Metastatic Lesions Of Colorectal Cancer Research Articles

The Clinical Potential of FBPA-PET/CT Imaging for Colorectal Cancer and its Liver Metastases Expressing LAT1.

18F-fluoro-deoxy-glucose positron emission tomography (18F-FDG PET) has become indispensable for staging colorectal cancer but has limitations. Thus, PET with a focus on metabolism other than glucose, mainly amino acid metabolism, has been developed. L-type amino acid transporter 1 (LAT1) is known to be a cancer-specific amino acid transporter and, although 4-Borono-2-(18)F-fluoro-phenylalanine (FBPA) has been reported to be useful as a probe for LAT1, the significance of LAT1 expression in colorectal cancer is ambiguous and implementation of 18F-FBPA-PET in colorectal cancer has not yet been reported. The aims of this study were to investigate the expression of LAT1 in primary lesions and metastatic lesions of colorectal cancer by immunohistochemical analysis and report the initial experience of performing 18F-FBPA-PET on colorectal cancer patients in clinical practice. There was a significant correlation between LAT1 protein expression in primary tumors and liver metastases. Furthermore LAT-1 expression was positively correlated with recurrence (p=0.033). We performed 18F-FBPA-PET on three rectal cancer patients and detected cancer. LAT1 protein is expressed not only in the primary colorectal tumor, but also in liver metastases. 18F-FBPA-PET can be safely performed in patients with colorectal cancer.

The dynamics of telomere length in primary and metastatic colorectal cancer lesions

Telomeric sequences, the structures comprised of hexanucleotide repeats and associated proteins, play a pivotal role in chromosome end protection and preservation of genomic stability. Herein we address telomere length (TL) dynamics in primary colorectal cancer (CRC) tumour tissues and corresponding liver metastases. TL was measured by multiplex monochrome real-time qPCR in paired samples of primary tumours and liver metastases along with non-cancerous reference tissues obtained from 51 patients diagnosed with metastatic CRC. Telomere shortening was observed in the majority of primary tumour tissues compared to non-cancerous mucosa (84.1%, p < 0.0001). Tumours located within the proximal colon had shorter TL than those in the rectum (p < 0.05). TL in liver metastases was not significantly different from that in primary tumours (p = 0.41). TL in metastatic tissue was shorter in the patients diagnosed with metachronous liver metastases than in those diagnosed with synchronous liver metastases (p = 0.03). The metastatic liver lesions size correlated with the TL in metastases (p < 0.05). Following the neoadjuvant treatment, the patients with rectal cancer had shortened telomeres in tumour tissue than prior to the therapy (p = 0.01). Patients with a TL ratio between tumour tissue and the adjacent non-cancerous mucosa of ≥ 0.387 were associated with increased overall survival (p = 0.01). This study provides insights into TL dynamics during progression of the disease. The results show TL differences in metastatic lesions and may help in clinical practice to predict the patient’s prognosis.

EGFR, HER2, and HER3 protein expression in paired primary tumor and lymph node metastasis of colorectal cancer

Due to the difficulty in sampling of metastatic tumors, patient selection is commonly based on results of primary tumor samples when metastatic samples are not available. However, due to tumor heterogeneity, metastatic tumors may be different from primary tumors in their phenotypes. The aim of this study was to investigate the expression of EGFR, HER2, and HER3 between primary and lymph node metastatic lesions of colorectal cancer. Paired primary tumors and lymph node metastases from 79 patients with colorectal cancer were retrospectively collected and analyzed for EGFR, HER2, and HER3 expression. High EGFR, HER2, and HER3 expression (2+ and 3+) was found in 64.2%, 66.0%, and 85.0% of primary tumors, and 56.8%, 46.0%, and 76.0% of lymph node metastases, respectively. Correlation rates between primary and metastatic lesions were 67.1%, 63.3%, and 74.7% for EGFR, HER2, and HER3, respectively. Stage IV tumors (with distant metastasis) had higher correlation rates of HER2 expression compared to stage III tumors (without distant metastasis) (P = 0.050). Moderate correlation rates in EGFR, HER2, and HER3 expression were observed between primary and metastatic lesions of colorectal cancer. Tumor stage or existence of distant metastasis could serve as potential predictive markers for the correlation of HER2 expression between primary tumors and lymph node metastases of colorectal cancer.

