Evaluation of Histopathological Heterogeneity After Preoperative Chemotherapy in Patients With Liver Metastases from Colorectal Cancer

Background: Patients with liver metastases from colorectal cancer (CRLMs) frequently receive chemotherapy prior to liver resection. Histopathological assessment of the resected specimen can evaluate the response to chemotherapy. This study analyzed the correlation between histopathological changes in the primary site and liver metastases. Patients and Methods: This study comprised 45 patients with resectable CRLMs at the Surgical Oncology Department of Gifu University School of Medicine from January 2006 to August 2015. Results: The study included 24 men and 21 women. The primary colonic tumor was located in the right side in 13 (28.9%) patients and the left side in 32 (71.9%) patients. We evaluated patients with metastatic colorectal cancer (31/45) after excluding those in whom histopathological heterogeneity between the primary and liver metastasis changed to grade 3 after chemotherapy. We compared the group which underwent hepatectomy after chemotherapy (n=25) with that underwent hepatectomy alone (n=6). In 16 (53.3%) out of 25 patients, histopathological heterogeneity of the liver metastasis was lost (p=0.04). Conclusion: Chemotherapy appears to change histopathological heterogeneity.Our study suggests that the change of intratumoral heterogeneity reect by the response of chemotherapy.


Background
Colorectal cancer is the third most common human malignant tumor and is one of the major causes of cancer mortality in the Western world. Metastatic tumors account for 40% to 50% of malignancies in newly diagnosed patients (1). The prognosis of metastatic colorectal cancer (mCRC) remains poor.
Among the treatment options for colorectal liver metastases (CRLM), liver resection is the most conducive to a cure, with 5-year overall survival rates of 29-48%. Even for initially unresectable CRLM, effective chemotherapy, along with targeted therapy, sometimes enables their resection (2,3). Promising treatments for CRLM include chemotherapy and molecular agents that target epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF). These reports suggest that the combination of targeted agents and chemotherapy can increase rates of liver resection and response, thus improving progression-free and overall survival of patients with CRLM. However, no studies have compared histopathological type by treatment with anti-VEGF agents and anti-EGFR agents for wild-type RAS liverlimited CRLM (4,5).

Patients And Methods
This study included 45 patients with CRLM con rmed to be resectable following neoadjuvant chemotherapy (NACT). Patients were treated with surgery alone or with surgery following FOLFOX alone or FOLFOX plus anti-EGFR (cetuximab or panitumumab) FOLFOX plus anti-VEGF (bevacizumab) as rst- Because an important consequence of intratumoral heterogeneity is potential differences in histopathology between primary tumors and their liver metastases, the histopathological pro le of the primary colorectal tumors prior to and after chemotherapy and that of the CRLMs resected postchemotherapy were assessed to investigate the changes between them (Figures 1 and 2).
In accordance with the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (6), the same methods were used to perform tumor assessment at baseline and subsequently every 8-12 weeks using torso contrast-enhanced computed tomography and liver contrast-enhanced magnetic resonance imaging. The tumor histopathological response rate was defined as the proportion of patients with grade Statistical analysis. For continuous variables, the data are summarized as the median with range and were compared using the non-parametric Mann-Whitney U-test, Kruskal-Wallis tests or the chi-square test. A p-value of less than 0.05 was considered to indicate statistical signi cance. Statistical analysis was performed using JMP12 software (SAS Institute Inc., Cary, NC, USA).

Results
The study comprised 45 patients with mCRC (24 men, 21 women; mean age = 61.9±9.4 years). Locations of primary tumor in the 45 patients were the right-sided colon in 13 (28.9%) patients and the left-sided colon in 32 (71.9%) patients. The group treated with surgery alone included nine patients, whilst those treated with surgery after FOLFOX alone or plus anti-VEGF or anti-EGFR included 12 patients each ( Table  I). The heterogeneity of the histopathology between the primary site and the liver metastasis was signi cantly different in the group treated with surgery alone compared with the other groups (p = 0.04).
However, there were no signi cant differences between the three groups. In addition, tub2 histopathology of appeared to be a predictive marker between primary site and posttreatment liver metastasis specimens from patients with CRLM because the histopathology forms disappeared after each chemotherapy treatment, except for tub2 (Figures 4-6). Though this study didn't showed data, we assume that Histopathological change greatly in uence long-term survival.We also evaluated patients with CRLM (31/45) after excluding the patients in whom histopathological heterogeneity changed to grade 3 in the liver metastasis after chemotherapy. This study compared the group which underwent hepatectomy after chemotherapy (n = 25) with that which underwent hepatectomy alone (n = 6). In all six of the latter patients, histopathological heterogeneity at the liver metastasis was maintained after therapy. However, in 16 out of the 25 patients (53.3%) who underwent hepatectomy after chemotherapy, histopathological heterogeneity of liver metastases was lost. We con rmed the loss of neoplastic cells by chemotherapy and homogeneity in liver metastases (p = 0.04).

