Progressive Acute Lower Extremity Ischemia Resulting From Cardiac Myxoma Embolization: A Case Report.

Cardiac myxomas are the most common primary benign tumors of the heart. The occlusion of peripheral arteries and complete obstruction of the abdominal aorta by a tumor embolus presents with distinct clinical manifestations. Herein, we present the case of a 38-year-old male with acute paresthesia, muscle weakness, erythematous, and violaceous changes in skin color localized to the dorsum of the left forefoot initially treated as cutaneous vasculitis. Further studies revealed the total occlusion of the terminal abdominal aorta by a saddle embolus from a cardiac myxoma. A multidisciplinary team consisting of cardiothoracic and vascular surgeons were involved in treating the patient, which resulted in full resolution of the case. This paper details the progression of acute bilateral limb ischemia to chronic limb threatening ischemia resulting from the total occlusion of the terminal abdominal aorta by a saddle embolus.

Circulating Polyploid Giant Cancer Cells, a Potential Prognostic Marker in Patients with Carcinoma.

Polyploid Giant Cancer Cells (PGCCs) have been recognized as tumor cells that are resistant to anticancer therapies. However, it remains unclear whether their presence in the bloodstream can be consistently detected and utilized as a clinical marker to guide therapeutic anticancer regimens. To address these questions, we conducted a retrospective study involving 228 patients diagnosed with six different types of carcinomas (colon, gastric, NSCLC, breast, anal canal, kidney), with the majority of them (70%) being non-metastatic. Employing a highly sensitive liquid biopsy approach, ISET®, and cytopathological readout, we isolated and detected circulating PGCCs in the patients' blood samples. PGCCs were identified in 46 (20.18%) out of 228 patients, including in 14.47% of 152 non-metastatic and 29.85% of 67 metastatic cases. Patients were subsequently monitored for a mean follow up period of 44.74 months (95%CI: 33.39-55.79 months). Remarkably, the presence of circulating PGCCs emerged as a statistically significant indicator of poor overall survival. Our findings suggest that circulating PGCCs hold promise as a reliable prognostic indicator. They underscore the importance of further extensive investigations into the role of circulating PGCCs as a prognostic marker and the development of anti-PGCC therapeutic strategies to improve cancer management and patient survival.

Pilot study to evaluate isolation by size of circulating tumour cells in canine oral melanoma.

Liquid biopsy for circulating tumour cell (CTC) detection is generally unexplored in veterinary medicine. Dogs with highly aggressive and heterogeneous tumours, such as oral malignant melanoma (OMM), could benefit from studies involving size-based isolation methods for CTCs, as they do not depend on specific antibodies. This pilot study aimed to detect CTCs from canine OMM using Isolation by Size of Epithelial Tumor Cells (ISET), a microfiltration methodology, followed by immunocytochemistry (ICC) with Melan-A, PNL2, and S100 antibodies. Ten canine patients diagnosed by histopathology and confirmed as OMM by immunohistochemistry were enrolled, their prognostic data was assessed, and blood samples were collected for CTC analysis. Results have shown the detection of intact cells in 9/10 patients. ICC has shown 3/9 Melan-A-positive, 3/9 PNL2-positive, and 8/9 S100-positive patients, confirming the importance of opting for a multimarker assay. A significant number of negative-stained CTCs were found, suggesting their high heterogeneity in circulation. Microemboli stained with either PNL2 or S100 were found in a patient with a high isolated cell count and advanced clinical stage. Preliminary statistical analysis shows a significant difference in CTC count between patients with and without lymph node metastasis (p < .05), which may correlate with tumour metastatic potential. However, we recommend further studies with more extensive sampling to confirm this result. This pilot study is the first report of intact CTC detection in canine OMM and the first application of ISET in veterinary medicine, opening new possibilities for liquid biopsy studies in canine OMM and other tumours.

TUMOR MARKERS EXPRESSION LEVELS IN GASTRIC CANCER PATIENT'S PERIPHERAL BLOOD BY RT-PCR ASSESSMENT.