Intratumor heterogeneity of positive lymph nodes provide a promising prognostic stratification factor for stage III colorectal cancer.

e15554 Background: Stage III colorectal cancer (CRC) is defined as patients with regional positive lymph nodes (pLN) but without distant metastasis, including stage IIIA, IIIB, IIIC. The prognosis of patients with stage III CRC varies dramatically, with 5-year survival rates ranging from 80 percent to 30 percent form stage IIIA to IIIC CRC, that does not exactly to be fully explained by the difference in the number of pLN. Therefore, other prognostic indicators need to be identified to improve and personalize prognosis prediction for patients with stage III CRC. Intratumor heterogeneity (ITH) widely exists between primary and metastatic lesions of CRC, however, there have been few studies focused on ITH of pLN. Therefore, this study aims to explore whether ITH of pLN could serve as a novel prognostic stratification factor for stage III CRC. Methods: We retrospectively analyzed the Poly-G genotype to reconstruct high-confidence phylogenetic trees for 20 CRC patients using 317 accurately scraped biopsy (134 primary tumors, 66 lymph node metastases and 37 normal samples). Root diversity score (RDS) index was used to reflect ITH of pLN. The association among Poly-G mutation rates, RDS index and clinical pathological data was analyzed. The area under the receiver operating characteristic (ROC) curves were calculated and the cut-off valve was confirmed. Results: Poly-G mutations accumulate in the occurrence and development of CRC. Poly-G mutation rate was correlated with tumor grade, N stage and microsatellite state (all P &lt; 0.05), and had a certain correlation trend with age (P = 0.066). The results of multivariate Cox regression analysis showed that ITH of pLN was an independent risk factor for recurrence and metastasis which lead to a poor progression free survival (PFS). The area under the curve (AUC) was 76%, and the cut-off valve of RDS index was 0.25. We also found ITH of pLN was not associated with substages of stage III CRC, which indicated that ITH of pLN and the number of metastases are two different dimensions. Conclusions: ITH of pLN may provide a promising prognostic stratification factor for the patients with stage III CRC. A higher ITH of pLN indicates that subclones with distant metastasis potential might more likely to exist in the primary tumors, which lead to a poor PFS including recurrence and distant metastasis. In addition, ITH of pLN is not related to the number of lymph node metastases in stage III CRC, suggesting that ITH of pLN is promising to serve as a supplementary strategy of TNM staging for patients with Stage III CRC.

Whole exome and transcriptome sequencing reveal clonal evolution and exhibit immune-related features in metastatic colorectal tumors

Liver is the most common site where metastatic lesions of colorectal cancer (CRC) arise. Although researches have shown mutations in driver genes, copy number variations (CNV) and alterations in relevant signaling pathways promoted the tumor evolution and immune escape during colorectal liver metastasis (CLM), the underlying mechanism remains largely elusive. Tumor and matched metastatic tissues were collected from 16 patients diagnosed with colorectal cancer and subjected to whole-exome sequencing (WES) and RNA sequencing (RNA-seq) for studying colorectal cancer clonal evolution and immune escape during CLM. Shared somatic mutations between primary and metastatic tissues with a commonly observed subclonal-clonal (S-C) changing pattern indicated a common clonal origin between two lesions. The recurrent mutations with S-C changing pattern included those in KRAS, SYNE1, CACNA1H, PCLO, FBXL2, and DNAH11. The main CNV events underwent clonal-clonal evolution (20q amplification (amp), 17p deletion (del), 18q del and 8p del), subclonal-clonal evolution (8q amp, 13q amp, 8p del) and metastasis-specific evolution (8q amp) during the process of CLM. In addition, we revealed a potential mechanism of tumor cell immune escape by analyzing human leukocytes antigens (HLA) related clonal neoantigens and immune cell components in CLM. Our study proposed a novel liver metastasis-related evolutionary process in colorectal cancer and emphasized the theory of neo-immune escape in colorectal liver metastasis.

Expression and prognostic value of epithelial-to-mesenchymal transition and cancer stem cellmarkersin primary lesions and liver metastases of colorectal cancers.

