Relapse In Colon Cancer Research Articles

Triune Nanomodulator Enables Exhausted Cytotoxic T Lymphocyte Rejuvenation for Cancer Epigenetic Immunotherapy.

Oncogene activation and epigenome dysregulation drive tumor initiation and progression, contributing to tumor immune evasion and compromising the clinical response to immunotherapy. Epigenetic immunotherapy represents a promising paradigm in conquering cancer immunosuppression, whereas few relevant drug combination and delivery strategies emerge in the clinic. This study presents a well-designed triune nanomodulator, termed ROCA, which demonstrates robust capabilities in tumor epigenetic modulation and immune microenvironment reprogramming for cancer epigenetic immunotherapy. The nanomodulator is engineered from a nanoscale framework with epigenetic modulation and cascaded catalytic activity, which self-assembles into a nanoaggregate with tumor targeting polypeptide decoration that enables loading of the immunogenic cell death (ICD)-inducing agent. The nanomodulator releases active factors specifically triggered in the tumor microenvironment, represses oncogene expression, and initiates the type 1 T helper (TH1) cell chemokine axis by reversing DNA hypermethylation. This process, together with ICD induction, fundamentally reprograms the tumor microenvironment and significantly enhances the rejuvenation of exhausted cytotoxic T lymphocytes (CTLs, CD8+ T cells), which synergizes with the anti-PD-L1 immune checkpoint blockade and results in a boosted antitumor immune response. Furthermore, this strategy establishes long-term immune memory and effectively prevents orthotopic colon cancer relapse. Therefore, the nanomodulator holds promise as a standalone epigenetic immunotherapy agent or as part of a combination therapy with immune checkpoint inhibitors in preclinical cancer models, broadening the array of combinatorial strategies in cancer immunotherapy.

579P A clinico-imaging predictive artificial intelligence model of relapse in colon cancer using baseline CT scans

579P A clinico-imaging predictive artificial intelligence model of relapse in colon cancer using baseline CT scans

Advanced Progression for the Heterogeneity and Homeostasis of Intestinal Stem Cells.

Current understanding of the leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) in intestinal stem cells (ISCs) is well established, however, the implications of ISC heterogeneity and homeostasis are poorly understood. Prior studies have provided important evidence for the association between heterogeneity of ISC pools with pathogenesis and therapeutic response of malignant disease. Leveraging the advantages of organoids and single cell RNA sequencing (scRNA-seq), glandular development has been simulated and cell heterogeneity has been clarified. Based on this research, several potential ISCs were identified, such as LGR5 + p27 + quiescent ISCs, LGR5 + Mex3a + slowly proliferating stem cells, and CLU + reverse stem cells. We also illustrated major factors responsible for ISC homeostasis including metabolism-related (LKB1, TGR5, HMGCS2), inflammation-related (IFB-b, IFN2, TNF), and Wnt signaling-related (CREPT, Mex3a, MTG16) factors. ISCs play complex roles in intestinal tumorigenesis, chemoresistance and occasional relapse of colon cancer, which bear discussion. In this review, we focus on novel technical challenges in ISCs fate drawing upon recent research with the goals of clarifying our understanding of complex ISCs, elucidating the integrated intestinal crypt niche, and creating new opportunities for therapeutic development.

Cell2Grid: an efficient, spatial, and convolutional neural network-ready representation of cell segmentation data.

Cell segmentation algorithms are commonly used to analyze large histologic images as they facilitate interpretation, but on the other hand they complicate hypothesis-free spatial analysis. Therefore, many applications train convolutional neural networks (CNNs) on high-resolution images that resolve individual cells instead, but their practical application is severely limited by computational resources. In this work, we propose and investigate an alternative spatial data representation based on cell segmentation data for direct training of CNNs. We introduce and analyze the properties of Cell2Grid, an algorithm that generates compact images from cell segmentation data by placing individual cells into a low-resolution grid and resolves possible cell conflicts. For evaluation, we present a case study on colorectal cancer relapse prediction using fluorescent multiplex immunohistochemistry images. We could generate Cell2Grid images at resolution that were 100 times smaller than the original ones. Cell features, such as phenotype counts and nearest-neighbor cell distances, remain similar to those of original cell segmentation tables ( ). These images could be directly fed to a CNN for predicting colon cancer relapse. Our experiments showed that test set error rate was reduced by 25% compared with CNNs trained on images rescaled to with bilinear interpolation. Compared with images at resolution (bilinear rescaling), our method reduced CNN training time by 85%. Cell2Grid is an efficient spatial data representation algorithm that enables the use of conventional CNNs on cell segmentation data. Its cell-based representation additionally opens a door for simplified model interpretation and synthetic image generation.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence

