Abstract
Dirty necrosis (DN) is a form of tumor necrosis (TN) with prominent neutrophil infiltration and cell detritus in the necrotic foci. This study aimed to characterize the clinicopathological features of DN in metastatic lung cancers of the colon and rectum (MLCRs). A total of 227 patients who underwent pulmonary metastasectomy and complete resection for MLCR were included in this study. TN was evaluated using digitally scanned resection specimens. These slides were immunostained for biomarkers of NETosis (citrullinated histone H3 [citH3] and myeloperoxidase [MPO]), and the area positive for citH3 and MPO was further quantified. TN was observed in 216 cases (95.2%), and 54 (25.0%) of these cases had DN. The presence of TN was not associated with a worse prognosis; however, patients with DN had a significantly shorter overall survival than those without DN (p < 0.01). Furthermore, the presence of DN was a poor prognostic factor in both the univariate and multivariate analyses. Immunohistochemical analysis revealed that the percentage of citH3‐positive and MPO‐positive areas in the DN‐positive cases was significantly higher than that in the DN‐negative cases (p < 0.01 and p < 0.01, respectively). In surgically resected MLCR, DN is the characteristic TN subtype associated with poor prognosis and neutrophil extracellular traps (NETs).
Keywords: colon and rectum, dirty necrosis, metastatic lung cancer, NETosis, tumor necrosis
This study aimed to characterize the clinicopathological features of dirty necrosis (DN) in metastatic lung cancer of the colon and rectum (MLCR). We reported that DN is a prognostic factor in MLCR and is associated with neutrophil extracellular traps (NETs). Thus, identification of DN in pathological diagnosis may assist in determining postoperative treatment strategies including the postoperative follow‐up period and additional chemotherapy.
Abbreviations
- citH3
citrullinated histone H3
- CRC
colorectal cancer
- DN
dirty necrosis
- H&E
hematoxylin and eosin
- MLCR
metastatic lung cancer of the colon and rectum
- MPO
myeloperoxidase
- NETs
neutrophil extracellular traps
- OS
overall survival
- RFS
recurrence‐free survival
- TA
tumor area
- TN
tumor necrosis
- TNA
tumor necrosis area
1. INTRODUCTION
Colorectal cancer (CRC) is one of the most common cancers and the leading cause of cancer‐related deaths worldwide. 1 The most common site of metastasis of CRC outside the abdomen is the lung, with 10%‐25% of all CRC patients having metastasis. 2 , 3 The characteristic pathology of metastatic lung cancer from the colon and rectum (MLCR) is glands lined with pseudostratified high columnar cells and extensive necrosis. 4
Histologically, sheet‐like and uniform aggregates of dead cells are used to define tumor necrosis (TN). TN is primarily caused by chronic ischemia within tumors due to vascular collapse, high interstitial pressure, and/or rapid tumor growth that exceeds blood supply. 5 In addition, TN is caused by dysregulated cell proliferation and nutrient imbalance. 6 TN is an important pathological feature and is associated with a higher risk of metastasis in stage I lung adenocarcinoma. 7 , 8 , 9 Although the number of metastases, carcinoembryonic antigen (CEA) levels, liver metastases, and mediastinal and hilar lymph node metastases have been reported as prognostic factors in MLCR, the presence of TN in MLCR has no prognostic impact. 10 , 11 , 12 , 13 , 14
The pathogenesis and forms of cell death and necrosis vary, and the detailed mechanisms remain unclear. 15 Several studies have focused on the quality of TN, including “dirty necrosis” (DN) defined as the presence of inflammatory cells and detached material in the glandular lumen. 16 Moreover, DN is an indicator of poor prognosis in renal cell carcinoma. 17 Furthermore, neutrophils use NETosis, a cell death mechanism unique to neutrophils for antimicrobial defense. During NETosis, the neutrophilic DNA, histones, and enzymes, including myeloperoxidase (MPO), form neutrophil extracellular traps (NETs), which bind to bacteria and viruses, trapping and killing them. 18 , 19 Neutrophils are not only activated by infection but also by abnormally activated molecules released into the tumor microenvironment. One of the neutrophil responses that is abnormally triggered during tumorigenesis is NETosis, wherein activated neutrophils expel their DNA and intracellular contents into a reticular structure called NETs. Recent studies have identified NETs in the peripheral blood of cancer patients, and the role of NETosis is gaining attention in the field of oncology.