Cancer stem cells (CSCs) and epithelial mesenchymal transition (EMT) markers are considered useful indicators associated with metastasis and prognosis of colorectal cancers (CRCs). However, only a few studies have focused on the expression of these useful markers in metastases. Metastasectomy is widely used in advanced CRCs, and thus the postoperative prognostic factors are worth investigating. The present study investigated the consistency and differences of target proteins between primary and metastatic lesions of colorectal cancer, and discussed the prognostic indicators following resection of colorectal liver metastases. Clinical data of 56 patients with liver metastases from colorectal cancer were collected and the expression levels of target proteins (Ki-67, CD133, CD44, Snail, E-cadherin and β-catenin) were detected in primary tumor and matched liver metastases via immunohistochemistry analysis. Paired comparison between both tissue types was performed. The prognostic values of the target proteins for resectable colorectal cancer liver metastases were assessed. No significant differences were observed between the primary tissues and metastatic tissues. The consistency rates of these protein expression levels ranged from 51.8–78.6%. The maximum diameter of the liver metastases was <5 cm. Low Snail expression in metastases was associated with a longer overall survival (OS) time following resection of colorectal liver metastases. Furthermore, N0 stage and low carcinoembryonic antigen levels were associated with a longer progression-free survival time. Notably, no significant differences were observed in expression levels of the target proteins between the primary tumors and liver metastases. Taken together, the results of the present study suggest that Snail expression in liver metastases may be used as a novel independent prognostic factor for OS following resection of colorectal liver metastases.

Выбор теста тромбоэластометрии для оценки эффекта профилактической дозы низкомолекулярных гепаринов

Цель исследования: изучить возможность использования тестов тромбоэластометрии (ТЭМ) для оценки эффекта профилактической дозы низкомолекулярных гепаринов (НМГ). Материалы и методы. В исследование включено 30 пациентов (14 мужчин и 16 женщин), которым выполнялась резекция печени в связи с метастатическим поражением колоректальным раком. Для профилактики тромботических осложнений в послеоперационном периоде использовали НМГ надропарин. У пациентов в первую неделю послеоперационного периода определяли активность антитромбина III (AT-III) и проводили ротационную ТЭМ, при которой исследовали 3 пробы крови: проба № 1 — утром натощак перед введением пациенту НМГ; проба № 2 — как и первая проба, но с добавлением в пробу НМГ in vitro до достижения целевой концентрации 0,5 МЕ / мл крови; проба № 3 — через 4 ч после подкожного введения НМГ. У 10 пациентов проведены тесты ТЭМ — INTEM и NATEM (нативный тест), в которых оценивали показатели CT, CFT, угол α, MCF; у 20 пациентов проведен только тест NATEM. Результаты. Средние показатели теста INTEM перед введением НМГ не выходили за рамки референтных значений, в тесте NATEM только показатель MCF превышал нормальные значения. После внесения в исследуемую пробу 0,5 МЕ / мл НМГ in vitro средние значения CT и CFT теста ТЭМ значимо удлинялись. Интервал CT теста INTEM был удлинен выше нормы в 2 наблюдениях, CFT теста INTEM превышал норму в 1 наблюдении. В тесте NATEM удлинение интервалов CT и CFT выше нормальных выявлено в 100% случаев. Показатели ТЭМ через 4 ч после введения НМГ были аналогичны результатам in vitro. Выявлены статистически значимые различия показателей нативного теста от активности AT-III, наибольшей чувствительностью и специфичностью из них обладали интервал CT и ΔCT. Заключение. Метод ротационной ТЭМ является перспективным прикроватным методом оценки антикоагулянтного эффекта профилактических доз НМГ. Использование этого метода в послеоперационном периоде позволит индивидуально дозировать антикоагулянты и повысить эффективность профилактики венозных тромбоэмболических осложнений. Objectives: to investigate the applicability of various types of thromboelastometry (TEM) to assess effects of low molecular weight heparins (LMWHs) administered in prevention doses. Patients/ Methods. The study included 30 patients (14 men and 16 women) who underwent liver resection due to metastatic lesions of colorectal cancer. LMWH (nadroparin) was used to prevent thrombotic complications in the postoperative period. In the first week of postoperative period antithrombin III (AT-III) activity was determined and rotational TEM was performed, in which 3 blood samples were examined: sample № 1 — in the morning, fasted, before LMWH administration; sample № 2 — like the first sample, but with in vitro LMWH addition to the sample to achieve the target concentration of 0.5 IU per 1 ml of blood; sample № 3–4 hours after subcutaneous LMWH administration. TEM tests — INTEM and NATEM (native test) were performed in 10 patients with CT, CFT, angle α, and MCF assessment; in 20 patients only NATEM test was performed. Results. The average values of INTEM test before LMWH administration were within the reference values, in NATEM test only MCF exceeded normal values. The average CT and CFT values in TEM test significantly increased after in vitro LMWH addition to the test sample. Elongation of CT interval in INTEM test was found in 2 cases, CFT in INTEM test exceeded the norm in 1 case. Elongation above normal of CT and CFT intervals in NATEM test was noticed in 100% of cases. TEM parameters in 4 hours after LMWH administration were similar to in vitro results. The statistically significant differences between native test parameters and AT-III activity were revealed; CT and ΔCT intervals had the greatest sensitivity and specificity. Conclusions. Rotational TEM is a perspective bedside method for anticoagulant effect assessment of LMWH prevention doses. Using this method in postoperative period will allow to dose anticoagulants individually and increase prophylaxis effectiveness of venous thromboembolic complications.