SummaryRecent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to damage-induced quiescent revival stem cells of the regenerating intestine. In addition, we identify negative regulators of cell cycle, downstream of p53, that we show are indicators of poor prognosis and may be targeted for qCSC abolition in both p53 wild-type and mutant tumors. These data support the temporal inhibition of downstream targets of p53 signaling, in combination with standard-of-care treatments, for the elimination of qCSCs and prevention of relapse in colon cancer.

Identification of an Immune Signature Predicting Prognosis Risk and Lymphocyte Infiltration in Colon Cancer.

Increasing studies have highlighted the effects of the tumor immune micro-environment (TIM) on colon cancer (CC) tumorigenesis, prognosis, and metastasis. However, there is no reliable molecular marker that can effectively estimate the immune infiltration and predict the CC relapse risk. Here, we leveraged the gene expression profile and clinical characteristics from 1430 samples, including four gene expression omnibus database (GEO) databases and the cancer genome atlas (TCGA) database, to construct an immune risk signature that could be used as a predictor of survival outcome and immune activity. A risk model consisting of 10 immune-related genes were screened out in the Lasso-Cox model and were then aggregated to generate the immune risk signature based on the regression coefficients. The signature demonstrated robust prognostic ability in discovery and validation datasets, and this association remained significant in the multivariate analysis after controlling for age, gender, clinical stage, or microsatellite instability status. Leukocyte subpopulation analysis indicated that the low-risk signature was enriched with cytotoxic cells (activated CD4/CD8+ T cell and NK cell) and depleted of myeloid-derived suppressor cells (MDSC) and regulatory T cells. Further analysis indicated patients with a low-risk signature harbored higher tumor mutation loads and lower mutational frequencies in significantly mutated genes of APC and FBXW7. Together, our constructed signature could predict prognosis and represent the TIM of CC, which promotes individualized treatment and provides a promising novel molecular marker for immunotherapy.

Transcriptome profiling reveals an integrated mRNA-lncRNA signature with predictive value of early relapse in colon cancer.

The purpose of our study was to develop a multigene signature based on transcriptome profiles of both mRNAs and lncRNAs to identify a group of patients who are at high risk of early relapse in stages II-III colon cancer. Firstly, propensity score matching was conducted between patients in early relapse group and long-term survival group from GSE39582 training series (N = 359) and patients were matched 1:1. Global transcriptome analysis was then performed between the paired groups to identify tumor specific mRNAs and lncRNAs. Finally, using LASSO Cox regression model, we built a multigene early relapse classifier incorporating 15 mRNAs and three lncRNAs. The prognostic and predictive accuracy of the signature was internally validated in 102 colon cancer patients and externally validated in other 241 patients. In the training set, patients with high risk score were more likely to suffer from relapse than those with low risk score (HR: 2.67, 95% CI: 2.07-3.46, P < 0.001). The results were validated in the internal validation set (HR: 2.23, 95% CI: 1.23-3.78, P = 0.003) and external validation (HR 1.88, 95% CI 1.42-2.48; P < 0.001) set. Time-dependent receiver operating curve at 1 year showed that the integrated mRNA-lncRNA signature [area under curve (AUC) = 0.742] had better prognostic accuracy than AJCC TNM stage (AUC = 0.615) in the entire 702 patients. In addition, survival decision curve analyses at 12 months revealed a good clinical usefulness of the integrated mRNA-lncRNA signature. In conclusion, we successfully developed an integrated mRNA-lncRNA signature that can accurately predict early relapse.

