 ml of xylene at 50°C for 3 minutes, then washed twice with 1 ml of 100% ethanol, and finally dried at 55°C for 30–45 minutes. Tissue was lysed in 210 µl of a mixture containing proteinase K and its corresponding buffer. The reaction was incubated in heat blocks at 55°C overnight and then for 1 hour at 90°C. Subsequently, 200 µl of each sample were dispensed onto plate 1, and 100 µl of a mixture of DNase and its corresponding buffer were deposited on plate 2, before automating nucleic acid extraction using the Genexus Purification System (Thermo Fisher). Finally, 20 µl of DNA and RNA (10 ng, quantified using Qubit) were deposited into a 96-well plate. This plate, containing the nucleic acids along with the reagents necessary for sequencing, was placed into the Ion Torrent™ Genexus™ Integrated Sequencer. This sequencer is a fully automated NGS system that integrates library preparation, template preparation, sequencing, and data analysis. The panel used was the Oncomine Precision Assay (OPA), which allows simultaneous detection of hotspot mutations (substitutions, insertions, and deletions), copy number variations (CNVs), and gene fusions across 50 cancer-related genes using the Ion Torrent GX5 Chip (Supplementary Table 2). Data were analyzed automatically using Oncomine™ Reporter software (Thermo Fisher). The results were filtered using the Variant Matrix Summary filter (version 5.16), which provides a summary of hotspot mutations including the amino acid changes and allelic frequencies of each variant. Furthermore, it indicates the number of readings of detected fusions and provides numerical values for CNVs. Only results from samples that met the quality parameters for FFPE samples recommended by the manufacturer were considered.

2.3. In silico study

A univariate Kaplan–Meier analysis was performed using the UCSC Xena Functional Genomics Explorer online tools at https://xena.ucsc.edu/. A multivariate analysis was carried out using the database available at https://kmplot.com/analysis/, accessed on April 15, 2024. These analyses were conducted to assess the prognostic value of RSPO3 gene expression in colorectal cancer. For UCSC Xena, the selected study was TCGA Colon and Rectal Cancer, Primary Tumor. Patients were categorized into low- or high-expression groups based on median dichotomization. Recurrence-free survival (RFS) and overall survival (OS) were used as endpoints for survival assessment.

2.4. Statistical analysis

All the values of clinical/biological, morphological, genetic, and evolutionary variables were collected in a database. Statistical significance of group differences between variables, including OS, was calculated using the appropriate tests within SPSS v.21 (IBM Corp., Armonk, NY, USA).

3.1. Patient characteristics

Overall, 53 patients diagnosed with aCRC (26 males and 27 females; median age of 73 years, ranging from 36 to 94 years) at the Nuestra Señora de Sonsoles Hospital (Avila; Spain) were studied. By histological type, all cases were adenocarcinomas, of whom 46 (87%) and 7 (13%) were classified as low and high grade, respectively. In all cases, histopathological grade was systematically confirmed in a second, independent evaluation by another experienced pathologist. The most relevant clinical and laboratory data for each aCRC patient studied are summarized in Supplementary Table 1.

All patients underwent complete surgical tumor resection. aCRC cases were more frequently located in the left colon and had a higher frequency of lymph node metastases (55% of cases) and abnormally higher CEA serum levels (median of 30 ng/ml, ranging from 0.6 to 825 ng/ml). As shown in Table 1, most patients had died by the close of the study (62%). Median follow-up was 53 months (range 2-133 months).

3.2. Frequencies and intratumoral patterns of genetic abnormalities detected by NGS

To identify new biomarkers in patients with aCRC, we performed NGS on 53 primary tumors from these patients. The genetic abnormalities found in primary tumors were most frequently mutations of TP53 (30/53 cases; 57%), KRAS (24/53 cases; 45%), PI3KCA (14/53 cases; 27%) and BRAF (8/53 cases; 15%), followed by RET mutations (5/53; 10%) and RSP03 fusions (4/53; 8%) (Fig. 1). Furthermore, NGS techniques revealed the presence of two or more tumor cell clones in 27 (51%) and 7 (13%) tumors, respectively; in each tumor, each of the clones represented ≥ 3% of cells in the sample (Supplementary Table 1). Supplementary Table 1 provides a detailed description of the different tumor cell clones present in the primary tumor of each of the patients studied. Detailed analysis of the frequency of genetic abnormalities detected in the different tumor cell clones identified in each tumor sample allowed us to speculate on the underlying patterns of intratumoral clonal evolution. Figure 1 summarizes the driver alterations and those acquired throughout the intratumoral clonal evolution detected for the most frequently altered genes. Clones containing driver genetic abnormalities, with a high minor allele frequency (MAF), in a sample were considered to be the ancestral tumor cell clone; in turn, those containing genetic alterations with a low MAF were considered secondary clones. As expected, the presence of a tumor cell clone with TP53 (42% of cases), KRAS (38%), and BRAF (13%) mutations, as well as PTPRK::RSPO3 fusions (8%) were the most frequently detected early aberration profiles. Additional clones acquiring new genetic abnormalities were detected in 34/53 tumors (Supplementary Table 1; Fig. 1) mainly involving PIK3CA (21%), TP53% (15%), RET (10%) and PTEN (8%) genes.

