Histopathological variables in liver metastases of patients with stage IV colorectal cancer: potential prognostic relevance of poorly differentiated clusters

Accepted Manuscript Histopathological variables in liver metastases of patients with stage IV colorectal cancer: potential prognostic relevance of poorly differentiated clusters

Simona Lionti, Luca Reggiani Bonetti, Stefania Bettelli, Andrea Spallanzani, Fabio Gelsomino, Valeria Barresi PII: DOI: Reference:

S0046-8177(18)30148-5 doi:10.1016/j.humpath.2018.04.019 YHUPA 4554

To appear in: Received date: Revised date: Accepted date:

27 February 2018 8 April 2018 12 April 2018

Please cite this article as: Simona Lionti, Luca Reggiani Bonetti, Stefania Bettelli, Andrea Spallanzani, Fabio Gelsomino, Valeria Barresi , Histopathological variables in liver metastases of patients with stage IV colorectal cancer: potential prognostic relevance of poorly differentiated clusters. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yhupa(2018), doi:10.1016/ j.humpath.2018.04.019

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ACCEPTED MANUSCRIPT Title: Histopathological variables in liver metastases of patients with stage IV colorectal cancer: Potential prognostic relevance of poorly differentiated clusters Running title: PDC in colorectal LM Authors: Simona Lionti1, Luca Reggiani Bonetti2, Stefania Bettelli3, Andrea Spallanzani3, Fabio

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Gelsomino3, Valeria Barresi1

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Affiliations: 1Department of Pathology in Evolutive Age and Adulthood “Gaetano Barresi”, University of Messina, Italy

Department of Laboratory Integrated Activities, Anatomic Pathology and Legal Medicine,

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University of Modena and Reggio Emilia, Modena, Italy

Division of Oncology, Department of Oncology and Hematology, University Hospital of Modena,

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Modena, Italy

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Address for correspondence:

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Dr Valeria Barresi Department of Pathology in Evolutive Age and Adulthood AOU Polyclinic G. Martino, Pad D Via Consolare Valeria 98125 Messina Italy Phone: +390902212537 e-mail: [email protected]

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Declarations of interest: none

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Key words: PDC; liver metastases; colorectal cancer; prognosis

ACCEPTED MANUSCRIPT ABSTRACT The prognosis of patients with colorectal liver metastases (LM) is mostly established on clinical variables or on the anatomic extent of colorectal cancer (CRC). Histopathological factors of LM which may actually reflect the biological aggressiveness of the tumor are not routinely considered to define the risk of worse clinical outcome in those patients. The number of poorly differentiated

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clusters (PDC) of neoplastic cells in primary CRC is associated with metastatic risk and bad

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prognosis, but PDC presence in LM has been barely analyzed thus far.

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We assessed PDC presence in the histological slides of surgically resected and synchronous LM in 63 patients with CRC, who had been not submitted to any neo-adjuvant treatments. Then we

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analyzed its association with patients' cancer specific survival (CSS) or progression free survival (PFS). The presence of PDC (P= 0.016) and PDC localization at tumor edge of LM (P = 0.0004)

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were significantly associated with shorter CSS. PDC presence at the periphery of LM and positive resection margin were independent prognostic variables for CSS. PDC localization at the tumor

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edge of LM was a significant (P= .0079) and independent prognosticator of shorter PFS. Our data suggest that PDC presence and peripheral localization in LM may be relevant to predict outcome and useful for clinical decision making in patients with colorectal synchronous LM.

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Key words: PDC; liver metastases; colorectal cancer; prognosis

ACCEPTED MANUSCRIPT INTRODUCTION In developed countries, colorectal cancer (CRC) is the third most common malignancy in males and the second in females [1]. This tumor mostly metastatizes to the liver, so that about half of the patients with CRC have liver metastases (LM) at the time of diagnosis (synchronous metastases) or develop LM during disease course (metachronous metastases) [2,3].

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Surgery represents a potentially curative treatment in patients with colorectal LM [4]. Indeed, over

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the years it has led to significant improvement in long-term outcome of patients with advanced

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CRC4, so that 5-year survival rate after surgery ranges between 46% and 58% in specialized centers [5]. However, despite complete surgical resection of LM and advances in adjuvant therapy and

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surgical approach, about 60-78% of patients develop disease recurrence [5,6].

