Yan-Jun Qu, Qian-Shi Zhang, Bo Wang, Feng Zhang, Evenki Pan, Chun-Yan Zhao, Si-Ye Liu, Li-Ping Fang

Abstract

Key Words: Multiple primary colorectal carcinoma; Next-generation sequencing;Cetuximab; RET fusion; Case report

INTRODUCTION

Colorectal cancer (CRC) is one of the most lethal and prevalent malignancies worldwide, with approximately half of CRC patients eventually developing metastatic CRC[1].However, the occurrence of multiple primary colorectal carcinoma(MPCC) is rare (between 1.1% and 8.1%)[2].MPCC is defined as the discovery of two or more primary colorectal carcinomas in an individual occurring either synchronously or metachronously[3].Preoperative detection of multiple primary cancers is important when planning treatment.Nevertheless, current diagnostic criteria may not identify all MPCC patients, leading to inappropriate treatment and follow-up plans[4].To reduce the rate of misdiagnosis or missed diagnosis, recent studies have proposed the use of molecular testing and genomic profiling[5].Herein, we report a case of MPCC that was initially missedviaimaging but was diagnosed using next-generation sequencing (NGS).The patient was found to have two tumors on the surface of the colorectum which had completely different mutation patterns.Furthermore, aKIF5B-RETfusion was identified following cetuximab resistance, which has not been previously reported in CRC.

CASE PRESENTATION

Chief complaints

A 63-year-old man presented with hematochezia and abdominal pain in June 2020.

History of present illness

He was diagnosed with colorectal carcinoma initially.

History of past illness

He had no major illnesses in the past.

Personal and family history

He and his family both had no history of cancer.

Physical examination

The patient's vital signs were normal.

Laboratory examinations

To search for an efficient therapeutic strategy, genomic DNA from the formalin-fixed paraffin-embedded sample of lesion A and circulating tumor DNA (ctDNA) from plasma were subjected to targeted NGS of 425 cancer-related genes (Nanjing Geneseeq Technology Inc.) (Figure 1A).A comparison of the genetic alterations in lesion A and B can be found in Supplementary Table 1.

Imaging examinations

The computed tomography (CT) scan showed two lesions (defined as A and B) on the liver.Endoscopic resection revealed that the two lesions were located on the surface of the colon, and only lesion A was removed (Figure 2).

Figure 1 Sequence of the patient’s multiline treatments and computed tomography images, the levels of colorectal cancer biomarkers,and allele frequencies of circulating tumor DNA alterations during treatments.A: Timeline of multiline therapies received by the patient; B: Computed tomography images of liver metastases during treatment.Lesions are indicated by the red circles.The number represents the change in the size of liver metastases compared with the previous image; C: The levels of the colorectal cancer biomarkers carcinoembryonic antigen and carbohydrate antigen 19-9 are shown by the blue and black lines, respectively.The four background colors represent each treatment line; D: The allele frequencies of circulating tumor DNA alterations are shown during cetuximab treatment.CEA: Carcinoembryonic antigen; CA19-9: Carbohydrate antigen 19-9; PR: Partial response; PD: Progressive disease.

FINAL DIAGNOSIS

He was diagnosed with stage Ⅳ (pT3N1M1) colorectal carcinoma with liver metastases in June 2020.

Figure 2 Schematic diagram of the use of targeted next-generation sequencing for the diagnosis of two primary colorectal tumors, and the corresponding treatment and efficacy.NGS: Next-generation sequencing; FFPE: Formalin-fixed paraffin-embedded; CRC: Colorectal cancer.

