With the development of tumor diagnosis and treatment technology, the incidence of multiple primary malignant tumors (MPMTs) has increased greatly (Kumagai et al., 2001). However, triple primary cancer is still a very rare finding, and its genetic change is not clear yet. In this study, we reported the case of a 70-year-old Chinese man with triple primary cancers of esophagus, stomach and right-sided colon and clarified the genetic abnormalities underlying them.

A 70-year-old male patient was admitted to our hospital complaining of choking while eating for more than 1 month on March 20, 2019. CT showed tumor signs in the middle of esophagus on March 6, 2019. Esophagogastroduodenoscopy demonstrated the patient was suffering from esophageal carcinoma and gastric cancer (Figures 1A,C). He had no family history of malignancy. Pathology examination confirmed they were locally advanced esophageal squamous cell carcinoma and adenocarcinoma of gastric horn (Figures 1B,D). On March 21, 2019, the patient underwent laparoscopic feeding jejunostomy and postoperative radiotherapy.

On November 15 and 18, 2019, esophagogastroduodenoscopy and abdominal CT showed that esophageal and gastric cancers were under control. However, CT indicated a tumor in the right-sided colon. On November 19, 2019, colonoscopy and pathological findings confirmed it was locally advanced moderately differentiated adenocarcinoma of colon (Figures 1E,F). Immunohistochemical stains revealed proficient mismatch repair proteins and positive expression of S-100, but negative expression of D2-40 or CD34. The patients underwent laparoscopic-assisted resection of right-sided colonic cancer and lysis of abdominal adhesions on November 22, 2019. By the time of submission of this manuscript, the patient had been well and no sign of recurrence or metastasis had been detected. The timeline was shown as Figure 2.

In order to seek possible personalized therapy strategies, the paraffin-embedded tissue blocks from malignant esophagus, stomach and right-sided colon, respectively, were subjected to next-generation sequencing (NGS) using a pan-cancer 599-gene panel [ChosenOne599, ChosenMed Technology (Beijing) Co. Ltd, Beijing, China] on July 20, 2020. NGS demonstrated the TMB value was 10.11 Muts/Mb, 11.37 Muts/Mb, 6.32 Muts/Mb in the tumor tissues of esophagus, stomach and colon, respectively, which ranked 29.58% (moderate), 6.45% (high) and 62.73% (low) in patients with corresponding malignancy. TP53 mutation was identified in all three tumor tissues. However, the mutation location was different from each other. In the esophagus cancer tissues, two TP53 variations were identified (NM_000546: exon6: c.580C>T: p.L194F and NM_000546: exon8: c.814G>A: p.V272M). In the gastric carcinoma tissues, there were also two TP53 alterations (NM_000546: exon4: c.375G>A: p.T125T and NM_000546: exon7: c.715A>G: p.N239D). In the right-sided colon carcinoma tissues, there was one TP53 mutation (NM_000546: exon8: c.821T>G: p.V274G) (Table 1).

At the same time, other important variations were also identified in the esophagus carcinoma tissues, including ATRX (NM_000489: exon21: c.5424T>G: p.Y1808^*), CHEK1 (NM_001114121: exon7: c.676dup: p.T226fs), copy number variations (CNVs) of CCND1 and FGF3/4/19. In the gastric carcinoma tissues, the same CHEK1 variation was also detected. In the colon carcinoma tissues, there were two APC variations (NM_000038: exon16: c.4468del: p.H1490fs and NM_000038: exon16: c.2621C>G: p.S874^*, Table 1).

MPMTs are referred to the presence at least two histologically distinct malignancies that are not caused by recurrence or metastasis in the same patient. For the diagnosis of two primary malignant tumors, the following conditions must be met: (Dranka-Bojarowska and Lewinski, 2019) (a) Pathology confirms that both are malignancies; (b) Malignancies must locate separately. If they are close, they must be separated by a healthy mucosa with an area of at least 2 cm. If they are formed in the same organ, the period of more than 5 years after diagnosis must pass; (c) The possibility that the second malignancy is caused by metastasis from the primary site is excluded.

There are two kinds of MPMTs: synchronous and metachronous MPMTs. The former is defined as the diagnosis of a second primary malignancy within 6 months of the first primary malignancy, while the latter refers to the second primary malignancy diagnosed over 6 months after the first primary one (Zhai et al., 2018). Studies revealed that MPMTs often occur in the digestive system, followed by digestive-respiratory system in China (Zhai et al., 2018). The patient in our study was firstly diagnosed as esophagus carcinoma, then as gastric adenocarcinoma within one month (synchronously), finally as colon cancer 8 months later (metachronously).

To date, the molecular pathogenesis of MPMTs remains elusive. Some studies speculated that the development of MPMTs was associated with unhealthy lifestyle, genetic susceptibility, side effects of chemotherapy and radiotherapy, weak immunity, etc. The patient had a long history of smoking and drinking, and it might be one of predisposing factors of his MPMTs.