Abstract 902: Cancer-stem-cell (CSC) marker, DCLK1-S, enhances invasive potential of cancer cells by phosphorylating/activating NFATc2: role of COL3A1 and SPARC in mediating metastatic effects of DCLK1-S/NFATc2

Abstract DCLK1 expression is critically required for colon carcinogenesis in mice, and for maintaining tumorogenic potential of human colon cancer cells (hCCCs) (Singh et al, 2016). Down-regulation of DCLK1, combined with chemoprevention, eliminates CSCs, and avoids colon cancer relapse (Kantara et al, 2014). We recently discovered that long (L) and Short (S) isoforms of DCLK1 (DCLK1-L/DCLK1-S) are transcribed by two separate promoters (5’(α)/IntronV(β)) in the hDCLK1-gene (O’Connell et al, 2015). During adenoma-carcinoma sequence of colon-tumorigeneses, L-isoform becomes silenced by DNA-methylation while S-isoform gets upregulated by many fold (O’Connell et al, 2015). S-isoform specifically imparts invasive potential to cancer cells, unlike L-isoform (Singh et al, 2016); others have similarly reported metastatic potential of DCLK1 expressing cancer cells (Ito et al, 2016). Thus the goal of our studies was to evaluate molecular/genetic pathways mediating invasive effects of DCLK1-S in cancer cells. Isogenic clones of HCT116-cells, wild-type or down-regulated for DCLK1-S (HCT-C/HCT-D), were subjected to next generation sequencing and pathways analysis. SPARC and COL3A1 emerged as two candidate genes/proteins, which were decreased/increased by several fold in response to loss/overexpression of DCLK1-S, respectively. We present data confirming a critical role of COL3A1 and SPARC in mediating metastatic effects of DCLK1-S expression in hCCCs. We additionally discovered that DCLK1-S functions as a specific kinase for the transcriptional factor, NFATc2, and phosphorylates 53SPPS56 motif of NFATc2, resulting in activation of NFATc2 and increased expression of COL3A1. Conclusions. Our novel findings, suggest for the first time, that DCLK1-S expression by colonic tumors in humans, mediates invasive potential of colon cancer cells by phosphorylation/activation of NFATc2, resulting in up-regulation of COL3A1/SPARC; the latter proteins re-model extracellular matrix, assisting unhindered invasion of colon cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Malaney O. Connell, Shubhashish Sarkar, Heidi Spratt, Steven Widen, Thomas G. Wood, Pomila Singh. Cancer-stem-cell (CSC) marker, DCLK1-S, enhances invasive potential of cancer cells by phosphorylating/activating NFATc2: role of COL3A1 and SPARC in mediating metastatic effects of DCLK1-S/NFATc2 [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 902. doi:10.1158/1538-7445.AM2017-902

5-fluorouracil resistant colon cancer cells are addicted to OXPHOS to survive and enhance stem-like traits.

Despite marked tumor shrinkage after 5-FU treatment, the frequency of colon cancer relapse indicates that a fraction of tumor cells survives treatment causing tumor recurrence. The majority of cancer cells divert metabolites into anabolic pathways through Warburg behavior giving an advantage in terms of tumor growth. Here, we report that treatment of colon cancer cell with 5-FU selects for cells with mesenchymal stem-like properties that undergo a metabolic reprogramming resulting in addiction to OXPHOS to meet energy demands. 5-FU treatment-resistant cells show a de novo expression of pyruvate kinase M1 (PKM1) and repression of PKM2, correlating with repression of the pentose phosphate pathway, decrease in NADPH level and in antioxidant defenses, promoting PKM2 oxidation and acquisition of stem-like phenotype. Response to 5-FU in a xenotransplantation model of human colon cancer confirms activation of mitochondrial function. Combined treatment with 5-FU and a pharmacological inhibitor of OXPHOS abolished the spherogenic potential of colon cancer cells and diminished the expression of stem-like markers. These findings suggest that inhibition of OXPHOS in combination with 5-FU is a rational combination strategy to achieve durable treatment response in colon cancer.