Abstract 781: Functional consequence of the p53 codon 72 polymorphism in colorectal cancer

Abstract Background: The codon 72 polymorphism in p53 has been implicated in colorectal cancer (CRC) and CRC health disparities. In this study, we determine the functional consequence of this polymorphism in CRC following both in-vitro cell based and in-vivo mouse model studies. Experimental Design: Plasmids (pCMV6) that express different phenotypes of p53 [p53 wild type (wt) at codon 72 (R72wt), R72wt with mutation at codon 273 cysteine (R72273Cys), p53 mutation at codon 72 (P72wt) and P72wt with mutation at codon 273 (P72273Cys)] were constructed. We selected a CRC cell line Caco2, which does not express p53 for in vitro studies. Severe combined immunodeficient mice were inoculated with CRC cells (HT29, SW480, and LS174) to establish tumor xenografts (tumors). Tumor angiogenesis was assessed in tumors by immunostaing for CD31. Sequencing analysis for codon 72 polymorphism of p53 was performed using genomic DNA purified from tumors. Western blot (WB) or immunostaining analyses for proteins of signaling mechanisms were performed to assess the functional consequence of P72 phenotype of p53. Results: Our study demonstrated that P72 tumors had well established vascularity, while R72 tumors had very poor vascularization. Indeed, the mean micro vessel density was higher in P72 tumors than in R72 tumors. WB analyses revealed that P72wt or mutant phenotypes effectively induced the activation of p38 and RAF/MEK/ extracellular signal-regulated kinase (ERK) MAP kinases. Up-regulation of phosphorylated SEK1/MKK4, an upstream kinase of p38 MAPK was associated with P72wt or mutant phenotypes. This activation was accompanied by up-regulation of phosphorylated-MAPKAPK-2, -Hsp27, and -CREB, downstream targets of p38 MAPK. Increased activation of CREB was found to be higher in tumors that exhibit P72 phenotype. Metastatic lesions of CRC expressed more phospho-CREB than non-metastatic lesions. Furthermore, suppression of RAF/MEK/ERK activation was significantly higher in cells that express R72wt phenotype compared to cells that express P72wt or mutant phenotypes. The expression of P72wt or mutant phenotypes displayed decreased expression of E cadherin and/or an increased expression of vimentin, fibrinectin, CD44, thereby promoting CRC metastasis. Conclusions: These findings offer significant novel insights into the mechanism by which P72 contributes to the aggressiveness of CRC. Because P72 is over-expressed in CRC, specifically in African-American patients, these studies suggest a role for P72 in cancer health disparities. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251. Citation Format: Venkat R. Katkoori, Upender Manne, Harvey Bumpers. Functional consequence of the p53 codon 72 polymorphism in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 781. doi:10.1158/1538-7445.AM2017-781

Predictors and prognosticators for survival with Yttrium-90 radioembolization therapy for unresectable colorectal cancer liver metastasis.