Next, we investigated the association between different alterations detected and the primary tumor's location. A significant correlation was observed between the genetic alterations detected in the ancestral clone and at the site of the primary tumor (Fig. 2). Thus, while alterations at the level of the TP53 and NRAS genes were more frequently observed in the left colon, BRAF mutations and RSPO3 fusions were detected more commonly in the right or transverse colon (p = 0.01). In addition, microsatellite instability (MSI) was most frequent in the right colon (11% in the right vs. 6% in the transverse vs. 2% in the left colon; p = 0.006). By contrast, no significant differences were found between the location of the primary tumor and other genetic anomalies observed in the ancestral clone of the patient's tumor sample.

3.3. Association between PTPRK::RSPO3 fusions in aCRC tumors and features of the disease

Of all the alterations detected, we have focused on the RSPO3 fusion because it could be a therapeutic target in this pathology and because its association with disease parameters is unknown in patients with aCRC. PTPRK::RSPO3 fusions were more often associated with the presence of small tumors, without reaching statistical significance (< 5 cm; p = 0.07), localized in the right colon (p = 0.01), of low histopathological grade (p = 0.05), and without microsatellite instability (p = 0.006) (Table 1). From a prognostic point of view, aCRC patients with PTPRK::RSPO3 fusions also showed a higher frequency of deaths (p = 0.04), which was associated with significantly shortened patient OS (median of 36 vs. 54 months, respectively; p < 0.001). By contrast, no significant differences were found between aCRC cases with and without the PTPRK::RSPO3 with respect to patient age, gender, lymph node involvement, and metastatic process (Table 1).

3.4. In silico validation of the prognostic impact of RSPO3 expression in two independent cohorts of CRC patients

It has been reported that RSPO3 fusion results in increased RNA transcript levels ^{11, 21, 22}, so we decided to evaluate whether these increases could influence OS and RFS in two independent series of consecutive CRC patients from the https://xena.ucsc.edu/ (354 and 376 CRC patients for RFS and OS, respectively) and https://kmplot.com/analysis/index.php?p=service (1167 and 814 CRC patients for RFS and OS, respectively) databases (accessed on 24 April 2024). The high RSPO3 expression was associated with significantly worse outcomes in terms of patient RFS and OS (p ≤ 0.05; Fig. 3).

The search for novel therapies in oncology is a medical need, especially for the treatment of advanced disease. In this context, aCRC exhibits elevated mortality mainly due to the failure of currently available antitumoral agents. The identification of new genetic alterations susceptible to being attacked with drugs in tumor samples from oncology patients is essential if the prognosis of their disease is to improve. NGS has revolutionized molecular tumor testing in oncology by offering the simultaneous assessment of many gene regions using formalin-fixed, paraffin-embedded clinical samples. Currently, NGS is performed in pathological anatomy services to determine molecular alterations that can be actioned with drugs, mainly in non-small cell lung cancer (NSCLC). As the impact of NGS on aCRC has not been thoroughly studied or defined, we initiated a study of NGS with the intention of identifying new potentially actionable molecular alterations in a subgroup of 53 patients with aCRC whose therapeutic options were limited.

Analysis of these patients revealed that the genetic alterations most frequently observed in primary tumors were TP53, KRAS, PI3KCA and BRAF, as reported in previous studies using NGS ^23–25. Interestingly, one type of alteration detected in our series was the group of PTPRK::RSPO3 rearrangements. This fusion and the PTPRK::RSPO2 fusion were initially identified in CRC by Seshagiri et al. using RNA-seq ¹¹. In our series, RSPO3 rearrangements were found in 8% of cases (4 out of 53), an identical proportion (5 out of 68 cases) to those found in the Seshagiri et al. study. However, lower incidences of 1.33% and 2.3% for the RSPO3 rearrangements in CRC were observed in two other studies, respectively ^{12, 22}. These discrepancies in the incidence of RSPO3 rearrangements in CRC could have arisen for several reasons, including the type of methodology used, and the number and stage of the histology samples analyzed in the various studies. In fact, while all the patients analyzed here by NGS were at an advanced stage of the disease (pT4a-b) and were consecutive cases, those analyzed by Shinmura et al. ²² and Hashimoto et al. ¹² using multiplex reverse-transcription PCR were selected cases. However, we recognize that one of the limitations of our study is the relatively small number of samples analyzed (n = 53), which probably explains why we were unable to find the RSPO2 rearrangements in our series. This fusion was detected at lower proportions than were the RSPO3 rearrangements in the three previous studies ^{11, 12, 22}: for example, Hashimoto et al. detected the RSPO2 fusion in one in five patients with RSPO3 rearrangements ¹².