In routine clinical practice, morphological features that may be considered DN are often observed in patients with MLCR, 20 but their prognostic significance has not been studied. Therefore, this study aimed to characterize the prognostic significance of DN in patients with MLCR and the relationship between DN and NETs.
2. MATERIALS AND METHODS
2.1. Case selection
We investigated 420 cases of pulmonary metastasectomy performed at the National Cancer Center Hospital East between 2011 and 2018. The inclusion criteria were as follows: (1) no gender and age restrictions, (2) metastatic lung cancer, (3) undergoing surgical treatment, and (4) with or without medical treatment. The exclusion criteria were as follows: (1) not MLCRs and (2) incomplete resection. A total of 227 patients were included in the study (Figure S1). All specimens were collected after comprehensive written informed consent had been obtained from the patients. This study was approved by the institutional review board of the National Cancer Center (IRB number 2021‐311) and conducted in accordance with the guidelines of the Declaration of Helsinki.
2.2. Clinicopathological characteristics
For each patient, the clinicopathological characteristics collected from the medical records include age, sex, smoking status, preoperative chemotherapy history, tumor diameter, single/multiple metastases, overall survival (OS), and relapse‐free survival (RFS). The median follow‐up was 23.2 months (range 0.9‐130.9 months).
2.3. Histopathological evaluation
All sectioned specimens were fixed with 10% formalin via infusion through the bronchial tree and embedded in paraffin. The tumors were sliced into 5‐mm‐thick slices, and serial 4‐μm sections were stained with hematoxylin and eosin (H&E). Pathological features were evaluated by two pathologists (Y. K. and G. I.). H&E slides of the largest tumor cross‐section were selected. The largest lesions were examined in patients with multiple metastases. These slides were digitally scanned and analyzed using an Aperio VERSA (Aperio ImageScope) to calculate the tumor necrosis area (TNA), tumor area (TA), and necrosis percentage (NP; TNA divided by TA) (Figure S2). TN was defined as the presence of microscopic necrosis, as described by Sengupta et al. 21 Briefly, TN was characterized histologically by homogeneous clusters and sheets of dead cells. Furthermore, we classified TN into DN (+) and DN (−) based on a previous study. 17 In brief, when TN with abundant inflammatory cells and cell detritus was observed in two or more different locations in the field view at 40 × magnification, it was defined as DN (+) (Figure 1A). DN (−) was defined as TN with only persisting cell outlines and TN with abundant inflammatory cells and cell detritus in only one location (Figure 1B). Cases with mixed DN+ and DN− were defined as DN+ (Figure S3).
FIGURE 1.
Dirty necrosis (DN) (+) and DN (−) tumor necrosis (TN) in metastatic lung cancer from the colon and rectum (MLCR). A, Dirty necrosis (DN) (+) in MLCR in low magnification. B, DN (+) in MLCR in middle magnification. C, DN (+) with abundant neutrophils and cell detritus in high magnification. D, DN (−) in MLCR in low magnification. E, DN (−) in MLCR in medium magnification. F, DN (−) in MLCR in high magnification. Only tumor cells showing coagulation necrosis are seen, without neutrophils and cell detritus
2.4. Immunohistochemistry
Tumor necrosis cases matched for background (Table S1) were stained with anti‐MPO antibody (A039829‐2, DAKO) and anti‐citrullinated histone H3 (citH3) antibody (Ab5103 Abcam) to evaluate NETosis. Immunostaining was performed using the standard protocol of the Benchmark ULTRA automated staining system (Roche/Ventana Medical Systems). Geminin staining (Leica) was also performed using the standard protocol of the Benchmark ULTRA automated staining system to compare the proliferation index of cancer cells around DN (+) and DN (−).
2.5. Evaluation of immunohistochemistry
For citrullinated histone H3 (citH3) and MPO, products showing positive images within necrosis were identified, and their areas were measured and evaluated. A virtual slide system prepared using Aperio VERSA (Aperio ImageScope) was used to measure the area. The percentage of geminin‐positive tumor cells was determined by calculating the average number of positive tumor cells per tumor in four high‐power microscopic fields of view containing at least 100 tumor cells (400×; 0.0625 mm2).