This critical review aims to explore predictive and prognostic biomarkers of Yttrium-90 (Y90) radioembolization therapy of colorectal liver metastases. A brief overview of established predictive and prognostic molecular and genetic biomarkers in colorectal cancer therapies will be discussed. A review of the literature on imaging modalities, genetic, metabolic and other molecular markers and the subsequent outcomes in post-Y90 treatment will be presented. How these biomarkers and future biomarker research can inform locoregional treatment decisions in the clinical setting of metastatic colorectal cancer lesions of the liver will be explored. There are opportunities for personalized cancer treatment in the setting of Y90 radioembolization. The ability to predict tumor response after Ytrium-90 radioembolization therapy can greatly impact clinical decision making and enhance treatment outcomes, therefore further research into the field is needed.

322. Comparative gene mutations between primary and peritoneal metastatic lesions of colorectal cancer

322. Comparative gene mutations between primary and peritoneal metastatic lesions of colorectal cancer

Assessment of metastatic colorectal cancer with hybrid imaging: comparison of reading performance using different combinations of anatomical and functional imaging techniques in PET/MRI and PET/CT in a short case series.

The purpose was to investigate the diagnostic performance of different combinations of anatomical and functional imaging techniques in PET/MRI and PET/CT for the evaluation of metastatic colorectal cancer lesions. Image data of 15 colorectal cancer patients (FDG-PET/CT and subsequent FDG-PET/MRI) were retrospectively evaluated by two readers in five reading sessions: MRI (morphology) alone, MRI/diffusion-weighted MRI (DWI), MRI/PET, MRI/DWI/PET; and PET/CT. Diagnostic performance of lesion detection with each combination was assessed in general and organ-based. The reference standard was given by histology and/or follow-up imaging. Separate analysis of mucinous tumours was performed. One hundred and eighty lesions (110 malignant) were evaluated (intestine n = 6, liver n = 37, lymph nodes n = 55, lung n = 4, and peritoneal n = 74). The overall lesion-based diagnostic accuracy was 0.46 for MRI, 0.47 for MRI/DWI, 0.57 for MRI/PET, 0.69 for MRI/DWI/PET and 0.66 for PET/CT. In the organ-based assessment, MRI/DWI/PET showed the highest accuracy for liver metastases (0.74), a comparable accuracy to PET/CT in peritoneal lesions (0.55), and in lymph node metastases (0.84). The accuracy in mucinous tumour lesions was limited in all modalities (MRI/DWI/PET = 0.52). PET/MRI including DWI is comparable to PET/CT in the evaluation of colorectal cancer metastases, with a markedly higher accuracy when using combined imaging data than the modalities separately. Further improvement is needed in the imaging of peritoneal carcinomatosis and mucinous tumours.

Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions

BackgroundColorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors.ResultsWe performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations.ConclusionsColorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0454-7) contains supplementary material, which is available to authorized users.

Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions

Colorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors. We performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations. Colorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.

Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer

Background:LINE-1 methylation level is a surrogate marker of global DNA methylation. LINE-1 methylation in primary colorectal cancers (CRCs) is highly variable and strongly associated with a poor prognosis. However, no study has examined LINE-1 methylation levels of metastatic CRCs in relation to prognosis or assessed the heterogeneity of LINE-1 methylation level within the primary CRCs.Methods:Pyrosequencing was used to quantify LINE-1 methylation level in 42 liver metastases, 26 matched primary tumours, and 6 matched lymph node (LN) metastases. KRAS, BRAF, and PIK3CA mutation status and microsatellite instability (MSI) status were also examined.Results:The distribution of LINE-1 methylation level in liver metastases was as follows: mean, 67.3; range, 37.1–90.1. Primary tumours showed LINE-1 methylation levels similar to those of matched liver and LN metastases. The difference in LINE-1 methylation level between superficial areas and invasive front areas was within 7.0 in all six cases evaluated. Prognostic impact of LINE-1 hypomethylation in liver metastases on overall survival was not observed. The concordance rate was 94% for KRAS, 100% for BRAF, 88% for PIK3CA, and 97% for MSI.Conclusion:Alteration of LINE-1 methylation level may occur in early CRC tumorigenesis, and the LINE-1 methylation level is relatively stable during CRC progression.

