The effect of tumor necrosis on postoperative survival of patients with solitary small hepatocellular carcinoma

Background: Small hepatocellular carcinoma (sHCC) is a special subtype of HCC with the maximum tumor diameter ≤ 3 cm and favorable long-term outcomes. Surgical resection or radiofrequency ablation offer the greatest chance for cure; however, many patients still undergo tumor recurrence after primary treatment. So far, there is no clinical applicable method to assess biological aggressiveness in solitary sHCC. Methods: In the present study, we retrospectively evaluated tumor necrosis of 335 patients with solitary sHCC treated with hepatectomy between December 1998 and 2010 from Sun Yat-sen University Cancer Center. Results: In the current study, the presence of tumor necrosis was observed in 157 of 335 (46.9%). Further correlation analysis showed that the presence of tumor necrosis in sHCC was significantly correlated with tumor size and vascular invasion (P = 0.026, 0.003, respectively). The presence of tumor necrosis was associated closely with poorer cancer-specific overall survival (OS) and recurrence-free survival (RFS) as evidenced by univariate (P < 0.001; hazard ratio, 2.821; 95% CI, 1.643-4.842) and multivariate analysis (P = 0.005; hazard ratio, 2.208; 95% CI, 1.272-3.833). More importantly, the combined model by tumor necrosis, vascular invasion and tumor size can significantly stratify the risk for RFS and OS and improve the ability to discriminate sHCC patients’ outcomes (P < 0.0001 for both). Conclusions: Our findings provide evidence that tumor necrosis has the potential to be a parameter for cancer aggressiveness in solitary sHCC. The combined prognostic model may be a useful tool for identifying solitary sHCC patients with worse outcomes. first necrosis in sHCC demonstrates that the presence of tumor necrosis is associated with poor survival, which is consistent with the results in other solid tumors listed above 26–30). This implies a relationship where increased tumor cell death indicates a more aggressive cancer. Coagulative necrosis is a common feature of solid tumors. Tumor microvessels are fragile and susceptible to hypoxia, which suggests that the degree of tumor necrosis reflects the level of intratumoral hypoxia 14). Measured experimentally with a polarographic needle, intratumoral hypoxia correlates with poor prognosis and sensitivity to radiotherapy and chemotherapy in solid tumors (13). Tumor necrosis has been reported as an indicator of a poor prognosis in a number of solid tumors. In breast cancer, tumor necrosis has been shown to correlate with increased tumor size, high-grade disease, negative estrogen receptor status, high microvessel density, and infiltrates of macrophages that express vascular endothelial growth factor (26, 31, 32). These findings suggest that, in rapidly growing tumors, a hypoxic environment that results in tumor necrosis stimulates angiogenesis due to the release of angiogenic growth factors from infiltrating macrophages.

Solitary sHCC is a special type of HCC with excellent long-term outcomes, for which surgical resection offers the optimal curative chance (7,8). Nevertheless, many patients still suffer from recurrence after primary resection and less is known about the factors correlated with aggressive biological phenotype of sHCC (5)(6)(7)(8). It may be ideal to identify patients at high risk of tumor recurrence and/or poorer outcome, and to target close follow-up or postoperative adjuvant therapies in these subpopulations (9,10). Currently, the known clinicopathological factors for sHCC enable the identification and screening the patients at high risk, however, the reliable factors remain ill-defined (11,12).
Tumor necrosis is a common pathological feature of solid tumors, which is found to be correlated with chronic ischemic injury due to the rapid growth of tumor (13)(14)(15). The extent of tumor necrosis reflects the level of intra-tumor hypoxia. Increased cellular hypoxia is linked to the increased metastatic potential and worse outcome in solid tumors, as well as resistance to radio-chemotherapy (13,16). To date, the clinical and prognostic implication of tumor necrosis in solitary sHCC remain elusive (17,18). In the present study, we assessed the prognostic value of tumor necrosis in solitary sHCC following hepatectomy and found that tumor necrosis can be regarded as a parameter for sHCC aggressiveness.

Case selection
Data were obtained from the 335 cases of the pathologically proven and non-distant-metastasis solitary sHCC between December 1998 and 2009 in Sun Yat-sen University Cancer Center (Guangzhou, China). The patients underwent surgical resection (not ablation or transplantation) as the first therapy course were included in this study. Cases were acquired following the eligibility criteria: (1) solitary sHCC (diameter ≤ 3 cm) only; (2) positive for HBV surface antigen; (3) with primary and curative hepatectomy; (4) absence of metastasis and residual disease; (5) without preoperative adjuvant therapy; having complete follow-up information..
We collected clinicopathologic data including patient age, gender, alfa-fetoprotein (AFP) level, alanine aminotransferase (ALT) level, tumor size, tumor capsule, histological differentiation, liver cirrhosis, vascular invasion and necrosis. These data are described in The presence of necrosis was carefully assessed on hematoxylin and eosin (H&E)-stained slides.
Necrosis consisted of homogenous clusters of sheets of dead cells, or coalescing groups of cells forming a coagulum, containing nuclear and cytoplasmic debris as previously described (19).
Coagulative tumor necrosis was found to be present without regard to the area of tumor involved, and the extent of involvement was not assessed. Vascular invasion in each HCC specimen was identified in several serial cross sections, defined as infiltration of vessel walls or the existence of tumor emboli (20). The criteria include the following: macroscopic and/or microscopic tumor emboli within the large capsular vessels, the central hepatic vein, or the portal vein (21).