2.6. Statistical analysis
All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University), which is a graphical user interface for R (The R Foundation for Statistical Computing, version 4.0.0). More precisely, it is a modified version of the R commander designed to add statistical functions frequently used in biostatistics. Associations between variables and the presence of TN were analyzed using Fisher's exact test (for categorical variables) and the Mann‐Whitney U test (for continuous variables compared between two groups). The Kaplan‐Meier method and log‐rank test were used to assess the statistical significance of the differences in outcomes. The influence of clinicopathological variables on OS and recurrence was assessed using the Cox proportional hazards model. The criterion for significance was a P value of <0.05.
3. RESULTS
3.1. Clinicopathological differences between DN (+) and DN (−)
Out of 227 tumors, 216 tumors had TN, while 11 tumors did not have TN. There were no significant differences in clinicopathological features between the two groups (Table 2). Of the 216 specimens with TN, 54 were DN (+) and 162 were DN (−). The occurrence of DN was significantly associated with age (<65 years old) and tumor size (>25 mm2) (p < 0.01 for both). Sex, smoking status, preoperative chemotherapy, CEA, and the number of tumors were not associated with the DN (Table 1).
TABLE 2.
Univariable cox hazard model analysis for overall survival (OS)
| Features | n | Hazard ratio | 95% CI | p value |
|---|---|---|---|---|
| MLCR pathological stage | ||||
| I‐III | 44 | Ref. | 0.194 | |
| IV | 183 | 1.520 | 0.808‐2.858 | |
| Nonpulmonary metastasis | ||||
| Negative | 119 | Ref. | <0.01 | |
| Positive | 108 | 5.254 | 2.689‐10.27 | |
| Hilar or mediastinal tumor‐infiltrated lymph nodes | ||||
| Negative | 222 | Ref. | 0.831 | |
| Positive | 5 | 0.806 | 0.111‐5.847 | |
| Time to metastasis (month) | ||||
| <16 | 110 | Ref. | 0.120 | |
| ≥16 | 117 | 0.644 | 0.369‐1.121 | |
| Preoperative chemotherapy | ||||
| No | 82 | Ref. | 0.098 | |
| Yes | 145 | 1.684 | 0.908‐3.124 | |
| Postoperative chemotherapy | ||||
| No | 116 | Ref. | <0.01 | |
| Yes | 111 | 12.15 | 4.819‐30.61 | |
| CEA | ||||
| Negative | 173 | Ref. | 0.216 | |
| Positive | 54 | 1.463 | 0.800‐2.673 | |
| Number of tumors | ||||
| 1 | 154 | Ref. | <0.01 | |
| ≥2 | 73 | 2.556 | 1.475‐4.430 | |
| Tumor area (mm2) | ||||
| <25 | 113 | Ref. | 0.916 | |
| ≥25 | 114 | 0.971 | 0.559‐1.685 | |
| Dirty necrosis | ||||
| Absent | 173 | Ref. | <0.01 | |
| Present | 54 | 2.166 | 1.226‐3.825 | |
Abbreviations: CEA, carcinoembryonic antigen; MLCR, metastatic lung cancer from the colon and rectum.
TABLE 1.
Association of dirty necrosis (DN) with clinicopathological features
| Features | DN (−) | DN (+) | p value |
|---|---|---|---|
| n = 162 | n = 54 | ||
| Sex | |||
| Female | 54 (33.3%) | 17 (31.5%) | 0.868 |
| Male | 108 (66.7%) | 37 (68.5%) | |
| Age | |||
| <65 | 60 (37.0%) | 32 (59.3%) | <0.01 |
| ≥65 | 102 (63.0%) | 22 (40.7%) | |
| Smoking status | |||
| Never | 54 (33.3%) | 17 (31.5%) | 0.868 |
| Current/former | 108 (66.7%) | 37 (68.5%) | |
| Preoperative chemotherapy | |||
| Negative | 65 (40.1%) | 14 (25.9%) | 0.077 |
| Positive | 97 (59.9%) | 40 (74.1%) | |
| CEA | |||
| Negative | 128 (79.0%) | 38 (70.4%) | 0.197 |
| Positive | 34 (21.0%) | 16 (29.6%) | |
| Tumor area (mm2) | |||
| <25 | 90 (55.6%) | 15 (27.8%) | <0.01 |
| ≥25 | 72 (44.4%) | 39 (72.2%) | |
| Number of tumors | |||
| 1 | 111 (68.5%) | 38 (70.4%) | 0.866 |
| ≥2 | 51 (31.5%) | 16 (39.6%) | |
Abbreviation: CEA, carcinoembryonic antigen.