It is well known that activating mutations in the KRAS (35% of cases), BRAF (8%) and NRAS (1–3%) genes are associated with poor response to anti-EGFR therapies, which rules out anti-EGFR-directed therapy as an option for these patients. In this context, Seshagiri et al. ¹¹ and Hashimoto et al. ¹² reported that samples showing RSPO rearrangements also presented somatic mutations on KRAS, BRAF, or NRAS genes (7/7 and 27/29 cases, respectively). This finding also emerged from our series, whereby RSPO3 rearrangements coexisted with KRAS mutations in around half of the patients analyzed. In such circumstances, patients with advanced disease who cannot be treated with anti-EGFR therapies could benefit from treatments with anti-RSPO therapies. Similarly, and consistent with previous observations that patients with RSPO3 rearrangements do not present microsatellite instability (MSI), our study also showed the same association. These findings are important from a therapeutic point of view because this subgroup of RSPO3-positive patients would not currently be susceptible to treatment with immunotherapy. However, this fusion has been proposed as a potential therapeutic target in CRC ¹¹. In fact, several works have demonstrated in preclinical models of CRC that the use of neutralizing antibodies that target RSPO3 fusions have an antitumoral effect ^{14, 26}. Moreover, the combination of RSPO3 antagonists with paclitaxel have a synergistic antitumoral effect in PDX models of CRC with RSPO3 fusions ¹⁵. In conclusion, these studies suggest that targeting RSPO3 fusions could be used as a possible treatment in this pathology. Furthermore, the possible use of effective drugs targeting the RSPO3 fusion would facilitate longer survival in this subgroup of patients in whom we have observed a high frequency of deaths and shorter OS than in RSPO3-negative cases. Thus, therapies targeting the RSPO3 fusion must also be assessed in more preclinical models of CRC that allow us subsequently to evaluate these outcomes in clinical trials in patients with CRC.

The pathogenesis of CRC depends on the location of the tumor, whereby molecular differences may exist between the right and left colon and rectum. Furthermore, it is well known that, in general terms, tumors in the left colon have a better prognosis than those in the right colon. This may be attributed to the molecular differences between them. In fact, in the present study, TP53 and NRAS were preferentially localized in the left side of the colon, whereas BRAF and MSI were more frequently detected in the right side. These findings are consistent with the results obtained by other authors who also analyzed these alterations in CRC using NGS ^{23–25, 27, 28}. Additionally, in the present study, the RSPO3 fusion was preferentially detected in the right and transverse sides of the colon. Similar results were obtained by Hashimoto et al. His group and ours observed a significant association of the presence of RSPO fusions when comparing the right colon versus the left colon plus rectum ¹². Therefore, identifying patients for targeted therapy and designing effective therapeutic strategies requires the location of the primary tumor to be considered.

In this study we also found an association between the presence of the RSPO3 rearrangements and small tumors (< 5 cm) of low histopathological grade with normal CEA values. These clinical parameters could also help identify patients with aCRC RSPO3 rearrangements. However, despite their potential clinical significance, previous reports lacked clinical data, so there is no clear information about the clinicopathological features of patients with RSPO3 rearrangements.

From a prognostic point of view, RSPO3 rearrangements were associated with significantly shorter OS. It is well known that the tumors with RSPO3 rearrangements show abnormally enhanced protein expression. In silico studies in consecutive sporadic CRC (pT1-pT4) cases showed that an increase in RSPO3 gene expression shows a negative influence on disease-free survival and OS. Unfortunately, no studies have evaluated the prognosis of RSPO3 rearrangements in patients with aCRC. To the best of our knowledge, this is the first study to demonstrate an association between RSPO3 rearrangements and clinical-pathological features in advanced disease patients.