Follow-up
After partial hepatectomy, patients were followed up by the Sun Yat-sen University Cancer Center, every 3 months by AFP and ultrasound or computed tomography or magnetic resonance imaging at least every 6 months for more than two years. The last date of follow-up is January 18th, 2014.

Patient characteristics
Our selection criteria identified 335 adult patients with resected solitary sHCC. All the patients were long-term carriers of HBV and treated with curative surgical resection, which was, in some cases, The relationship between tumor necrosis and patients' survival: univariate analysis Assessment of survival of sHCC patients revealed that some clinical pathological parameters indicated a significant impact of prognosis, such as tumor size (P = 0.001) and vascular invasion (P < 0.001, Table 2), which was reported in our previous study (22). The result demonstrated that patients with tumor necrosis displayed a poor overall survival ( Table 2; Fig. 2A) and recurrence-free survival ( Fig. 2B) than patients without tumor necrosis (P < 0.0001). for OS (Fig. 4, P < 0.0001) and RFS (Fig. 4, P < 0.0001) in our study based upon a combination of tumor necrosis, tumor size and vascular invasion. that in the non-cirrhotic patients with HCC tended to be well to moderately differentiated, may presented with certain areas of necrosis but did not demonstrate the relative prognosis (18). Thus, to the best of our knowledge, data regarding the incidence and prognostic impact of necrosis in HCC, are scarce and limited. This study has characterized, for the first time, tumor necrosis in sHCC and demonstrates that the presence of tumor necrosis is associated with poor survival, which is consistent with the results in other solid tumors listed above (13)(14)(15)(26)(27)(28)(29)(30). This implies a relationship where increased tumor cell death indicates a more aggressive cancer. Coagulative necrosis is a common feature of solid tumors. Tumor microvessels are fragile and susceptible to hypoxia, which suggests that the degree of tumor necrosis reflects the level of intratumoral hypoxia (13,14). Measured experimentally with a polarographic needle, intratumoral hypoxia correlates with poor prognosis and sensitivity to radiotherapy and chemotherapy in solid tumors (13). Tumor necrosis has been reported as an indicator of a poor prognosis in a number of solid tumors. In breast cancer, tumor necrosis has been shown to correlate with increased tumor size, high-grade disease, negative estrogen receptor status, high microvessel density, and infiltrates of macrophages that express vascular endothelial growth factor (26,31,32). These findings suggest that, in rapidly growing tumors, a hypoxic environment that results in tumor necrosis stimulates angiogenesis due to the release of angiogenic growth factors from infiltrating macrophages.

Discussion
HCC is characterized by a tendency for vascular invasion that is believed to be a strong predictor of outcome following hepatic resection and liver transplanation of HCC in multiple studies (23,24). In the previous study, our data showed that vascular invasion had an adverse impact on long-term survival in patients with sHCC and presence of vascular invasion led to a significant decrease in OS and RFS at 5 years. The association of tumor necrosis and vascular invasion is consistent with studies in breast cancer and malignant mesothelioma. It was observed microvessel hot spots were situated away from areas of tumor necrosis in these two neoplasms (26,33). It is possible to explain this apparently paradoxical relationship by rapid tumor growth outstripping the vascular supply, causing ischemic damage to the microvasculature and thereby increased tumor necrosis. Tumor size is a wellknown risk factor for poor survival following hepatectomy of HCC, We found that tumor size > 2.5 cm was correlated with a worse OS or RFS even in the patients with tumors ≤ 3 cm in our previous study (22). In breast cancer, tumor necrosis correlated with increasing tumor size (26), while the association between T stage and necrosis remains unclear in other solid tumors. In our present study, the association of tumor necrosis and tumor size is similar to the studies in breast cancer. It was confirmed that increasing mass was associated with hypoxia in the experimental murine allograft model (34).
The reported OS rates for patients with HCC following resection varies, with five-year OS rates ranging       The proposed prognostic model successfully stratified risk for survival prediction of patients with sHCC (log-rank test). Using this model, these sHCC patients were stratified into four groups: risk 1, n = 89; risk 2, n = 132; risk 3, n = 90; risk 4, n = 24. A, The RFS curves of the three groups were significantly different (P < 0.0001). B, The OS curves of the three groups were significantly different (P < 0.0001).