Introduction

Colorectal cancer is one of the most common malignancies worldwide, and dirty necrosis was first described as a characteristic histopathological feature in colorectal carcinomas. In metastatic foci, dirty necrosis is a helpful morphologic finding in determining the tumor origin1.

Tumor necrosis is a prognostically important finding that has been described in many types of cancer. Tumor necrosis is a coagulation necrosis associated with rapid tumor growth and insufficient vascular support of tumor cells2. Dirty necrosis is a special type in which neutrophil leukocytes and necrotic debris are observed in the gland lumens1. It is also known to have prognostic significance in some types of cancer, such as renal cell carcinomas and glioblastomas3.

Neutrophil leukocytes are cells with a very short life span. Even if their life span is prolonged in the presence of infection and inflammation, they die by apoptosis after a while. However, in the case of inadequate clearance, apoptotic neutrophils undergo secondary necrosis, leading to the onset of inflammation and the immune process4. Tumor-associated neutrophils (TANs) can have anti-tumoral (N1) and pro-tumoral (N2) effects. N1 TANs are neutrophils that cause a cytotoxic effect on tumor cells by releasing free oxygen radicals through NADPH oxidase activity. N2 TANs are generally described as pro-tumorigenic cells, often by promoting angiogenesis and facilitating tumor progression5.

Neutrophils also utilize an antimicrobial defense mechanism known as NETosis. In addition to fighting infections, neutrophils also use NETosis to target tumors and are commonly found in the tumor microenvironment6. In vitro studies have shown that NETosis is associated with N2 neutrophils, which have protumorigenic effects7. For this reason, it is associated with poor prognosis in many types of cancer, such as gastrointestinal, cervical and breast cancers8,9,10.

In a study in kidney tumors, the presence of dirty necrosis was shown to be associated with NETosis. NETs are involved in tumor progression by protecting cancer cells from T lymphocytes and NK cells in the tumor microenvironment11. In a recent study, ın gastric cancer suggest that NET-related gene expression may also have prognostic value, identifying both high- and low-risk groups12.

Gastric cancer is one of the most common cancers in the world and is still one of the leading causes of cancer deaths. Despite molecular and therapeutic studies, the 5-year survival rate is about 20%13.

Dirty necrosis is a finding associated with TAN and NETosis. NETosis is a poor prognostic finding in many cancer types. However, there is no morphological study evaluating the prognostic significance of dirty necrosis in gastric cancer.

The objective of this study is to determine the prognostic relevance of tumor necrosis and dirty necrosis in gastric cancer through an analysis of clinicopathological parameters.

Materials and methodology

We included 187 patients who underwent subtotal or total gastrectomy operation for gastric cancer at Eskisehir Osmangazi University Faculty of Medicine Hospital between 2013 and 2024. Patients who received neoadjuvant chemotherapy and had primary distal esophageal tumors were excluded. Neuroendocrine tumors and lymphoid neoplasms were excluded. Demographic data such as gender and age were recorded.

Histopathologic analysis

The slides with tumor tissue were re-evaluated by two pathologists (NSS and OFM) and the presence of tumor necrosis and dirty necrosis were evaluated. Dirty necrosis and tumor necrosis were evaluated similarly. All H&E-stained slides containing tumor tissue were reviewed at 10× and 20× magnification. In poorly cohesive and poorly differentiated carcinomas, the presence of dirty necrosis was assessed in the tubular areas of the tumor. In addition, cases with dirty necrosis were classified as rare and extensive. The presence of dirty necrosis in a few gland lumens was classified as rare, while the presence of dirty necrosis in almost every gland lumen was classified as extensive (Fig. 1a–c). The presence of tumor-infiltrating lymphocytes (TILs) was evaluated and classified as sparse or prominent. Sparse TILs were defined as scattered infiltration involving approximately < 10% of the tumor area, whereas prominent TILs were defined as dense infiltration involving ≥ 10% (Fig. 2a–c).

Fig. 1
figure 1

(a) Rare dirty necrosis (x100, H&E). (b) Extensive dirty necrosis (x100, H&E). (c) Extensive dirty necrosis (x100, H&E).

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Pathological prognostic parameters of the tumors, tumor histological type (according to WHO and Lauren classification), tumor size, tumor grade, pathological T stage, pathological N stage, presence of LVI-PNI, Her2 status were recorded.