Colon Cancer Cells Escape 5FU Chemotherapy-Induced Cell Death by Entering Stemness and Quiescence Associated with the c-Yes/YAP Axis

Metastasis and drug resistance are the major limitations in the survival and management of patients with cancer. This study aimed to identify the mechanisms underlying HT29 colon cancer cell chemoresistance acquired after sequential exposure to 5-fluorouracil (5FU), a classical anticancer drug for treatment of epithelial solid tumors. We examined its clinical relevance in a cohort of patients with colon cancer with liver metastases after 5FU-based neoadjuvant chemotherapy and surgery. We show that a clonal 5F31 cell population, resistant to 1 μmol/L 5FU, express a typical cancer stem cell-like phenotype and enter into a reversible quiescent G0 state upon reexposure to higher 5FU concentrations. These quiescent cells overexpressed the tyrosine kinase c-Yes that became activated and membrane-associated upon 5FU exposure. This enhanced signaling pathway induced the dissociation of the Yes/YAP (Yes-associated protein) molecular complex and depleted nuclear YAP levels. Consistently, YES1 silencing decreased nuclear YAP accumulation and induced cellular quiescence in 5F31 cells cultured in 5FU-free medium. Importantly, YES1 and YAP transcript levels were higher in liver metastases of patients with colon cancer after 5FU-based neoadjuvant chemotherapy. Moreover, the YES1 and YAP transcript levels positively correlated with colon cancer relapse and shorter patient survival (P < 0.05 and P < 0.025, respectively). We identified c-Yes and YAP as potential molecular targets to eradicate quiescent cancer cells and dormant micrometastases during 5FU chemotherapy and resistance and as predictive survival markers for colon cancer.

Doublecortin-like kinase 1 exhibits cancer stem cell-like characteristics in a human colon cancer cell line.

Colon cancer stem cells (CSCs) are implicated in colorectal cancer carcinogenesis, metastasis, and therapeutic resistance. The identification of these cells could help to develop novel therapeutic strategies. Doublecortin-like kinase 1 (DCLK1) has been viewed as a marker for gastrointestinal stem cells that fuel the self-renewal process, however others view them as a marker of Tuft cells or as an enteroendocrine subtype. The purpose of this study was to use a colon cancer cell line to identify and characterize the stem-like characteristics of the DCLK1+ cell population. To enrich stem-like cells, HCT116 cells (derived from colon adenocarcinomas) were cultured using serum-free media to form spheres under both normal oxygen and hypoxia condition. DCLK1 transcript expression in the adherent parental cells and spheroids was quantified using quantitative real time reverse transcription- polymerase chain reaction [(q)RT-PCR]. DCLK1 protein expression was determined using flow cytometry. Self-renewal capability from adherent parental cells and spheroids was determined using extreme limiting dilution analysis (ELDA). Under both normal oxygen and hypoxia condition, the adherent parental cells were composed of cells that express low levels of DCLK1. However, spheroids exhibited an increased frequency of cells expressing DCLK1 on both mRNA and protein levels. Cells derived from spheroids also possess stronger self-renewal capability. The higher fraction of DCLK1+ cells exhibited by spheroids and hypoxia reflects the stem-like characteristics of these cells. DCLK1 may represent an ideal marker to study and develop effective strategies to overcome chemo-resistance and relapse of colon cancer.

How long should adjuvant chemotherapy be used to prevent relapse in colon cancer? A personal case report

How long should adjuvant chemotherapy be used to prevent relapse in colon cancer? A personal case report

Effect of the ADAPT strategy on dormant CD133+ colon cancer stem cells (CSC) and molecular complete remission measured by peripheral blood mononuclear (PBMC) CD133 mRNA.