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The identification of prognostic markers, able to discriminate patients with LM who will have adverse clinical course from those who will have good prognosis, might be useful to design

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individualized therapies after surgery. So-called clinical risk scores, integrating several clinico-

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pathological variables, have been proposed to predict the clinical course of patients with CRC and LM [7-9]. Interestingly, adjuvant chemotherapy seems to have different efficacy in groups of patients stratified by those clinical scores [10]. However, most scoring systems include clinical

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variables or factors associated with the anatomic extent of the primary tumor and LM (i.e. the stage

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of the primary tumor, the size, number and resection margin of LM or the presence of extrahepatic metastases), while the histopathological features of LM – which may be more strictly associated with the biological aggressiveness and metastatic potential of the tumor- are barely considered. Grade of differentiation represents a relevant prognostic factors in CRC, according to World Health Organization (WHO) Classification [11]. In addition, it is highly considered for clinical decision making in the management of patients with CRC [12].

ACCEPTED MANUSCRIPT Park et al. [13] found that tumor dedifferentiation in LM is a significant and independent prognostic factor, associated with high recurrence risk, in patients with CRC. However their definition of dedifferentiation – i.e. clusters of tumor cells with large solid sheet, cribriform pattern, spindle cell morphology, trabecular or palisading pattern or extensive single cells in more than 50% of the tumor - appears to be quite arbitrary and subjective, which could limit the reproducibility and

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prognostic relevance of this histological feature in the prediction of recurrence risk of patients with

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colorectal LM [13]. In the last years, a novel grading system, based on the counting of clusters of at

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least five neoplastic cells and with no glandular formation - so-called poorly differentiated clusters (PDC)- was shown to be highly reproducible and relevant for the prediction of prognosis and risk of

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metastatization in CRC [14,15]. PDC seem to have biological significance similar to that of tumor budding [16]. However, by definition they are formed of at least five neoplastic cells, whereas

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tumor budding foci are isolated tumor cells or clusters of less than five tumor cells [16]. In addition, PDC can be found both within the tumor stroma and at its edge, while the presence of tumor

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budding is assessed only at the invasive front of CRC [16].

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Interestingly, grading based on PDC in primary CRC was demonstrated to be a significant and independent prognostic factor, associated with higher recurrence risk at extra-hepatic sites and with

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shorter disease-free survival, in patients with CRC and submitted to surgical resection of LM [17].

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To the best of our knowledge, the prognostic relevance of PDC in LM of CRC was only analyzed in one study thus far [18]. However, that study considered both cases with synchronous or metachronous metastases. In addition, all the patients had received peri-operative chemotherapy (pre- and/or post-operative) [18].

ACCEPTED MANUSCRIPT This study aimed to investigate PDC presence in LM, its eventual association with other clinicopathological variables and its prognostic role in patients with CRC and submitted to hepatectomy

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for colorectal synchronous LM.

ACCEPTED MANUSCRIPT MATERIALS AND METHODS In this study we considered 63 cases (45 males; 18 females; median age: 64 years; age range: 39-86 years) of pathological Tumor Node Metastasis (pTNM) stage IV CRCs with surgically resected liver metastases (LM), which were present in the Tumor Registry of Colorectal Cancer of the University of Modena and Reggio Emilia, Italy, between 2007 and 2013.

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In all cases, patients had received surgery for primary tumor and LM at the same time, to reduce

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hospital stay. None of them had been submitted to neo-adjuvant therapies. In all patients, all

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metastatic nodules were resected. After surgery, all patients had been submitted to adjuvant treatments, with different protocols.

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All surgical specimens had been fixed in formalin for 24 hours at room temperature and paraffin embedded. In the primary tumor, pathological staging had been performed according to the TNM

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classification system [11], while histological grading had been assessed according to the WHO criteria [11]. Based on their localization, primary CRCs were subdivided into three groups: (1)

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CRCs located in the right colon, including caecum and ascending and transverse colon; (2) CRC

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located in the left colon, including descending and sigmoid colon; and (3) CRC located in the rectum.

