Gastric cancer (GC) is the fifth most diagnosed malignancy worldwide with over 1 million estimated new cases annually (1). Due to its intratumoral and intertumoral huge heterogeneity, several classifications have been proposed to categorize different morphological subtypes of GC. The most commonly used classifications are those published by the Japanese Gastric Cancer Association (JGCA) (2), the World Health Organization (WHO) (3), and Lauren (4). However, a categorization into a few macro groups is burdened by excessive internal heterogeneity, resulting in conflicting evidence about the ability of the histopathological phenotype to predict patient prognosis or response to therapy. In 2019, the European Chapter of the International Gastric Cancer Association (IGCA) proposed the adoption of the recent fifth edition of the WHO classification for each newly diagnosed GC (5, 6). The latest classification recognized five main histological subtypes of GC: tubular, papillary, mucinous, poorly cohesive, and mixed adenocarcinomas. The latter account for 6%–27% of all GCs and are characterized by the coexistence of two or more distinct histological components: glandular (tubular/papillary) and signet ring cell (SRC)/poorly cohesive. Recently, the percentage of SRC was proposed as an independent prognostic factor of cancer-related survival in a poorly cohesive histotype, and high amounts of SRC were related to lower recurrence and mortality rates, better prognosis, and most advantageous clinicopathological features (6–8). Surprisingly, clinicopathological and prognostic aspects of mixed histotype have not been deeply investigated. Available data suggest that patients with mixed adenocarcinomas had a poorer prognosis (9, 10) and higher incidence of lymph node metastasis than those with only one component. Furthermore, to the best of our knowledge, no previous research has studied the connection between the amount of SRCs and biological as well as prognostic differences of mixed GC.

We conducted this study to compare the clinicopathological features and the prognostic differences of mixed GC according to the percentage of SRCs.

A retrospective analysis was performed through a prospectively maintained database of adult patients from the Division of Surgical Oncology at the University of Siena, Italy. These patients all underwent upfront surgery for GC between 1995 and 2016, and their tissue samples were available for research at an institutionally approved tissue bank. Only patients with confirmed “mixed” gastric carcinoma, according to the last WHO classification (3), submitted to surgical treatment with curative purpose, were recruited for the analysis. All enrolled patients were then reclassified into the three groups according to the percentage of cells with signet ring features as proposed by the European Chapter of IGCA (6): Group 1 (presenting ≤10% of SRCs), Group 2 (presenting <90% but >10% of SRCs), and Group 3 (presenting ≥90% of SRCs).

Electronic medical records and pathological reports were thoroughly analyzed to acquire information related to age, sex, tumor size, location, depth of invasion, perineural/lymphovascular invasion, lymph node metastases, and TNM (8th edition) according to the American Joint Committee on Cancer Staging Manual (11).

Our results showed that a high percentage of SRCs in mixed-type GC was linked to the most favorable clinicopathological characteristics as well as better survival outcomes, providing new insight into the evidence of the prognostic value of SRC also in mixed-type GC.

GCs exhibit a highly heterogeneous nature in terms of histological type and differentiation (1, 15). In the age of tailored surgery, it has been of greater interest not only to merely classify the tumor from a histological point of view but also to establish a pathological classification system with an independent prognostic peculiarity pursuing personalized clinical management. Although several classification systems for GC have been proposed over the years (3, 4, 16–21), the question of which of them may be relevant in the clinical setting has not yet been answered. Additionally, it would appear that the 50% SRC threshold proposed in the previous classifications has been vague. The World Health Organization (WHO) classification system is recognized as one of the most detailed among all classification systems and is employed by several researchers investigating the pathological and prognostic aspects of GC (1). Additionally, the 5th update of WHO classification has led to a marked improvement in GC knowledge. The mixed type has been described as a distinct histopathological entity that includes both glandular (tubular/papillary) and SRC/poorly cohesive components. Similarly, the Lauren classification, with a comparable significance to that provided by WHO, also identifies mixed type as a distinct category with a mixture of intestinal and diffuse components (4, 22). Evidence gathered over the last years has shown conflicting results, and the prognostic impact of this histotype is still debated among the scientific community, with some authors describing the mixed type as a predictor of poor prognosis and high risk of lymph node metastasis (23–25) and others reporting similar outcomes compared with other histotypes (26, 27).