e14153 Background: The median survival for patients with unresectable metastatic colorectal cancer (CRC) is ~2 years with modern chemotherapy which yields only 5-10% complete responses (CR) including metastasectomy. Recurrences after CR are very common thanks to presence of dormant CSC that are best targeted by our proposed two-step ADAPT strategy: activate from dormancy and potentiate targeting. We examine this strategy in various CRC models and reviewed the impact on stemess including CD133 mRNA, a circulating CSC marker that predict colon cancer relapse. Methods: Different CRC models (in vitro and in vivo) were interrogated similar to clinical ADAPT treatment protocol using capecitabine (or 5FU) plus celecoxib. We also conducted IRB approved retrospective review of unresectable metastatic CRC patients treated ADAPT therapy and in those who also had PBMC CD133 mRNA measured. Results: Contrary to 5FU, which eliminates proliferating CRC cells via apoptosis but also stimulates stemness, celecoxib preferentially deplete CD133+ colon cells and exert potent stemness inhibition via rapid tumor necrosis by perturbing hypoxia and energy metabolism via CA-IX. Following response to first-line chemotherapy, ADAPT strategy plus radiation improved CR or near CR rate to 49/126 (40%) in unresectable CRC patients whose median survival had reached 92.7 months (95% CI, 53.5 months - not reached). Paradoxically, none surgical CR patients (n= 16) enjoyed 100% 5-year relapse free survival compared to 42% of surgical patients (p = 0.04). The PBMC CD133 mRNA in five long-term CR patients were 0.0024, 0.29, 0.5, 0.56, 2.96 respectively, all below previously reported cutoff value of 4.79 for recurrence and far below CD133 mRNA levels (28, 375, 3997, 15662, 83240) in none CR patients. Conclusions: ADAPT plus radiation preferentially targets colon CSC via hypoxia/CA-IX and improves clinical CR rate and molecular CR as measured by PBMC CD133 mRNA. We are actively interrogating the effects of ADAPT strategies in a phase II study funded by Gateway in CRC patients and in genetic CRC animal models.

A subpopulation of CD24+ cells in colon cancer cell lines possess stem cell characteristics

Cancer stem cells (CSCs) have been shown to contribute to the resistance and relapse in a range of cancer types such as breast cancer and glioma. However, colon cancer stem cells remain poorly characterized. Here we reported that CD24+ subpopulation in colon cancer cell lines HCT116 and SW480 exhibited cancer stem cell-like characteristics. Using flow cytometry candidate CSCs markers were selected after initial screening of known CSCs markers from other types of cancer on colon cancer cell lines HCT116, SW480 and HT29. CD24 was expressed in the minority of bulk cell population of HCT116 and SW480 cell lines. Moreover, functional tests demonstrated that CD24+ cells exhibited enhanced chemotherapy-resistance, self-renewal and tumorigenic capacity both in vitro and in vivo, compared to CD24- subpopulations. These results suggest that CD24+ subpopulation in colon cancer cell lines HCT116 and SW480 exhibits CSCs like characteristics, and represents a nice model to study and develop effective strategies to overcome chemo-resistance and relapse of colon cancer.

APPLICATION of EUTOS SCORE IN CHRONIC Myeloid LEUKEMIA AFFECTING VERY Elderly (>75 years) PATIENTS