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At gross examination of LM, the external surface of each surgically resected nodule had been inked; then, nodules smaller than 15 mm in diameter had been completely embedded, while one section

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every 5 mm had been taken from nodules bigger than 15 mm in diameter. For each case we noticed the number and size of metastatic nodules, tumor border growth pattern (infiltrative or pushing) [19] and the status of microscopic margin. In particular, margin was considered to be positive (R1) when normal tissue from the edge of metastasis measured 5 mm or less [20,21]. Based on the number of metastatic nodules, cases were subdivided into two groups: 1) those having 1 to3 LM and 2) those having 4 or more LM [22]. In addition, we classified the cases into two

ACCEPTED MANUSCRIPT groups based on the size of the largest nodule and by using 5 cms of diameter as the cut-off value [23]. Finally, when more than one LM was present, cases were considered to have infiltrative tumor border configuration and R1 status when at least one nodule had those microscopic characteristics. We reviewed the histological slides of all LM in order to assess PDC counting. In addition, we reviewed the clinical records to retrieve data on patients follow-up, including occurrence of

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recurrences and death from CRC or from other causes.

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The study has been performed according to the Declaration of Helsinki and the study-design has

Comitato Etico della Provincia di Modena - Italy).

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PDC counting

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been approved by local ethics committee (RIF. PROTOCOL. N. n 1343/C.E. – April 11, 2017;

PDC were defined according to the original definition provided by Ueno and coll. [24]. For each

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case, PDC counting was carried out on haematoxylin and eosin (H&E) slides of all metastatic nodules. In detail, each slide was first scanned at low power magnification to identify the area with

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the highest number of PDC. Then, the clusters were counted using a ×20 objective lens (i.e. a

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microscopic field with a major axis of 1 mm), with a Zeiss microscope (Carlo Zeiss, Oberkochen Germany) [16]. Finally, the highest number of PDC found in all analyzed slides of each case was

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recorded as PDC counting and considered for statistical analyses.

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PDC counting was also performed on the histological slides of the primary CRC of each case. Statistical analyses

For statistical analyses, LM were subdivided into PDC positive (PDC> 0) and PDC negative (PDC: 0). Fisher exact and Chi-squared tests were used to assess the statistical correlations between the presence of PDC in LM, or the development of recurrences (absent vs present) and the other clinico-pathological parameters (site, pT, pN and WHO grade of the primary tumor; number, size,

ACCEPTED MANUSCRIPT tumor border configuration and margin status of LM; presence of synchronous metastases at other sites, development and site of recurrences and death from CRC). Spearman correlation test was applied to analyze the statistical correlation between PDC counting in the primary tumor and in corresponding LM. Cancer specific survival (CSS) and progression free survival (PFS) were assessed by the Kaplan-

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Meier method, with the date of primary surgery as the entry data. CSS was characterized as the

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length of survival to death from CRC. Patients who died of diseases independent from CRC were

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censored. PFS represented the time between surgery and detection of recurrences, either distant or local. The Mantel-Cox log-rank test was applied to assess the strength of association between CSS

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or DFS and each of the parameters (site, pT, pN and WHO grade of the primary tumor; number, size, tumor border configuration, PDC presence and margin status of LM, presence of synchronous

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metastases at other sites) as a single variable.

Multivariate analyses (Cox regression model) were used to determine the independent effect of each

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variable on CSS and PFS.

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P < 0.05 was considered to be statistically significant. Statistical analysis was carried out using

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MedCalc 12.1.4.0 statistical software (MedCalc Software, Mariakerke, Belgium).

ACCEPTED MANUSCRIPT RESULTS PDC were identified in 51/63 (81%) LM (Figure 1a). In the majority of cases (44/51), PDC counting was lower than 5 (mean number: 3), while only 7 LM had 5 to 7 PDC. PDC counting in LM was significantly correlated with that in primary CRC (r= 0.586; P < 0.0001). In 5 cases, PDC were localized in the center of metastatic tumor; in 18, they were found both at the

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center (Figure 1b) and at the tumor edge, while in 28 cases PDC were confined at the tumor edge of

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the metastasis (Figures 1c and 1d). The presence of PDC in LM was significantly more frequent in cases showing higher pT stage (P= 0.01) and infiltrative tumor border configuration of LM (Figure

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2) (P=0.006). In addition, it was significantly associated with development of recurrences in the

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liver, as compared with recurrences at other sites (P=.002), and with death from CRC as well (P=0.008) (Table 1). In 19 (30%) LM resection margin was positive.