3.2. OS and RFS based on the presence of necrosis
Overall, 130 patients (57.3%) had recurrence and 51 (22.5%) died; of these deaths, 41 (41/51; 80.4%) were from disease‐related causes. In all patients, the 5‐year OS rate was 54.5% in the absence of necrosis and 51.4% in the presence of necrosis (p = 0.726) (Figure S4A). The 5‐year recurrence‐free survival (RFS) rate was 18.1% in the absence of necrosis and 32.4% in the presence of necrosis (p = 0.198) (Figure S4B). Overall, the presence of necrosis was not associated with a worse prognosis or a significantly higher risk of recurrence.
We investigated the prognosis based on the TN percentage. When median values were used, there were no significant differences in OS and RFS (Figure S4C,D), but when cutoff values based on ROC analysis were used, a smaller TN ratio was associated with a poorer prognosis for both OS and RFS (Figure S4E,F).
3.3. Prognostic impact of DN
Figure 2 shows the effect of the TN subclassification on OS and RFS. Tumors with DN had significantly shorter OS than those without DN (p < 0.01) (Figure 2A). There was no significant difference in the RFS of DN (−) and DN (+) patients (p = 940) (Figure 2B). In the case of patients with a single tumor, patients with DN (+) tended to have shorter OS than those with DN (−) (p = 0.067) (Figure 2C). Moreover, in the case of patients with a single tumor, there was no significant difference in the RFS of DN (−) and DN (+) patients (p = 0.442) (Figure 2D).
FIGURE 2.
Overall survival (OS) and recurrence‐free survival (RFS) curves for patients without tumor necrosis (TN) and with and without dirty necrosis (DN). A, OS curves for patients without TN and those with and without DN. The black line shows the group without TN, the red line shows that without DN, and the green line shows that with DN. B, RFS curves for patients without TN and with and without DN. The black line shows the group without TN, the red line shows that without DN, and the green line shows that with DN. C, OS curves for patients without TN and those with and without DN in a single tumor. The black line shows the group without TN, the red line shows that without DN, and the green line shows that with DN. D, RFS curves for patients without TN and those with and without DN in a single tumor. The black line shows the group without TN, the red line shows that without DN, and the green line shows that with DN
Tables 2 and 3 show the relationship between unfavorable clinicopathological factors and OS in univariate and multivariate analyses. Nonpulmonary metastasis, postoperative chemotherapy, the number of tumors (≥2), and DN were significantly associated with shorter OS. Multivariate analysis revealed that nonpulmonary metastasis, postoperative chemotherapy, number of tumors (≥2), and DN were significant independent prognostic factors (p ≤ 0.01, p < 0.01, p = 0.015, and p < 0.01, respectively) for OS. MLCR pathological stage, hilar or mediastinal tumor‐infiltrated lymph nodes, time to metastasis, preoperative chemotherapy, CEA level, and TA were not independent prognostic factors for OS (p = 0.443, p = 0.561, p = 0.667, p = 0.440, p = 0.200, and p = 0.488, respectively). Table S3 shows the relationship between DN and unfavorable clinicopathological factors, but there was no association between them.
TABLE 3.