TREATMENT

As the patient presented with intestinal bleeding, endoscopic resection was performed to alleviate his symptoms.Based on the identification ofKRASG12D with a mutation allelic frequency (MAF) of 41.9% identified in lesion A, the patient was administered XELOX plus bevacizumab (oxaliplatin 130 mg/m2on day 1, capecitabine 1500 mg/m2twice daily for 14 d, bevacizumab 7.5 mg/kg day 1) every 3 wk as first-line treatment.The patient achieved an initial partial response(PR) with sustained response ongoing for 11 mo.In January 2021, the tumor was evaluated as progressive disease (PD),and second-line chemotherapy was initiated with irinotecan (180 mg/m2day 1), raltitrexed (3 mg/m2day 1) and bevacizumab (5 mg/kg day 1) every 2 wk.Unfortunately, the liver lesion size increased by 35% compared to baseline,indicating a PD (Figure 1B).In April 2021, both plasma and lesion B were subjected to NGS and four identical mutation types were identified, with noKRASmutations (Supplementary Table 1).A comparison of genomic alterations between lesion A and B revealed completely different mutation landscapes.Furthermore, immunohistochemistry illustrated significant differences between the two lesions (Supplementary Figure 1), which confirmed the diagnosis of MPCC.As the patient had wild-typeKRAS, he was treated with irinotecan (180 mg/m2day 1), raltitrexed (3 mg/m2day 1), plus cetuximab (500 mg/m2day 1), a monoclonal antibody that blocks the epidermal growth factor receptor (EGFR), every 2 wk in April 2021.Plasma ctDNA sequencing and CT scans were conducted every 2 wk and 2 mo, respectively (Figure 1B and C).Two months later, it was observed that the size of liver metastases had decreased by 75% compared to the previous examination and ctDNA had rapidly decreased to less than 1% (Figure 1B and D).Moreover, the tumor markers for CRC, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) also significantly decreased to normal levels (Figure 1C), indicating a PR.

OUTCOME AND FOLLOW-UP

In August 2021, stable disease was observed with a 4% decrease in liver metastasis size compared to the previous examination, and no significant increase in CEA and CA19-9 Levels was observed (Figure1B and C).However, the allele frequencies (AFs) of ctDNA alterations in plasma samples were considerably elevated (Figure 1D and Supplementary Table 1).One month later, the tumor size had increased by 30% compared to the previous month, with a significant increase in CEA and CA19-9 levels (Figure 1B and C), indicating a PD.Due to the occurrence ofKIF5B-RETfusion (MAF = 18.5%), we recommended the use of pralsetinib, a selective RET inhibitor, but the patient refused.Consequently, the fourth-line chemotherapy with XELOX and bevacizumab was administered.Two months later, the tumor size had increased by 21% (Figure 1B).Unfortunately, the patient later passed away due to hepatic failure in November 2021.

DISCUSSION

MPCC was initially discovered by Warren and Gates in 1941[6].Despite its rarity, the occurrence of MPCC is showing an upward trend[2].Due to a lack of understanding regarding MPCC and limited diagnostic techniques, it is always challenging to distinguish between multiple primary cancers and tumor metastasis.The emergence of NGS has already changed the landscape of cancer studies and is now widely used in the diagnosis of multiple primary cancers[7,8].In our case, lesions A and B, were initially misdiagnosed as a primary lesion with metastasis due to their similar features.Thus,only tumor A and plasma samples were subjected to targeted NGS, revealing a completely different mutation pattern.Further targeted NGS on lesion B showed that these two tumor lesions, A and B, did not share any mutations.Through genetic profiling, it was confirmed that lesions A and B were independent primary lesions.Of note, the molecular variations identified by NGS aided in the diagnosis of both primary tumors.It was also hypothesized that tumor B may have played a role in the development of liver metastases, but the patient declined a liver biopsy.Subsequent NGS supported this hypothesis, as there was a strong correlation between tumor B and plasma ctDNA.