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The median follow-up of the total patients was 40 months (range: 2–107 months), that of alive patients was 50 months (range: 16-107 months), while that of died of patients was 23 months

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(range: 2-79 months). During the follow-up time, 55 (87%) patients developed recurrences and 38

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(60%) died of disease. In detail, recurrences were: diffuse in 7 patients, hepatic in 31, pulmonary in 13 and peritoneal in 3. Development of recurrences was significantly associated with localization of

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PDC at the tumor edge of LM (P =0.0004) (Table 2). When we analyzed the prognostic value of PDC on CSS or PFS, we compared the Kaplan-Meier

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curves of: 1) patients with PDC in LM vs patients without PDC in LM; 2) patients with PDC at the periphery and center or only at the periphery of LM vs patients with PDC only at the center of LM or without PDC in LM. Univariate analyses showed that the presence of PDC (P =0.016) (Figure 3A), their localization at tumor edge of LM (P =0.0004) (Figure 3B), positive resection margin of LM (P=0.03) (Figure 3C), development of recurrences (P=0.001) and recurrences in sites other than liver (P=0.005) were significant prognostic factors associated with shorter CSS (Table 3).

ACCEPTED MANUSCRIPT The presence of nodal metastases (pN+) (P= 0.04) and PDC presence at the edge of LM (P= 0.07) (Figure 3D) were significantly associated with shorter PFS (Table 4). PDC presence at the periphery of LM and positive resection margin were significant and independent prognostic variables for CSS (Table 3). PDC presence at the periphery of LM was the

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only significant and independent prognostic variable for PFS (Table 4).

ACCEPTED MANUSCRIPT DISCUSSION In this retrospective study, we reviewed the clinical records and the histological slides of a series of colorectal synchronous LM in the aim: 1) to define whether prognostic parameters in other studies maintained their relevance in our patients and 2) to investigate the potential prognostic value of PDC counting in LM. In order to have a homogeneous cohort of tumors, we included only

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synchronous LM which had been resected at the same time of the primary CRC and which had been

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not submitted to any neo-adjuvant treatments. Indeed, pre-operative chemoradiotherapy may alter

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tumor morphology and patients outcome [25,26]. In addition, there is consensus that patients with

represent distinguished prognostic groups [4].

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hepatic synchronous and metachronous colorectal metastases have dissimilar outcome and thus

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Differently from that previously noted [22,27,28], a number of LM higher than 3 and maximum LM diameter higher than 5 cms were not significant for CSS and DFS length in our study. However,

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previous studies considered heterogeneous cohorts, composed of both synchronous and

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metachronous LM [27,28]. In addition, the inclusion of patients submitted to neo-adjuvant treatments might have increased the relevance of LM size as a prognostic factor in previous analyses [23]; indeed, if effective, therapy reduces tumor size [23], and LM diameter may just

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reflect treatment efficacy rather than being itself prognostically relevant.

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In line with that reported by other authors [5], we found that the development of recurrences, and especially of extra-hepatic recurrences, during the follow-up had significant prognostic impact in patients with CRC and synchronous LM. In addition, we confirmed previous evidence that tumor border < 5 mm from the cutting edge is significantly correlated with shorter CSS [21] and this was an independent prognostic variable in our cohort of patients.

ACCEPTED MANUSCRIPT However, the novelty of this study relates to the analysis of PDC in colorectal LM. Indeed, to the best of our knowledge, only one study quantified and assessed the prognostic significance of PDC in surgically resected colorectal LM. However, in that study, the cohort was widely heterogeneous as it included both synchronous and metachronous LM, which can have different prognosis [4]. In addition, the use of pre-operative chemoradiotherapy might have altered the morphological aspect

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of LM and appropriate quantification of PDC.

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PDC were firstly described by Ueno and coll. [24] in primary CRC both at the invasive front and

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within the tumor stroma. Then they were evidenced in nodal metastases [29] and this study describes their presence also in colorectal LM. PDC can be considered a morphological hallmark of

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epithelial mesenchymal transition (EMT), a process by which neoplastic cells lose their epithelial properties and acquire the mesenchymal potential to migrate through extracellular matrix and to

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metastatize [30]. That PDC represent neoplastic clones with higher metastatic potential is suggested by several factors. First, primary CRC with high number of PDC have higher probability of having

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nodal metastases [14], and even nodal micrometastases [31]. Second, when PDC and main tumor

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mass have different biomolecular profile, nodal metastases may have the same genetic mutations as

disease [32].