Abstract 1686The Eutos score has recently been published as a new prognostic score in CML because of its prognostic ability and its simplicity in the imatinib era. It maintains the percent of basophils and the spleen size and it eliminates three other laboratory values and the variable of the age, that is usually important in the prognosis of almost all the diseases. This score was able to predict the probability of achieving a complete cytogenetic response (CCgR) within 18 months, which is the most solid and confirmed surrogate marker of survival in CML treated with imatinib. Thus we reviewed the cases of CML affecting individuals > 75 years and we apply the Eutos score and compare results with classic Sokal risk. We collected 221 patients from 29 of the major Italian hematological centers for diagnosis and treatment of CML. Because of the lack of data for the calculation of the scores in 84 patients, 137 patients were evaluable. The M: F ratio was 70: 67, the median age was of 78 years (range 75–89). Patients aged >75 years as expected showed frequently comorbidities (cardiovascular, respiratory, renal, oncologic ones). The distribution according to the Sokal score was: 3 patients in the low risk group, 95 patients in the intermediate risk group and 39 patients in the high risk group. As expected, there were only few patients in the low Sokal risk group aged >75 years in demonstration of the importance of the age in this score. Calculating Eutos score we have identified only 6 patients with a high score, whereas the remaining 131 patients were in the low risk group. The group with high Eutos risk score included 5 males and 1 female; their median age was 77 years (range 75–78), at the onset their median white blood cell count was 33.840/mmc (range 29.500–300.000), platelet count was 1.443.000/mmc (range 453.000–4.849.000), their median level of hemoglobin was 8.8 g/dl (range 7.7–13.9). All of them showed percent of basophils>12%, whereas at physical examination splenomegaly was usually moderate with no hepatomegaly. Due to the small sample of patients in the high Eutos score statistical analysis was not feasible but we tried to correlate baseline informations to the outcome. Not all the high Eutos risk patients were in the high Sokal risk group (3 patients were in the high Sokal risk group and 3 were in the intermediate one) none was in the low Sokal risk group. All but one patients in the high risk Eutos score were treated with imatinib in early chronic phase of CML. Then we considered the complete cytogenetic response (CCyR) at 18 months, performed through conventional cytogenetic analysis on bone marrow cells by G-banding technique. This has been chosen for the validation of the Eutos score as a solid early surrogate marker of outcome. The overall rate of CCyR was 100% in the low Sokal risk group, 63% in the intermediate Sokal risk group and 69% in the high Sokal risk group; the same type of response was 67% in the low Eutos risk group and 50% in the high Eutos risk group (Table 1). In the high Eutos risk group, 2 patients achieved CCyR, 1 patient achieved MMR, 2 patients did not achieve it and 1 patient was not evaluable. Two patients died because of comorbidities (relapse of colon cancer and cardio-respiratory insufficiency) at 19 and 39 months of follow up. Resistance to imatinib was registered in 17 out of 131 patients (13%) in the low Eutos risk and in 3 out of 6 patients (50%) in the high Eutos risk. In our series of CML patients >75 years, high Eutos score was infrequent (4.38%). Moving from Sokal to Eutos score, patients were largely reallocated as low risk Eutos score. It is not clear whether the low frequency of high risk Eutos might be attributed to a early diagnosis of CML in this subset of patients with age-related comorbidities requiring frequent laboratory exams. In view of the ability to predict CCyR at 18 months, Eutos score validation in the setting of elderly patients with CML could be relevant to clinical practice.Table 1Distribution of patients according Sokal and Eutos scoreN° patients% CCyRSOKALLow risk3100%Intermediate risk9563%High risk3969%EUTOSLow risk13167%High risk650% Disclosures:Russo Rossi:Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

Anti-proliferative effect of levamisole on human myeloma cell lines in vitro

Levamisole has been employed as an immunomodulatory agent in conjunction with 5-fluorouracil in the treatment of colon cancer relapse. At high doses, levamisole has been shown to have both anti-cancer and immunosuppressive activities. In vitro, levamisole has been shown to potentiate the anti-proliferative effect of 5-fluorouracil in several types of tumor cell lines; however, its mechanism of cytotoxic action and its molecular targets in cells remains to be elucidated. Here, the effect of levamisole on the proliferative response of the human multiple myeloma cell lines RPMI 8226 and U266B1 was studied in vitro. Treatment of both lines with varying concentrations of levamisole for 48 and 72 h in culture resulted in a significant inhibition of proliferation (unstimulated) in a dose-dependent manner, as assessed by an 3-[(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide dye assay. Furthermore, measurements of cell viability (using a trypan blue dye exclusion assay) clearly showed that the levamisole was cytotoxic. The preliminary evaluation of the mechanism of this cytotoxic effect revealed that this drug induced apoptosis in the myeloma cells, as evidenced by increases in the levels of DNA fragmentation, release of cytochrome c into the cytoplasm, and the activation of caspase-3 activity in the cells. The results of these studies strongly suggest that levamisole could be a potent anti-myeloma agent and might be considered in the treatment of multiple myeloma in the future.

Multiple Tumor Marker Elevation in Androgen Ablation-Refractory Prostate Cancer with Long-Term Response to Metronomic Chemotherapy: A Case Report

Outcomes in hormone-refractory prostate cancer are very poor. The time from progression to death is only 12-19 months. We present the case of a 69-year-old man with hormone-refractory prostate cancer and bone metastases treated with metronomic chemotherapy (cyclophosphamide based). He had had a colon adenocarcinoma ten years before. The atypical features of this case were an unusually long-lasting response to metronomic chemotherapy and an increase in serum levels of some non-prostate-specific tumor markers (CEA and CA 19-9) that was not related to a relapse of colon cancer. We hypothesize a potential role of hypoxia inducing CA 19-9 and CEA expression in tumor cells, which may predict the development of progressive resistance to antiangiogenic therapies.