Fig. 2
figure 2

(a) Case without TIL (x200, H&E); (b) case with sparse TIL (x200, H&E); prominent TIL and extensive dirty necrosis (x200, H&E)

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Of the patients, 122 (65.2%) were male and 65 (34.8%) were female. The mean patient age was 64.3 ± 11.6 years (min 22- max 88). According to the WHO classification, 48 (25.8%) patients were poorly cohesive, and 138 (74.2%) patients were non-poorly cohesive. In the non-poorly cohesive group, 1 case was hepatoid, 1 case was medullary, 5 cases were mucinous and the remaining 131 cases were tubular histologic subtype. Tumor locations were: antrum in 82 cases (55.1%), corpus in 80 cases (42.8%), and the entire stomach in 4 cases (2.1%). The mean tumor size was 5.66 ± 3.88 cm (min 0-max 26 cm). According to pathological tumor staging, there were 21 (11.2%) pT1, 19 (10.2%) pT2, 68 (36.4%) pT3, and 79 (42.2%) pT4 cases. According to pathologic node staging, there were pN0 43 (23%), pN1 25 (13.4%), pN2 37 (19.8%), and pN3 82 (43.8%) cases. 44 of the pN3 cases were pN3a and 38 were pN3b.

In addition, the associations of dirty necrosis and tumor necrosis with recurrence-free survival and overall survival were also evaluated.

Statistical analysis

Fisher’s exact test, continuity correction, and Pearson chi-square tests were performed to evaluate the statistical association between the clinicopathological variables and dirty necrosis and tumor necrosis. Univariate and multivariate analyses were performed by using the Cox proportional hazards regression model. For multivariate analysis, backward stepwise (wald) method was utilized. Survival curves were calculated using the Kaplan–Meier method and were examined for statistical significance using the log-rank test. A P-value of less than 0.05 was considered statistically significant.

All methods used in this study were carried out in accordance with relevant guidelines and regulations. Ethics approval was obtained from the Non-Drug Clinical Research Ethics Committee of Eskişehir Osmangazi University Faculty of Medicine (protocol number: E-2024-37). Informed consent was obtained from all participants and/or their legal guardians.

Results

Dirty necrosis was detected in 74 (39.6%) cases. It was observed as rare in 38 (51.34%) and extensive in 36 (48.66%) of these cases. Tumor necrosis was found in only 18 (9.6%) cases.

Thirty-seven (19.8%) cases relapsed, and 112 (59.9%) patients died due to the disease.

When the relationship between clinicopathological data and dirty necrosis was evaluated, it was found to be associated with tumor type (p < 0.05), tumor grade (p < 0.05), pN stage (p = 0.001), perineural invasion (p = 0.022), TIL (p < 0.05) and ex status (p < 0.05). According to these statistically significant results, the presence of dirty necrosis was associated with lower tumor grade, less perineural invasion, lower pN stage, and lower ex rate, and was directly associated with significant TIL (Table 1).

Table 1 Relationship between dirty necrosis and clinicopathologic features in gastric carcinomas.
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Furthermore, a significant association with recurrence-free survival was found, and accordingly, cases with dirty necrosis were associated with significantly longer recurrence-free survival (p < 0.001). Similarly, overall survival was significantly higher in patients with dirty necrosis (p < 0.001).

In our initial analysis, the presence of dirty necrosis appeared to be a favorable prognostic indicator. However, we hypothesized that the inclusion of poorly cohesive carcinoma cases might have influenced this finding. To clarify this, we performed a subgroup analysis excluding patients with the poorly cohesive type. We found it to be a better prognostic factor, showing significant associations with the tumor grade (p < 0.001), pN stage (p = 0.013), TIL relationship (p < 0.001), and ex status (p = 0.023).

Also the survival analysis was performed after excluding the poorly cohesive subtype and we found longer recurrence-free survival (p < 0.001) and longer overall survival (p = 0.01). When the survival analysis was restricted to cases diagnosed between 2013 and 2021 to ensure adequate follow-up, overall survival (OS) was significantly longer in patients with dirty necrosis compared with those without dirty necrosis (median OS: 74.9 vs. 23.2 months, p < 0.001). Similarly, recurrence-free survival was longer in patients with dirty necrosis compared with those without dirty necrosis; however, the difference did not reach statistical significance (p = 0.111) (Fig. 3a–d).

Fig. 3
figure 3

(a, b) Kaplan-Meier survival curves of categorized dirty necrosis, positive and negative, showed a significant separation for recurrence-free survival and overall survival (p< 0.001; p=0.001, log-rank test). (c, d) Kaplan–Meier survival curves of categorized dirty necrosis, positive and negative in gastric carcinoma cases (2013–2021). A significant separation was observed for overall survival (p = 0.01, log-rank test), whereas recurrence-free survival showed a trend toward longer survival in the dirty necrosis–positive group, but this did not reach statistical significance (p = 0.111, log-rank test).

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Also we found that dirty necrosis was associated with TIL levels; cases with prominent TIL tended to have more extensive dirty necrosis (p < 0.001).