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those observed in PDC [32], which suggests that PDC may represent the source of metastatic

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Interestingly, we found PDC in at least one LM in most of our patients. Since in the majority of LM, PDC counting was lower than 5, cases were split into PDC positive and negative for statistical analyses. PDC presence was more frequent in LM from CRCs having higher pT stage. In addition, the number of PDC in LM was strongly correlated with that in primary CRC. PDC presence in LM was not associated with extra-hepatic disease. On the other hand, high number of PDC in primary CRC was significantly associated with extra-hepatic disease in patients with

ACCEPTED MANUSCRIPT colorectal liver metastases [17]. If PDC are actually the part of the tumor with the highest metastatic potential, it is tempting to speculate that extra-hepatic disease synchronous with LM is originated from the primary tumor rather than from LM. This study showed for the first time that the patients with PDC in LM had significantly shorter CSS compared to the patients without PDC in LM. Hence, the evaluation of PDC presence in LM may

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be relevant to predict the clinical course of patients with CRC and synchronous surgically resected

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LM. However, we noticed that PDC had not the same localization in all of our cases. Indeed, in 5

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cases (10%), PDC were found only at the center of the tumor, in 18 (35%) they were both at the center and periphery of LM, while in 28 (55%) PDC were found only at the periphery of metastatic

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tumor. We may hypothesize that PDC originate in the tumor center and that they successively migrate towards tumor periphery to invade surrounding tissue, so that they are more frequent in this

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zone of the tumor. However, an alternative hypothesis is that PDC in the center of LM represent residual neoplastic cells with EMT after metastatization. Indeed, it is known that EMT is a

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reversible process and that tumor cells need to undergo mesenchymal epithelial transition (MET)-

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i.e. to recover epithelial properties- in order to proliferate and to produce a metastatic tumor; then, some cells in the metastatic mass undergo EMT and re-acquire invasive and metastatic properties

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[33]. Thus, PDC at the center and periphery of the tumor may have different significance, the latter

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being associated with higher possibility of invasion and metastatization. In accordance with this hypothesis, none of the patients with PDC only at the center of LM died during the follow-up. In addition, when we lumped together cases without PDC in LM and those with PDC only at the center of LM, their CSS was significantly longer than that of cases with PDC at the periphery of LM; moreover, the presence of PDC at the periphery of LM was an independent prognostic variable at multivariate analysis for CSS. Finally, localization of PDC at the periphery of LM, but not the

ACCEPTED MANUSCRIPT sole PDC presence, was significantly associated with the development of recurrence and with shorter PFS in our patients. In addition, it was a negative and independent prognostic factor for PFS. Recently, Fonseca and coll. [34] reviewed the prognostic relevance of histopathological factors observed in surgical specimens of colorectal LM. Of note, those authors did not discuss on the

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prognostic impact of histological grading of LM [34]. However, this study demonstrates for the first

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time that a grading system based on the presence of PDC in LM is associated with worse prognosis,

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similarly to that observed in primary CRC [17] and in nodal metastases as well [30]. This issue warrants further investigation in other cohort of patients. Indeed, if confirmed, PDC presence and,

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above all, their localization in LM may be used to stratify patients on their risk of bad prognosis in terms of CSS and PFS. As such, this feature may be added to the histopathological report as a

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relevant information useful to predict patients prognosis and for clinical decision making.

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ACCEPTED MANUSCRIPT TABLES Table 1. Statistical correlations between PDC presence in LM and the various clinicopathological parameters. . Parameter

PDC in LM absent (n= 12) present (n= 51)

20 19 12

0 2 10 0

1 1 30 19

6 6

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3 9

9 42 30 21 42 9

9 3

42 9

5 7 0

15 24 12

9 3

15 36

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9 3

40 11

3 9

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36 15

11 1

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M F Site primary tumor Right Colon Left colon rectum pT 1 2 3 4 pN 0 N+ Grade primary tumor LG HG Number LM 1-3 >4 Diameter of largest LM ≤5 cm >5 cm Site LM right lobe left lobe bilateral Tumor border configuration LM pushing infiltrative Resection Margin LM R0 R1 Synchronous metastases at other sites absent present Recurrences absent present Recurrences

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Sex

ACCEPTED MANUSCRIPT hepatic 2 30 non hepatic 7 16 Death from CRC absent 9 16 present 3 35 M: males. F: females. LM: liver metastasis. PDC: poorly differentiated clusters. LG: low-grade. HG: high

AC

CE

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D

MA

NU

SC

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grade.

