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.