Abstract 648: Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer.

Abstract Background: Cancer cells exhibit two types of DNA methylation changes: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Genome-wide DNA hypomethylation plays an important role in genomic instability and carcinogenesis. As LINE-1 or L1 retrotransposon constitutes a substantial portion of the human genome, the level of L1 methylation is regarded to be a surrogate marker of global DNA methylation. In Colorectal cancer (CRC), L1 methylation in primary tumor has been shown to be highly variable, and L1 hypomethylation is strongly associated with poor prognosis. Nonetheless, no study has examined L1 methylation level in metastatic lesions [liver, lung, and lymph node (LN)] from CRCs, and assessed the heterogeneity of L1 methylation level within CRC tumor. Methods: A total of 65 patients with liver and/or lung metastases from CRC who were undergoing hepatic and/or lung resection at Kumamoto University Hospital were enrolled in this study. The bisulfite-pyrosequencing technology was utilized to quantify the L1 methylation level in 60 liver metastases, matched 26 primary tumors and matched 6 LN metastases, and 6 paired lung metastatic and primary tumors. The heterogeneity within primary tumor (superficial area vs. invasive area) was also evaluated (n=6). Results: The distribution of the L1 methylation level in liver metastases (n=60) was as follows: mean, 68.9; median, 71.0; SD, 10.8; range, 37.1-90.1 (all in 0-100 scale). L1 methylation level of liver metastases was not associated with sex, age at hepatic resection, tumor stage of primary lesion, or tumor size. L1 methylation level of liver metastases was significantly related with that of matched primary tumors (n=26; Pearson correlation coefficient r=0.71, p=0.0001). LN metastases showed similar L1 methylation level of matched primary tumors and liver metastases (n=6); the distribution of the L1 methylation level in matched 6 LN metastases was as follows: mean, 71.0; SD, 9.37; range, 59.3-81.9. In addition, lung metastases and matched primary tumors exhibited similar L1 methylation level; the distributions of the L1 methylation level in lung metastases and matched primary tumors (n=6) were as follows: mean, 65.6 and 71.3; range, 55.7-82.2 and 66.1-79.6, respectively. Interestingly, the heterogeneity of L1 methylation level was not observed; the difference between superficial area and invasive area was within 7 in all 6 cases. Conclusions: Liver, lung and LN metastases showed similar L1 methylation level with primary tumors. In addition, L1 methylation level of superficial area was roughly in accordance with that of invasive area; thus supporting the absence of heterogeneity of L1 methylation level within one tumor. These results may suggest that cancer cells acquire “global DNA hypomethylation” at the early stage of tumor development, and it is preserved through the process of cancer invasion and metastasis. Citation Format: Asuka Murata, Yoshifumi Baba, Masayuki Watanabe, Hironobu Shigaki, Shiro Iwagami, Yasuo Sakamoto, Yuji Miyamoto, Hideo Baba. Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 648. doi:10.1158/1538-7445.AM2013-648

Colorectal Liver Metastasis After 90Y Radioembolization Therapy: Pilot Study of Change in MDCT Attenuation as a Surrogate Marker for Future FDG PET Response

The purpose of this study was to investigate whether changes in attenuation and size of liver metastatic lesions of colorectal cancer at MDCT 1 month after (90)Y radioembolization treatment are predictive of response at FDG PET 3 months after treatment. Twenty patients with colorectal liver metastasis consecutively treated with (90)Y radioembolization underwent triphasic MDCT of the liver at baseline and 1 and 3 months after treatment and FDG PET at baseline and 3 months after treatment. Percentage change in tumor attenuation at MDCT (volumetric attenuation), tumor size at MDCT (according to Response Evaluation Criteria in Solid Tumors [RECIST] and World health Organization [WHO] criteria), and volume-weighted maximum standardized uptake value at FDG PET were evaluated. The correlation between FDG PET response 3 months after treatment and response according to RECIST, WHO criteria, and attenuation 1 month after treatment was evaluated. Only 13.3% of patients with FDG PET findings of response 3 months after treatment were identified according to RECIST and WHO criteria 1 month after treatment. According to attenuation criteria at 1 month, however, 53.3% of patients with an FDG PET response at 3 months were identified. A strong association was found between FDG PET response at 3 months and response based on attenuation criteria (odds ratio, 12.4; 95% CI, 0.58-265.3; p = 0.05). Early changes in the attenuation of liver metastatic lesions of colon cancer after (90)Y radioembolization treatment may be predictive of future response at FDG PET.