In summary, we found a high incidence of RSPO3 rearrangements in advanced disease (8% of cases) of CRC, and a strong association between the presence of RSPO3 rearrangements and patients with small, low histological grade tumors, with normal CEA levels, and that were preferentially located on the right side of the colon. We also showed that abnormal expression of RSPO3 has an adverse influence on OS, making it an important marker for therapeutic decision-making. Additional prospective studies in larger series of patients are required to confirm the clinical utility of this biomarker.

CRC

Colorectal cancer

aCRC

Advanced colorectal cancer

Overall survival

CEA

Carcinoembryonic antigen

dMMR

Mismatch repair-deficient

NTRK

Neurotrophic receptor tyrosine kinase

MSI-H

High degree of microsatellite instability

RFS

Recurrence-free survival

FFPE

Formalin-fixed, paraffin-embedded

AJCC

American Joint Committee on Cancer

OPA

Oncomine precision assay

CNV

Copy number variation

MAF

Minor allele frequency

NSCLC

Non-small cell lung cancer

Data availability: The datasets used and analyzed during the current study available from the corresponding author on reasonable request.

Author Contributions: Conceptualization, M.A., J.C.M, and J.M.S.; Methodology, M.B.R., E.F., and M.R.; Validation: M.A., R.T., J.C.M.; Formal analysis, R.T., E.F.; Investigation, J.M.S., J.C.M.; Writing original draft preparation, R.T., M.A., J.C.M, and J.M.S.; Writing review and editing, J.C.M., J.M.S., and M.A.; Supervision, M.A. and E.F.; Funding acquisition, M.A. and M.R. All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding: This work has been partially supported by grant from the Gerencia Regional de Salud de Castilla y León, Valladolid, Spain.

Institutional Review Board Statement: All procedures were done in accordance with the Declaration of Helsinki and approved by the Local Ethics Committee of the University Hospital of Ávila (Ávila, Spain) under reference 2024_05_1657.

Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Conflicts of Interest: The authors declare no conflict of interest.

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Table 1

Clinical and pathological characteristics of advanced colorectal cancer (aCRC) patients with (n = 4) and without (n = 49) PTPRK-RSPO3 fusion at diagnosis.
Variable	PTPRK-RSPO3 Fusion Yes (n = 4) No (n = 49)
Age (years)*	72 (65–90)	73 (36–94)	NS	73 (36–94)
Gender
Female	2 (50%)	25 (51%)	NS	27 (51%)
Male	2 (50%)	24 (49%)	NS	26 (49%)
Site of primary tumor
Right colon Transverse colon	2 (50%) 2 (50%)	18 (37%) 1 (2%)	0.01	20 (38%) 3 (6%)
Left colon	0 (0%)	26 (53%)	0.01	26 (49%)
Rectum	0 (0%)	4 (8%)		4 (7%)
Histopathological grade
Low	3 (75%)	43 (88%)	0.05	46 (87%)
High	1 (25%)	6 (12%)	0.05	7 (13%)
Lymph node involvement
pN0	2 (50%)	22 (45%)		24 (45%)
pN1	1 (25%)	14 (28%)	NS	15 (28%)
pN2	1 (25%)	13 (27%)	NS	14 (27%)
Metastasis
Yes	2 (50%)	16 (33%)	NS 0.07	18 (34%)
No Size (cm) <5 ≥5	2 (50%) 3 (75%) 1 (25%)	33 (67%) 28 (57%) 21 (43%)	NS 0.07	35 (66%) 31 (58%) 22 (42%)
Microsatellite instability
Yes	0 (0%)	10 (20%)	0.006	10 (19%)
No	4 (100%)	39 (80%)	0.006	43 (81%)
CEA serum levels (ng/ml)*	6.7 (0.7–23)	31 (0.6–825)	0.009	30 (0.6–825)
Number of deaths**	3 (75%)	30 (61%)	0.04	33 (62%)
OS (months)**	36 (2–98)	54 (2-133)	< 0.01	53 (2-133)

Results expressed as number and percentage of cases, or *as median (range). CEA: carcinoembryogenic antigen; OS: overall survival; NS, no statistically significant differences found for any group comparisons (p>0.05).

No competing interests reported.

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20 Aug, 2024

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Incidence of RSPO3 rearrangements in advanced colorectal cancer patients detected by NGS and their relationship with disease characteristics

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Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Patients and samples

2.2. Next-generation sequencing study

2.3. In silico study

2.4. Statistical analysis

3. Results

3.1. Patient characteristics

3.2. Frequencies and intratumoral patterns of genetic abnormalities detected by NGS

3.3. Association between PTPRK::RSPO3 fusions in aCRC tumors and features of the disease

4. Discussion

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1