ACCEPTED MANUSCRIPT Table 2. Statistical correlations between recurrences of CRC and the various clinicopathological parameters. Parameter

Recurrences

P

Absent (n= 8)

Present (n= 55)

6 2

39 16

1 6 1

21 17 17

0 0 6 2

1 3 34 17

Sex

CE

AC

SC

RI

PT

1

0.053

0.542

0.169

31 24

1

7 1

46 9

1

7 1

44 11

1

4 3 1

16 28 11

0.49

3 5

9 46

0.169

3 1 1

2 17 27

0.0004

4 4

20 35

0.466

6 2

38 17

1

7

42

D

5 3

NU

9 46

MA

3 5

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M F Site primary tumor Right Colon Left colon rectum pT 1 2 3 4 pN pN0 pN+ Grade primary tumor LG HG Number LM 1-3 >4 Diameter of the largest LM ≤5 cm >5 cm Site LM right lobe left lobe bilateral PDC LM absent present PDC localization in LM Center center/tumor edge tumor edge Tumor border configuration LM pushing infiltrative Resection margin LM R0 R1 Synchronous metastases at other sites absent

ACCEPTED MANUSCRIPT present 1 13 0.671 M: males. F: females. LM: liver metastasis. PDC: poorly differentiated clusters. LG: low-grade. HG: high grade. Bold character indicates significant variables.

Table 3. CSS analysis in 63 patients with CRC showing synchronous LM

Variables

n

Died of disease (n) (%)

Univariate HR (95% CI)

P

Multivariate HR (95% CI) P

Sex 27 11

60 61

1 1.2 (0.6-2.7)

22 23 18

16 11 11

73 48 61

1 0.4 (0.2-1) 0.6 (0.2-1.4)

1 3 40 19

1 2 23 12

100 67 58 63

1 0.4 (0.01-15.7) 0.3 (0.01-8) 0.4 (0.01-12.3)

0.5

12 51

7 31

58 61

1 1.6 (0.8-3.3)

0.212

36 27

23 15

64 56

1 0.8 (0.4-1.6)

0.671

32 6

60 60

1 0.9 (0.4-2.3)

0.996

51 12

29 9

57 75

1 1.8 (0.8-4.2)

0.07

20 31 12

11 18 9

55 58 75

1 1 (0.5-2.2) 1.5 (0.6-4)

0.544

12 51

3 35

25 69

1 3.7 (1.7-7.8)

0,.164

17

3

17

1

46

35

76

6.1 (3.1-12)

0.0004

24 39

11 27

46 69

1 1.8 (0.9-3.4)

0.09

44

25

57

1

CE

AC

RI

0.473

0.139

SC

NU

MA

D

53 10

PT

45 18

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M F Site primary tumor Right Colon Left colon rectum pT 1 2 3 4 pN pN0 pN+ Grade primary tumor LG HG Number LM 1-3 >4 Diameter of the largest LM ≤5 cm >5 cm Site LM right lobe left lobe bilateral PDC absent present PDC localization in LM Absent/ at the center At the center and periphery/at the periphery Tumor border configuration LM pushing infiltrative Margin LM R0

0.1 (0.04-0.4) 1

1

0.001

ACCEPTED MANUSCRIPT 19

13

68

1.9 (0.8-4.2)

0.039

49 14

30 8

61 57

1 0.9 (0.4-2)

0.93

8 55

0 38

0 69

/ /

0.001

CE

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D

MA

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SC

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grade. Bold character indicates significant variables.

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2.5 (1.2-5.3) 0.01

8 0 0 1 3 1 33 1 52 37 71 2 (0.4-10) 0.005 liver metastasis. PDC: poorly differentiated clusters. LG: low-grade. HG: high

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R1 Synchronous metastases at other sites absent present Recurrences absent present Recurrences absent hepatic other sites M: males. F: females. LM:

ACCEPTED MANUSCRIPT Table 4. PFS analysis in 63 patients with CRC showing synchronous LM

n.