Heterogeneity of KRAS Status May Explain the Subset of Discordant KRAS Status Between Primary and Metastatic Colorectal Cancer

KRAS status is a useful predictive marker for anti-epidermal growth factor receptor antibody therapy. This study aimed to examine the concordance rate of KRAS mutation status between corresponding primary and metastatic colorectal cancer lesions, and also among multiple metastatic tumors. Furthermore, we examined the heterogeneity of KRAS mutations with respect to discordant KRAS status between primary and metastatic tumors. This study was retrospective in design. Forty-three patients with primary tumors and 113 metastatic tumors were studied. The KRAS mutational status was determined by the peptide nucleic acid clamp real-time polymerase chain reaction TaqMan assay. We also performed sequencing analysis to validate the KRAS mutational status. When KRAS status differed between primary and metastatic tumors, we examined the heterogeneity of KRAS status within individual primary tumors by microdissecting multiple samples in each patient. The frequency of KRAS mutations in primary tumors was 34.9%. A high concordance rate of KRAS (88.4-91.7%) mutations was observed between primary and metastatic tumors. All 5 cases (11.6%) with discordant KRAS status had heterogeneous KRAS status in primary tumors. However, in 10 concordant cases all microdissected areas showed an identical KRAS mutational status within each patient. The KRAS mutational statuses in all multiple liver and/or lung metastatic tumors were the same as those of the primary tumor. We could not validate KRAS status in microdissected samples by the direct sequence method that was used in the present study, because the quantity of DNA was not sufficient to perform direct sequencing. KRAS status in a primary site may be used for selecting patients who would benefit from anti-epidermal growth factor receptor therapy. However, KRAS status can be heterogeneous within a primary tumor, and thus different parts of such tumors should be examined for KRAS status to correctly predict the KRAS status in metastatic lesions.

Localization and Density of Immune Cells in the Invasive Margin of Human Colorectal Cancer Liver Metastases Are Prognostic for Response to Chemotherapy

Analysis of tumor-infiltrating lymphocytes (TIL) in primary human colorectal cancer (CRC) by in situ immunohistochemical staining supports the hypothesis that the adaptive immune response influences the course of human CRC. Specifically, high densities of TILs in the primary tumor are associated with good prognosis independent of other prognostic markers. However, the prognostic role of TILs in metastatic CRC lesions is unknown, as is their role in response or resistance to conventional chemotherapy. We analyzed the association of TIL densities at the invasive margin of CRC liver metastases with response to chemotherapy and progression-free survival in a set of 101 large section samples. High-resolution automated microscopy on complete tissue sections was used to objectively generate cell densities for CD3, CD8, granzyme B, or FOXP3 positive immune cells. A predictive scoring system using TIL densities was developed in a training set and tested successfully in an independent validation set. TIL densities at the invasive margin of liver metastases allowed the prediction of response to chemotherapy with a sensitivity of 79% and specificity of 100%. The association of high density values with longer progression-free survival under chemotherapy was statistically significant. Overall, these findings extend the impact of the local immune response on the clinical course from the primary tumor also to metastatic lesions. Because detailed quantification of TILs in metastatic lesions revealed a strong association with chemotherapy efficacy and prognosis, we suggest that the developed scoring system may be used as a predictive tool for response to chemotherapy in metastatic CRC.