Studies demonstrated cancer develops from the accumulation of mutations in oncogenes and tumor suppressor genes (Califano et al., 1996). It is in line with this study, as the NGS data demonstrated the TMB value was middle to high, i.e., 10.11 Muts/Mb, 11.37 Muts/Mb, 6.32 Muts/Mb in the tumor sample of esophagus, stomach and colon, respectively, which ranked 29.58, 6.45, and 62.73% in the patients with corresponding malignancy.

Study of Dranka-Bojarowska and Lewinski (2019) revealed that genetic background, especially mutation of tumor suppressor gene TP53, BRCA1-associated protein 1 (BAP1) (Cheung et al., 2015) or partner and localizer of BRCA2 (PALB2) (Schrader et al., 2016) played a crucial role in the development of MPMTs. Germline TP53 mutation is closely associated with Li-Fraumeni Syndrome, characterized by a high frequency of various malignancies (Consul et al., 2020). In the patient, five different TP53 mutations were detected in the patient's three tumors samples. Currently, clinical trial on Adavosertib (AZD1775, NCT02448329) targeted against TP53 variations is ongoing.

In addition, ATRX variation (NM_000489: exon21: c.5424T>G: p.Y1808^*) was also detected in the esophagus carcinoma tissues. According to the annotations of OncoKB, the ATRX mutation may be pathogenic. Previous studies have shown that loss of ATRX is associated with a variety of tumors such as sarcomas, hepatic angiosarcomas, gliomas and so on (de Wilde et al., 2012; Marinoni et al., 2014; Qadeer et al., 2014; Leeper et al., 2015; Liau et al., 2015a,b,c; Rodriguez et al., 2016; Singhi et al., 2017). There is currently no targeted therapy against the ATRX mutation.

The patient harbored CHEK1 (NM_001114121: exon7: c.676dup:p.T226fs) in the tissues of esophagus carcinoma and gastric carcinoma (Table 1), Previous studies have shown that CHEK1 mutation was associated with multiple malignancies, including lung cancer, prostate cancer, cervical cancer, and colorectal cancer (Gali-Muhtasib et al., 2008; Mazumder Indra et al., 2011; Al Nakouzi et al., 2014; Liu et al., 2015). According to the annotations of OncoKB, the CHEK1 mutation may lead to loss of protein function, which is possibly pathogenic. However, no targeted therapy against the CHEK1 mutation has been approved.

Besides, the patient also harbored CNVs of CCND1 and FGF3/4/19 (Table 1). CCND1 often co-amplified with FGF3/4/19. A study reported that 86.7% (26/30) of the patients with esophageal carcinoma showed positive expression of CCND1 through immunohistochemistry (Hu et al., 2016). Clinical trials on Palbociclib (NCT04439201) and Abemaciclib (NCT03356223) targeted against CCND1 amplification are ongoing.

In the colon carcinoma tissues, two APC alterations were detected (Table 1). According to the annotations of OncoKB, both APC mutations could cause protein loss of function, abnormal Wnt/β-catenin signaling pathway and promote the development of colon cancer. To date, No targeted drug against these two APC mutations have been approved.

Germline mutations were not mentioned in this study because none of the patient's family members had malignant tumors.

The treatment strategies for MPMTs depend on some factors, such as the tumor stage, patient's age and comorbidities. Furthermore, a multidisciplinary team management approach is necessary for personalized therapy. In metachronous MPMTs, the treatment always involves sequential therapy for each tumor, whereas in synchronous MPMTs, individualized and single treatment is decided upon after appropriate evaluation and consensus is reached by multidisciplinary team (Vogt et al., 2017). The patient underwent surgery and postoperative radiotherapy for synchronous esophageal and gastric carcinomas. Then, he underwent laparoscopic-assisted resection of right-sided colonic cancer and lysis of abdominal adhesions. By the time of submitting this manuscript, the patient had been well and no sign of recurrence or metastasis had been detected.

To the best of our knowledge, this case is the first one to clarify the genetic abnormalities of triple primary cancers of esophagus, stomach and colon in a Chinese patient. It may contribute to understanding the molecular pathogenesis of multiple primary digestive malignancies and providing valuable treatment strategies for the similar patients in the future. The limitation of this study is that NGS-based multi-gene panel couldn't identify all genetic predispositions like epigenetic modifications and other CNVs.

All of the data supporting the findings in this study are available upon reasonable request from the corresponding author (Yuedong Wang).

The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Affiliated Hospital Zhejiang University School of Medicine. Written informed consent for participation in the study, publication of clinical details and images was obtained from the patient.

YW: conceptualization. XZ: writing-original draft. SC and EM: conducting bioinformatics analysis. LH and KS: writing-review and editing. All authors contributed to the article and approved the submitted version.

SC and EM are employees at ChosenMed Technology. 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.