Variables

Recurrences n. (%)

Univariate HR (95% CI)

P

0.517

Multivariate HR (95% CI) P

39 16

87 89

1 1.2 (0.6-2.2)

22 23 18

21 17 17

95 74 94

1 0.5 (0.3-1) 1 (0.5-2.2)

1 3 40 19

1 3 34 17

100 100 85 89

1 0.9 (0..06-13.1) 0.5 (0.05-6.3) 0.9 (0.08-10.9)

12 51

9 46

75 90

36 27

31 24

53 10

46 9

AC

center/tumor edge tumor edge Tumor border configuration LM pushing infiltrative

SC

0.0402

86 89

1 1.1 (0.6-1.8)

0.705

87 90

1 1 (0.4-2)

0.965

NU

1 1.9 (1,1-3,5)

MA

D

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0.316

51 12

44 11

86 92

1 1.3 (0.6-2.6)

0.336

20 31 12

16 28 11

80 90 92

1 1.6 (0.9-2.8) 1.6 (0.7-3.5)

0.225

12 51

9 46

75 90

1 1.5 (0.8-2.8)

0.217

17 46

11 44

65 96

1 2.2 (3.3-3.9)

0.0079

24 39

20 35

83 90

1 1.1 (0.6-1.9)

0.603

44

38

86

1

Margin LM R0

0.109

RI

45 18

CE

M F Site primary tumor Right Colon Left colon rectum pT 1 2 3 4 pN pN0 pN+ Grade primary tumor LG HG Number LM 1-3 >4 Diameter bigger LM ≤5 cm >5 cm Site LM right lobe left lobe bilateral PDC absent present PDC localization in LM

PT

Sex

0.1 (0.040.4) 1

0.014

ACCEPTED MANUSCRIPT R1 19 17 Synchronous metastases at other sites absent 49 42 present 14 13 M: males. F: females. LM: liver metastasis.

89

1.4 (0.7-2.7)

0.147

86 1 93 1 (0.5-1.9) 0.918 PDC: poorly differentiated clusters. LG: low-grade. HG: high

AC

CE

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D

MA

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SC

RI

PT

grade. Bold character indicates significant variables.

ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. A. Poorly differentiated clusters (PDC) in liver metastases (LM) from colorectal cancer (CRC). PDC were formed by at least 5 neoplastic cells in the absence of glandular formation (Haematoxylin and eosin stain; original magnification, x200). B. PDC at the periphery of LM (arrows) (Haematoxylin and eosin stain; original magnification, x100).

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Higher magnification of PDC indicated by black arrow in the inset (Haematoxylin and eosin

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stain; original magnification, x400). C. PDC (circle) in the centre of LM (Haematoxylin and

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eosin stain; original magnification, x 400). D. Poorly differentiated (according to World Health Organization grading) LM showing PDC (arrow) (Haematoxylin and eosin stain;

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original magnification, x100).

Figure 2. A. LM with pushing tumor border configuration (Haematoxylin and eosin stain;

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original magnification, x200). B. LM with infiltrative tumor border tumor border configuration (Haematoxylin and eosin stain; original magnification, x100).

D

Figure 3. Kaplan-Meier curves showed that (A) cancer specific survival (CSS) of the

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patients with no PDC in LM was significantly longer compared to that of the patients with PDC in LM (P= 0.0164), (B) CSS of the patients with no PDC in LM or PDC only at the

CE

center of LM was significantly longer compared to that of the patients with PDC at the periphery of LM (P <0.001) (C) CSS of patients with negative margin of resection (R0) in

AC

LM was significantly longer compared with that of patients with positive margin of resection (R+) in LM (P= 0.03), (D) progression free survival (PFS) of the patients with no PDC in LM or PDC only at the center of LM was significantly longer compared to that of the patients with PDC at the periphery of LM (P=0.0079).

ACCEPTED MANUSCRIPT HIGHLIGHTS

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

The prognosis of patients with colorectal LM is commonly established on clinical features. Low attention is given to histopathological features of LM as prognostic factors. The number of PDC in primary CRC is correlated with prognosis and metastatic disease. This study shows that PDC presence and peripheral localization in synchronous colorectal LM is associated with bad prognosis. Assessment of PDC counting in synchronous LM may be useful to assess prognosis and for clinical decision making in patients with stage IV CRC.

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   

Figure 1

Figure 2

Figure 3