Tumor necrosis was found to be associated with tumor size. Accordingly, the incidence of tumor necrosis increased with an increasing tumor size (p = 0.003). Similarly, a significant correlation was also shown with the presence of TIL, and the likelihood of tumor necrosis increased in cases with prominent TIL (p = 0.001).

The results of univariate analysis in terms of overall survival and recurrence-free survival are shown in Tables 2 and 3. Accordingly, in univariate analyses, the presence of dirty necrosis has a significantly positive effect on both survival and recurrence-free survival risk (HR = 0.466, p < 001 and 0.453, p < 0.001). The presence and extent of TIL also had a positive effect on survival and recurrence-free survival (HR = 0.589, p = 0.008 and HR = 0.558, 0 = 0.003).

Table 2 Cox regression analysis for recurrence-free survival in gastric carcinoma.
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Table 3 Cox regression analysis for overall survival in gastric carcinoma.
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Histological type, tumor size, tumor grade, pTN stage, LVI, and PNI were found to increase both recurrence-free survival risk and overall survival risk. Age and gender of the patients and HER2 status had no effect. The presence of tumor necrosis did not affect survival. There was no prognostic difference between rare or extensive dirty necrosis.

In the multivariate analysis for RFS and OS, including all significant covariates, only pN stage remained as an independent prognostic factor (HR = 2.28, 95% CI: 1.47–3.53, p < 0.001).

Discussion

Tumor necrosis is a factor that negatively affects patient survival in many tumor types, such as colorectal cancer, renal cell cancer, breast cancer and gastrointestinal system tumors14. Increased tumor size is associated with hypoxia-induced death of tumor cells and is also responsible for tumor resistance to therapy15. Among the known prognostic findings of gastric cancers are patient age, lymph node status, and depth of tumor invasion16. A study of esophageal adenocarcinoma did not show any effect of tumor necrosis on survival17. In colorectal cancer, it was found to be an independent risk factor for both overall survival and recurrence-free survival14. In this study, there was a correlation between tumor necrosis and tumor size in gastric cancer, but no significant results were found with other data or in survival analysis.

Dirty necrosis is a different type of necrosis compared to tumor necrosis caused by the death of tumor cells due to hypoxia. It is a necrosis in the center of adenocarcinoma glands accompanied by neutrophil leukocytes. It is associated with neutrophil leukocytes and NETs in the tumor microenvironment in some types of cancer3. NET release from neutrophils can occur as a result of necropitosis of neutrophils18. The death patterns of cells cannot be recognized morphologically, and cell culture studies should be performed. In a study investigating cell death types and prognosis in gastric cancers, propitosis, apoptosis, and necropitosis-related genes are divided into two groups in terms of survival analysis, and there is a significant survival difference between the two groups19. Although detailed studies are still needed in this regard, it can be concluded that not all types of cell necrosis are bad. Our study was morphological and had some limitations in this respect. However, we have also shown that dirty necrosis in the tumor has a significant effect on the patient’s recurrence-free survival and overall survival.

We can associate this with cell death genes belonging to the good prognostic group. However, there is another very important point that we found in this study. When we look at the literature, there are studies in which the presence of TIL in gastric cancers is a good prognostic finding20,21. In our study, we have shown that TIL and dirty necrosis are seen together. In this case, the presence of TIL may be one of the reasons why dirty necrosis is a good prognostic finding. However, there are also studies showing that TIL has no effect on prognosis22,23.

Another interesting finding in our study is that the presence of dirty necrosis was also found to be associated with perineural invasion and pN stage. Tumor cells escape from the tumor microenvironment by neural invasion and contribute to tumor progression. However, less perineural invasion and lower pN stage were observed in the presence of dirty necrosis24. In our study, we suggest that the association of dirty necrosis with TIL creates a microenvironment that prevents perineural invasion of tumor cells. In support of this, studies on PNI and TIL in colorectal carcinomas have shown that high TIL cases have low PNI25.

This is the first study to associate tumor necrosis and dirty necrosis with TIL. Our study suggests that these two morphologic findings support each other and lead to longer survival of patients. However, our study also has limitations. For example, it is a morphologic study, and additional examinations to support NETosis would have strengthened our data. Also, the prognostic effect of tumor necrosis may not have been shown due to the small number of cases.

Gastric cancer is still a type of cancer with a high mortality risk in the world, and there is a need for studies that will affect the survival of these patients. With immunotherapy, cell types in the tumor microenvironment have also gained importance. There is a need to optimize the evaluation of TIL in gastric cancer. In addition, this study also shows that not all necrosis may have a poor prognostic meaning. Therefore, we think that gastric cancer cases with dirty necrosis and TIL should be investigated with further studies.