Multivariable cox hazard model analysis for overall survival (OS)
| Features | n | Hazard ratio | 95% CI | p value |
|---|---|---|---|---|
| MLCR pathological stage | ||||
| I‐III | 44 | Ref. | 0.443 | |
| IV | 183 | 0.774 | 0.402‐1.490 | |
| Nonpulmonary metastasis | ||||
| Negative | 119 | Ref. | <0.01 | |
| Positive | 108 | 3.633 | 1.757‐7.508 | |
| Hilar or mediastinal tumor‐infiltrated lymph nodes | ||||
| Negative | 222 | Ref. | 0.561 | |
| Positive | 5 | 0.546 | 0.071‐4.212 | |
| Time to metastasis (month) | ||||
| <16 | 110 | Ref. | 0.667 | |
| ≥16 | 117 | 0.874 | 0.474‐1.612 | |
| Preoperative chemotherapy | ||||
| No | 82 | Ref. | 0.440 | |
| Yes | 145 | 0.768 | 0.393‐1.501 | |
| Postoperative chemotherapy | ||||
| No | 116 | Ref. | <0.01 | |
| Yes | 111 | 8.132 | 3.104‐21.31 | |
| CEA | ||||
| Negative | 173 | Ref. | 0.200 | |
| Positive | 54 | 1.500 | 0.806‐2.790 | |
| Number of tumors | ||||
| 1 | 154 | Ref. | 0.015 | |
| ≥2 | 73 | 2.101 | 1.157‐3.814 | |
| Tumor area (mm2) | ||||
| <25 | 113 | Ref. | 0.488 | |
| ≥25 | 114 | 1.245 | 0.670‐2.312 | |
| Dirty necrosis | ||||
| Absent | 173 | Ref. | <0.01 | |
| Present | 54 | 2.503 | 1.351‐4.638 | |
Abbreviations: CEA, carcinoembryonic antigen; MLCR, metastatic lung cancer from the colon and rectum.
3.4. The association of NETosis with DN
We performed citH3 and MPO staining as biomarkers of NETosis in DN (−) and DN (+) cases. Figure 3A, B shows citH3 and MPO staining in DN (−) and DN (+), respectively. A positive image of citH3 and MPO was conspicuous in the DN (+) cases, and granular and reticular cell disintegration products were abundantly observed. Although not as evident as that in the DN (+) cases, the DN (−) cases also showed cell disintegration products that were positive for citH3 and MPO. The area of citH3‐and MPO‐positive debris within the necrotic area of tumors in the DN (−) and DN (+) groups was measured and compared as a percentage of TNA. The median citH3‐positive area percentage in the DN (−) group was 3.7% (0‐54.98), and that in the DN (+) group was 15.7% (0.99‐94.75). The median MPO‐positive area percentage in the DN (−) group was 5.3% (0.01‐52.29), and that in the DN (+) group was 17.0% (0.59‐96.08). The median citH3‐ and MPO‐positive percentages in the DN (+) group were significantly greater than those in the DN (−) group (p < 0.01 and p < 0.01, respectively) (Figure 3C).
FIGURE 3.
Immunohistochemical analysis of citrullinated histone H3 (citH3) and myeloperoxidase (MPO). A, Immunohistochemical expression of citH3 and MPO in dirty necrosis (DN) (−). B, The immunohistochemical expression of citH3 and MPO in DN (+). C, Comparison of citH3‐ and MPO‐positive area percentages between tumors with DN (−) and DN (+) (Mann‐Whitney U test; p < 0.01 and p < 0.01, respectively)
3.5. Proliferative activity of metastatic CRC cells around TN with and without DN
Geminin staining was performed to compare the proliferation index of cancer cells around TN with and without DN. The median geminin‐positive percentage of cancer cells around DN (−) was 20.2% (0%‐48.39%), and that around DN (+) was 22.2% (0%‐53.49%), with no significant difference between the two groups (Figure 4A,B,C).
FIGURE 4.
Immunohistochemical analysis of geminin. A, Immunohistochemical expression of geminin‐positive cancer cells around tumor necrosis (TN) without dirty necrosis (DN). B, The immunohistochemical expression of geminin‐positive cancer cells around TN with DN. C, Comparison of geminin‐positive cancer cells around TN percentage with and without DN (Mann‐Whitney U test; p = 0.221)
4. DISCUSSION
This is the first study to investigate the prognostic impact of DN on MLCR. The presence of TN did not affect prognosis, but MLCRs with DN had a worse prognosis than those without DN, and TN with DN was associated with NETosis.
Studies focusing on histological qualitative findings of TN have been reported in MLCR, and Flint et al. showed that DN is also present in lung metastases from MLCR. 20 In this study, we first divided TN into DN (+) and DN (−) based on qualitative differences and then evaluated the clinicopathological characteristics of both.