Prediction potential of IR-micro spectroscopy for colon cancer relapse

This study aimed to determine the potential of IR-spectroscopy to diagnose abnormality in histologically normal resection margins for predicting relapse in colon cancer patients. The present study evaluates potential abnormal crypt proliferation in histologically normal resection margins. Resection margins of 10 colon cancer patients (adenocarcinoma) (27 biopsies in all), found completely normal by standard histology were re-evaluated. Fourier transform infrared microscopy (FTIR-MSP) was performed on the longitudinal sections of the crypt, and spectral data collected from the base, middle and top portion of crypts. Absorbance in the region 900-1185 cm(-1) arising from carbohydrates and nucleic acids was found to be the most effective variate for such evaluation. In total 225 crypts were classified after assessing the levels of abnormality observed by the above technique. The abnormal biopsies detected using the above optical method was correlated with a relapse in the patient's history. Patients who had a relapse had at least one abnormal biopsy (crypt) based on the present methodology. Among the patients, the only case without a relapse was also the case where no abnormal crypts were found in any biopsies from the resection margins. The agreement between the biopsy status, as determined by the optical methodology, and the relapse of colonic malignancy based on the patients' medical files, establishes the translational nature of FTIR-MSP for medical purposes and hints at future clinical evaluation of the biopsies using this technique to determine more precisely the zone of excision during anastomosis.

Predictive factors of early relapse in UICC stage I–III colorectal cancer patients after curative resection

To predict the clinicopathologic factors for early relapse of UICC stage I-III colorectal cancer (CRC) patients undergoing curative resection and thus to identify a subgroup of patients who are at high risk for postoperative early relapse. Between January 2001 and June 2007, a total of 778 UICC stage I-III CRC patients who underwent a radical resection and regular follow-up were retrospectively analyzed. Of these 778 CRC patients, 521 colon cancer and 257 rectal cancer cases were analyzed, respectively, to determine the predictors of early relapse postoperatively. These 778 patients were followed-up intensively, and their outcomes were investigated retrospectively. Out of 521 colon cancer patients, postoperative relapse after primary resection was found in 142 (27.3%) patients, and 77 (54.2%) of 142 recurrent colon cancer patients were classified as postoperative early relapse. Meanwhile, among 257 rectal cancer patients, postoperative relapse was found in 68 (26.5%) patients and 44 (64.7%) of 68 recurrent rectal cancer patients were identified as postoperative early relapse. Forty-nine (63.6%) of 77 early relapsed colon cancer patients were stage III, and likewise, 26 (59.1%) of 44 early relapsed rectal cancer patients were stage III. Univariately, postoperative early relapse of colon cancer patients was significantly correlated with the presence of vascular invasion (P < 0.001), perineural invasion (P < 0.001), high postoperative carcinoembryonic antigen (CEA) level (P = 0.001), and type of surgery (P = 0.016). Using a Cox proportional hazards analysis, the presence of vascular invasion (P = 0.033), perineural invasion (P = 0.005), and high postoperative CEA levels (P = 0.001) were demonstrated to be independent predictors of postoperative early relapse of colon cancer patients, while in rectal cancer patients, both vascular invasion (P = 0.039) and perineural invasion (P = 0.008) were statistically significant predictors of early relapse by univariate analysis. Using a Cox proportional hazards analysis, only perineural invasion (P = 0.043) was an independent factor. Early relapse cases had significant lower overall survival rates than non-early relapse cases either in colon cancer (P < 0.001) or in rectal cancer (P = 0.0091) patients. This study suggests that vascular invasion, perineural invasion, and postoperative CEA level may be significant factors for postoperative early relapse of colon cancer; while only perineural invasion is considered to be a significant predictor in rectal cancer patients. Identification of these high-risk UICC stage I-III CRC patients of early relapse is important, and thus could help to define patients with this tumor entity for an enhanced follow-up and therapeutic program.