Discussion
The present study describes the histopathological patterns of response of CRLMs to preoperative NACT followed by liver resection. To our knowledge, this is the rst report to focus on change of histopathological heterogeneity comparing between primary tumor sites and CRLMs.
Over the past decade, NACT has been widely recommended for the management of initially resectable CRLMs with the aim of inducing tumor shrinkage to identify optimal candidates for subsequent surgical removal. The assessment of tumor regression has been gradually used to quantify the histopathological response to NACT and has served as an early parameter predicting prognosis (7,8).
Discrepancies in the tumor regression patterns in response to different chemotherapy regimens have been revealed in previous literature. Rubbia-Brandt et al. reported that an oxaliplatin-based regimen improved histopathological response compared with 5-uorouracil-, and irinotecan-based regimens (9,10). In terms of monoclonal antibodies, a bevacizumab-containing regimen provided a better histopathological response than chemotherapy alone or in combination with cetuximab (11,12). Poultsides et al. published a large retrospective analysis of 366 patients (68% treated preoperatively and 32% not) who underwent CRLM resection. In that study there was no increase in the degree of necrosis after chemotherapy (13). Nevertheless, it should be noted that only 69 out of 249 (28%) patients received bevacizumab as part of the preoperative treatment, and the results in terms of necrosis for that subgroup were not reported. In other experience, increase in necrosis seemed to be due to a bevacizumab-related effect (14,15). Additionally, the tumor histopathological response rate was defined as the proportion of patients with grade ≥Ib.
Taken together, all of the above-reported observations of reduced viable cells, brosis, and necrosis from a pathological perspective explained the typical pattern of CLMs detected using computed tomography scanning in patients receiving chemotherapy and bevacizumab. Before treatment, the lesions showed different types of enhancement, a heterogeneous degree of attenuation, and ill-de ned borders that were transformed into hypo-attenuated and homogeneous metastases with well-de ned borders after treatment (16). Such histological and morphological characteristics strengthen the hypothesis that the RECIST criteria are not completely adequate for evaluating response in patients receiving bevacizumab (17).
In contrast, the anti-EGFR agent cetuximab in combination with chemotherapy has been reported to increase the response rate and yield a good curative hepatectomy. In the PEAK (18), FIRE-3 (19) and CALGB/SWOG 80405 (20) randomized controlled trials performed to compare bevacizumab and anti-EGFR therapy for the progression of recurrent CRC, anti-EGFR was also con rmed to have a positive effect on survival extension in the presence of wild-type RAS.
Recently, the multicenter, randomized, phase II ATOM trial from Japan was designed to evaluate the efficacy and safety of mFOLFOX6 plus bevacizumab and mFOLFOX6 plus cetuximab in patients with liver-limited metastasis from wild-type all-RAS CRC. After study treatment followed by surgical resection of tumors with R0/R1 status, the median progression-free survival of the bevacizumab-treated arm was 6.5 months [95% con dence interval (CI) = 4.0-13.6 months], whereas that of the cetuximab-treated arm was 13.8 months (95% CI = 8.4 months-not reached; hazard ratio = 0.610, 95% CI = 0.298-1.245). Of the 57 tumors for which the histopathological analysis was assessable, the histopathological response rate (grade 1b/2/3) was 66.6% (20/30) in the bevacizumab-treated arm and 92.6% (25/27) in the cetuximabtreated arm (p = 0.0229) (16), indicating that the rate tended to be better in the cetuximab-treated arm (21). Falcão et al. reported three categories of tumor growth: (a) Replacement growth pattern, in which the tumor permeates between the liver hepatocytes without disrupting the normal architecture; (b) desmoplastic growth pattern, in which the tumor is separated from the liver parenchyma by a band of brous tissue that contains tumor-in ltrating lymphocytes; and (c) pushing growth pattern, in which the tumor expands and compresses the surrounding hepatocytes. They reported the pushing growth pattern to be an independent risk factor for reduced survival (22).
Recently, a tumor-heterogeneity concept that considers a single tumor to consist of many tumor cell subclones has become an important topic in cancer genomics (23). It is hypothesized to play a critical role in the progression of many cancer types and is a major obstacle to precision cancer therapy. During this process, sub-clones continuously arise via genomic mutation. The presence of sub-clones has been shown to adversely affect outcome in chronic lymphocytic leukaemia, head and neck cancer, and lung adenocarcinoma. However, the full complement of factors that lead to tumor heterogeneity during CRC progression is unknown.
Several promising CRLM treatments have been reported, including chemotherapy and molecular agents that target EGFR and VEGF. Anti-EGFR drugs resulted in high response and resection rates in the CELIM phase II trial and other studies for initially unresectable CRLM with wild-type KRAS (24 25).. Anti-VEGF regimens, such as mFOLFOX6 or CAPEOX plus bevacizumab, have also shown high response and resection rates in phase II studies (26).. These reports suggest that the combination of targeted agents and chemotherapy can increase the response rate.
A previous study reported a higher pathological response rate to bevacizumab than for cetuximab. This study suggested that the loss of intratumoral heterogeneity markedly affects the response to chemotherapy.

Conclusion
In conclusion, the present study highlighted marked differences between pre and posttreatment specimens from sites in patients with mCRC. tub2 Histopathology appeared to be a predictive marker in specimens comparing primary site and CRLMs posttreatment because histopathology types other than tub2 disappeared after chemotherapy treatment. Each NACT agent had an acceptable safety pro le. In the near future, we expect results from further study to expand the indication for NACT.

Availability of data and materials
The datasets used during this study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The present study was conducted in accordance with the World Medical Association Declaration of Helsinki and was approved by the Ethics Committee of Gifu University (Approval number: 28-508; March 23, 2017). As this study was a retrospective study and did not include any potentially identi able patient data, informed consent was not obtained from the enrolled patients. The institutional review board gave the ethics approval for this retrospective study.