Surgical intervention has long been the ideal choice for cancer patients[9], but not for patients with metastatic lesions.Our patient presented with intestinal bleeding, and we opted for surgery to ease his symptoms.For those patients who cannot undergo surgery, radiotherapy and chemotherapy are the primary methods for disease control[10].In addition,targeted therapy is an alternative approach that has proven to be effective in prolonging the overall survival rate of CRC patients[1].The first targeted agent for CRC approved by the Food and Drug Administration was cetuximab, a monoclonal antibody that blocks EGFR, in 2004[11].The efficacy of anti-EGFR therapy is dependent on the mutational status of downstream signaling molecules of the EGFR pathway, such asKRAS,NRAS,PIK3CA, andBRAF.Patients with aKRASwild-type tumor are more likely to respond to this therapy[12].In our patient, lesion B and ctDNA showed wildtypeKRAS, while lesion A, which was removed, had aKRASG12D mutation.Therefore, cetuximab was administered and the patient benefited from this treatment, with a decrease in liver tumor size, a reduction in AF of ctDNA, and lower serum tumor markers (CEA and CA19-9).Initially, the patient was found to have drug resistance by NGS, followed by serum tumor markers and a CT scan.Previous reports indicate that serial ctDNA profiling can detect disease progression earlier than CT scans[13].Additionally, continuous monitoring of ctDNA can provide a more accurate understanding of the tumor, which can improve personalized treatment decisions[14].To the best of our knowledge, this is the first timeKIF5B-RETfusion has been discovered in a CRC patient with resistance to cetuximab.The emerging RET fusion variant is a significant driver gene for drug resistance in multiple progressive cancers, such as non-small cell lung cancer.Zhuet al[15] reported that the emergence of theKIF5B-RETfusion gene may cause acquired resistance to EGFR-tyrosine kinase inhibitors in EGFR-mutant lung adenocarcinomas[15].Hence, we propose that thisKIF5B-RETfusion gene may be a novel factor contributing to acquired resistance to cetuximab inKRASwild-type CRCs.Nevertheless, the patient declined treatment with pralsetinib, a targeted RET inhibitor.

The limitations of the single case presentation in this study should be noted.While theKIF5B-RETfusion is a possible resistance mechanism to cetuximab, more pre-clinical research and clinical data are required to confirm its potential.NGS is a powerful tool that can provide valuable insights into an individual's genetic composition[16].It can help identify genomic variations potentially linked to certain diseases or conditions, allowing for earlier diagnosis and selection of more effective treatment[17].In this particular case, NGS played a pivotal role in diagnosing MPCC and offered direction for its treatment.

CONCLUSION

In summary, we report the rare case of a 63-year-old male patient with MPCC diagnosed through genetic profiling.The patient was treated with cetuximab based on wild-typeKRASidentified on the lesion and later developed novel mutations includingKIF5B-RETfusion, which provides a possible resistance mechanism to anti-EGFR therapy.This case highlights the necessity of using genetic testing for identifying primary tumors and the importance of longitudinal ctDNA profiling, which may trigger the development of effective therapeutic strategies.

FOOTNOTES

Author contributions:Qu YJ, Zhang QS, Wang B and Zhang F contributed equally to this work; All authors contributed to data analysis and drafting or revising the manuscript; All authors agreed on the journal to which the article is submitted, provided final approval of the version to be published, and agreed to be accountable for all aspects of the study.

Informed consent statement:This research was approved by the Ethics Committee of The Second Hospital of Dalian Medical University.Written informed consent to publish the clinical details and images were obtained from the patient.

Conflict-of-interest statement:Authors Evenki Pan and Chunyan Zhao are employed by Nanjing Geneseeq Technology Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

CARE Checklist (2016) statement:The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016).

Open-Access:This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers.It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial.See: https://creativecommons.org/Licenses/by-nc/4.0/

Country/Territory of origin:China

ORCID number:Yan-Jun Qu 0000-0001-7336-1778; Qian-Shi Zhang 0000-0003-3336-9934; Bo Wang 0000-0003-4172-9271; Evenki Pan 0000-0001-9214-5108; Li-Ping Fang 0000-0001-5848-5627.

S-Editor:Fan JR

L-Editor:A

P-Editor:Yu HG