Kuroe et al. reported a significant correlation between the presence of DN and poor prognosis in renal cell carcinoma. 16 Similarly, in the current study, we found a significant association between DN and unfavorable outcomes in MLCR. The significant correlation between the presence of DN and poor prognosis could be limited to a few types of tumors, such as renal cell carcinoma and MLCR, or this correlation could be valid in all tumor types. Thus, the specificity of this correlation to tumor types needs to be investigated in the future.
Neutrophils migrate to inflammatory sites in response to inflammatory stimuli caused by bacterial infection and form NETs. NETosis is the phenomenon of trapping pathogens by forming a mesh‐like structure. 22 In NETosis, PAD4 is activated by calcium ion binding, which converts histone arginine to citrulline residues. Thus, NETs can be detected by screening for the expression of citH3. 23 , 24 , 25 H&E images of DN (+) show neutrophil infiltration and accumulation of cellular decay products. Furthermore, reticular structures were observed around the cellular decay products. 19 Therefore, we focused on NETs, which have been implicated in cancer progression and metastasis 26 and examined their relationship with TN. In the present study, we found that the citH3‐positive and MPO‐positive regions overlapped and DN (+) tumors had significantly larger citH3‐positive and MPO‐positive regions than DN (−) tumors, indicating that DN (+) tumors had larger areas exhibiting NETs. NETs have been reported to attenuate the efficacy of chemotherapy through the activity of a variety of associated proteins and factors in the tumor microenvironment, 27 which supports the finding of shorter OS but no significant impact on RFS in MLCR patients with DN (+) (Figure 2).
Rayes et al. demonstrated that circulating NET levels are elevated in advanced esophageal, gastric, and lung cancer patients compared with local cancers and healthy controls and established circulating NET levels as a tumor‐induced prognostically significant biomarker. 28 Although an association between systemic inflammation and poor prognosis has been demonstrated by Chang et al. and Kuroe et al., 29 , 30 the present study found no association between systemic inflammation and the presence of DN (Table S4). This may be because MLCR has a smaller tumor volume than the primary tumor.
Yazdani et al. reported that NETs are associated with an increase in mitochondrial function, therefore, with the activation of tumor cell metabolism, favoring tumor growth. 31 Albrengues et al. reported that laminin remodeling by NETs induces the proliferation of dormant cancer cells by activating integrin α‐3β‐1 signaling. 32 Therefore, we examined the proliferative capacity of cancer cells around DN (+) and DN (−) using geminin expression. However, no significant differences were found between the groups. Both studies by Yazdani et al. and Albrengues et al. were animal studies, and the results may differ between animal and clinical studies.
This study has some limitations. First, in the current study, it was not possible to examine the correlation between the presence of DN in primary sites and metastatic sites in all cases. Although not in all cases, we examined the correlation between the presence of DN in the primary tumor where the primary site was available for this study and the presence of DN in the corresponding metastasis (n = 95); however, no significant correlation was found between the presence of DN in the primary tumor and metastatic tumor (data not shown). This may be due to differences in the microenvironment in the primary and metastatic lesions. Second, this was a retrospective study, and a prospective study with a large sample size is needed to validate the results of this study.
In this study, we found that DN in MLCR is a prognostic factor and is associated with NETs. Thus, identification of DN in pathological diagnosis may assist in determining postoperative treatment strategies including the postoperative follow‐up period and additional chemotherapy.
CONFLICT OF INTEREST
Dr. Genichiro Ishii is an editorial board member of Cancer Science.
ETHICAL APPROVAL
Approval of the research protocol by an Institutional Reviewer Board. All work included in the manuscript was performed at National Cancer Center, Kashiwa, Chiba, Japan. The research was approved by the Institutional Review Board of the National Cancer Center (Approval No. 2021‐311). No personally identifiable information was included in the manuscript.
Supporting information
Figure S1.
Figure S2.
Figure S3.
Figure S4.
Tables S1–S4.
ACKNOWLEDGMENTS
This study was supported by JSPS KAKENHI (21H02931).
Konishi Y, Taki T, Nakai T, et al. Clinicopathological features and prognostic impact of dirty necrosis in metastatic lung cancers from the colon and rectum. Cancer Sci. 2023;114:2169‐2177. doi: 10.1111/cas.15647
[Corrections made on 14 March 2023, after first online publication: The 4th author’s name has been corrected in this version.]
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Supplementary Materials
Figure S1.
Figure S2.
Figure S3.
Figure S4.
Tables S1–S4.