Predictive value of contrast enhancement on computed tomography for the responses to oxaliplatin-based chemotherapy with bevacizumab for hepatic metastases from colorectal cancer.

e14105 Background: The addition of bevacizumab (Bev) to chemotherapy is associated with improved survival by anti-angiogenic mechanism in colorectal cancer (CRC). Therefore, the antitumor effect of Bev may correlate with the degree of the contrast-enhancement (CE) of targets reflecting tumor microvessels on the computed tomography (CT). The purpose of this study was to determine whether responses to chemotherapy with bevacizumab correlated with the degree of CE of hepatic metastatic lesions of colorectal cancer (CRC). Methods: Thirty one patients with hepatic metastases from CRC who underwent at least three course of mFOLFOX6 or XELOX therapy as the first-line chemotherapy between September 2006 and April 2010 were analyzed retrospectively. The patients were divided into two groups with or without Bev (17 in the Bev group and 14 in the non-Bev group). CT scans were performed during the late arterial phase following intravenous injection with a nonionic contrast medium at a dose of 600 mgI/kg, using a multi detector CT. Using an imaging control system, the diameter and mean CT values of metastatic lesions before chemotherapy were determined in each patient. The degree of CE was evaluated by the ratio of the CT value of hepatic metastases in the late arterial phase by in the noncontrast phase (i.e., CE ratio), whereby the patients were divided into the high or low CE group by the median CE ratio. Results: There was significant correlation between the CE ratio and the tumor shrinkage rate in the Bev group (R2 =0.3817, p=0.0082), whereas no such relationship was noted in the non-Bev group. In the high CE group (n=16), the tumor shrinkage rate increased to 35.9% in the Bev group from 8.6% in the non-Bev group (p=0.0716). In the low CE group, however, no significant difference was noted. Conclusions: The present study demonstrated the correlation between the CE ration and the tumor shrinkage in patients treated with Bev. The results suggest that the CE ratio on CT scanning in hepatic metastatic lesion can be a predictive value for the response to chemotherapy with Bev, although generalization of the CE ratio and the prospective study should be need.

Monitoring early responses to irradiation with dual-tracer micro-PET in dual-tumor bearing mice

To monitor the early responses to irradiation in primary and metastatic colorectal cancer (CRC) with (18)F-fluorothymidine ((18)F-FLT) and (18)F-fluorodeoxyglucose ((18)F-FDG) small-animal position emission tomography (micro-PET). The primary and metastatic CRC cell lines, SW480 and SW620, were irradiated with 5, 10 and 20 Gy. After 24 h, the cell cycle phases were analyzed. A dual-tumor-bearing mouse model of primary and metastatic cancer was established by injecting SW480 and SW620 cells into mice. micro-PET with (18)F-FLT and (18)F-FDG was performed before and 24 h after irradiation with 5, 10 and 20 Gy. The region of interest (ROI) was drawn over the tumor and background to calculate the ratio of tumor to non-tumor (T/NT) in tissues. Immunohistochemical assay and Western blotting were used to examine the levels of integrin β(3,) Ki-67, vascular endothelial growth factor receptor 2 (VEGFR2) and heat shock protein 27 (HSP27). The proportion of SW480 and SW620 cells in the G(2)-M phase was decreased with an increasing radiation dose. The proportion of SW480 cells in the G(0)-G(1) phase was increased from 48.33% ± 4.55% to 87.09% ± 7.43% (P < 0.001) and that of SW620 cells in the S-phase was elevated from 43.57% ± 2.65% to 66.59% ± 7.37% (P = 0.021). In micro-PET study, with increasing dose of radiation, (18)F-FLT uptake was significantly reduced from 3.65 ± 0.51 to 2.87 ± 0.47 (P = 0.008) in SW480 tumors and from 2.22 ± 0.42 to 1.76 ± 0.45 (P = 0.026) in SW620 tumors. (18)F-FDG uptake in SW480 and SW620 tumors was reduced but not significantly (F = 0.582, P = 0.633 vs F = 0.273, P = 0.845). Dose of radiation was negatively correlated with (18)F-FLT uptake in both SW480 and SW620 tumors (r = -0.727, P = 0.004; and r = -0.664, P = 0.009). No significant correlation was found between (18)F-FDG uptake and radiation dose in SW480 or SW620 tumors. HSP27 and integrin β(3) expression was higher in SW480 than in SW620 tumors. The T/NT ratio for (18)F-FLT uptake was positively correlated with HSP27 and integrin β(3) expression (r = 0.924, P = 0.004; and r = 0.813, P = 0.025). (18)F-FLT is more suitable than (18)F-FDG in monitoring early responses to irradiation in both primary and metastatic lesions of colorectal cancer.