Our group previously reported that the percentage of SRCs is inversely related to tumor aggressiveness in poorly cohesive GCs confirming the role of SRC pattern as an independent predictor of survival (8). By extending the concept of this new evidence to mixed histology, we can postulate that the inconsistent findings of previous studies (23–28) could be explained by the heterogeneity of SRC components. To the best of our knowledge, our study is the first to evaluate the clinicopathological aspects and prognostic outcomes of mixed-type GCs according to SRC percentages. Nonetheless, the retrospective design and the small sample size could affect our results in terms of bias.

As discussed, several studies showed that mixed histotype is associated with aggressive behavior, such as tumor size, lymphatic invasion, and lymph node metastasis (23, 29, 30). On the other hand, Zhong et al. (26), in a retrospective study on a total of 298 patients, stated that histological mixed type was not an independent risk factor for lymph node metastasis and was not associated with more aggressive characteristics in comparison with other histotypes. The same results were also obtained by Min et al. (27) on a cohort of 1577 patients.

In an attempt to explain the discrepancies arising from previous studies, this paper introduces new and intriguing evidence as regards the mixed-type GCs. By evaluating histopathological characteristics and prognoses, we observed that compared with those with more than 10% of SRCs, cancer patients with less than 10% of SRCs were younger and were more frequently associated with increased size, depth of invasion, nodal involvement, and advanced pathologic stage at diagnosis. Additionally, our data highlighted a distinctive tendency toward a survival difference according to SRC percentage between the two groups of mixed-type GC. Definitively, according to this research, the SRC pattern was an independent factor predicting prognosis in mixed-type GC patients.

Molecular mechanisms underlying the unfavorable behavior are still unclear. Anyway, it has been postulated that more aggressive clinicopathological as well as morphological findings of mixed-type cancers (10, 31) could be attributed to the angiogenetic process and cell proliferation, to cytosine-phosphate-guanine (CpG) island hypermethylation (32), or to the unregulated expression of proteins such as Ki-67, E-cadherin, and VEGF proteins, which were involved in proliferation (10), adhesion, and angiogenesis activities (10, 28). Interestingly, some researchers argued that mixed type would undergo transitional events leading to phenotype transformation along with cancer progression (33). Based on this finding, further research aiming at SRC amount comparison between endoscopic biopsies and resected specimens could be conducted.

We provide compelling evidence suggesting that distinct clinicopathological and prognostic findings are associated with the amount of SRCs in mixed-type gastric tumors. The significance of these different SRC percentages has not been investigated deeper, and our focus on the clinical outcome rather than genetic alterations could represent a limit for our study. Nevertheless, our data have confirmed, through undeniable evidence, that the SRC pattern is an independent predictor of survival also in mixed-type GC. Results demonstrate that a lower SRC proportion is associated with poorer OS. Thus, the proportion of SRCs can be considered a marker of differentiation.

In conclusion, the percentage of SRCs is inversely related to aggressive behavior and poor prognosis in mixed-type GCs, highlighting the role of SRC amount as an independent predictor of survival. Further studies are needed to investigate specific genetic and molecular profiles underlying the SRC effects to reach a clearer interpretation of these findings.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Institutional Review Committee of the University of Siena. The patients/participants provided their written informed consent to participate in this study.

Study concepts: LMar, FR, MA, and KP. Study design: LMar, FR, OS, and KP. Data acquisition: LMar, MA, LR, LMal, and KP. Quality control of data and algorithms: VS, MC, LMal, RP, IB, and OS. Data analysis and interpretation: LMar, DM, and MA. Statistical analysis: LMar, LR, RP, and IB. Manuscript preparation: LMar, DM, FR, VS, LR, RP, MA, and MC. Manuscript editing: LR, LC, RP, MA, and OS. Manuscript review: LMar, MA, MC, VS, LR, LC, IB, and KP. All authors contributed to the article and approved the submitted version.

The 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.

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