BACKGROUND: Hematological recurrence is the second most frequent cause of failure in the treatment of gastric cancer. The detection of circulating tumor markers in peripheral blood by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method may be a useful tool to predict recurrence and determine the patient's prognosis. However, no consensus has been reached regarding the association between the tumor markers level in peripheral blood and its impact on patient survival. AIMS: To evaluate the expression of the circulating tumor markers CK20 and MUC1 in peripheral blood samples from patients with gastric cancer by qRT-PCR, and to verify the association of their expression levels with clinicopathological characteristics and survival. METHODS: A total of 31 patients with gastric adenocarcinoma were prospectively included in this study. CK20 and MUC1 expression levels were analyzed from peripheral blood by the qRT-PCR technique. RESULTS: There was no statistically significant (p>0.05) association between CK20 expression levels and clinical, pathological, and surgical features. Higher MUC1 expression levels were associated with female patients (p=0.01). There was a correlation between both gene levels (R=0.81, p<0.001), and CK20 level and tumor size (R=0.39, p=0.034). CONCLUSIONS: CK20 and MUC1 expression levels could be assessed by qRT-PCR from total peripheral blood samples of patients with gastric cancer. CK20 levels were correlated to MUC1 levels as well as to tumor size. There was no difference in disease-free survival and overall survival regarding both genetic markers expression in this series.

Molecular biomarkers in prostate cancer tumorigenesis and clinical relevance.

Prostate cancer (PCa) is the second most frequent type of cancer in men and assessing circulating tumor cells (CTCs) by liquid biopsy is a promising tool to help in cancer early detection, staging, risk of recurrence evaluation, treatment prediction and monitoring. Blood-based liquid biopsy approaches enable the enrichment, detection and characterization of CTCs by biomarker analysis. Hence, comprehending the molecular markers, their role on each stage of cancer development and progression is essential to provide information that can help in future implementation of these biomarkers in clinical assistance. In this review, we studied the molecular markers most associated with PCa CTCs to better understand their function on tumorigenesis and metastatic cascade, the methodologies utilized to analyze these biomarkers and their clinical significance, in order to summarize the available information to guide researchers in their investigations, new hypothesis formulation and target choice for the development of new diagnostic and treatment tools.

The Possible Role of Matrix Metalloprotienase-2 in the Relapse in Patients with Stage II Colon Cancer Treated by Curative Surgery.

AIMS: To determine the association of micro-metastatic matrix metalloproteinase-2 (MMP-2) expression,  the absolute lymphocyte count (ALC)) and outcome in stage II colon cancer. MATERIALS AND METHODS: A single centre, prospective observational study, one month post-surgery blood for ALC, circulating tumour cell (CTC) detection and a bone marrow biopsy for micro-metastasis detection were obtained.  CTCs were detected using differential gel centrifugation and immunocytochemistry with anti-CEA and anti-MMP-2, the bone marrow biopsy for the detection of micro-metastasis was processed as for CTCs . At each follow-up  ALC and CTC counts were determined. Bone marrow sampling was repeated if the ALC decreased by >10%, at relapse or at the end of the study period. Three MRD subgroups were defined, Group I MRD negative, Group II only positive for micro-metastasis and   Group III in which CTCs were detected. RESULTS: One hundred and eighty one patients  participated; 105 (58%) patients formed Group 1, 36 (20%) formed Group II  and 40 (22%)  formed Group III for a median follow-up of 4 years .  Of Group I 3/105 (3%), Group II 16/36 (44%) and Group III 34/40 (84%) patients relapsed. The ALC was significantly higher in Groups I and II, the expression of MMP-2 and MMP-2 score in Group II was significantly lower than in Group III patients. A low ALC was associated with a higher expression of MMP-2 in the micro-metastasis and presence of CTCs. CONCLUSIONS: Patients with stable ALCs did not relapse; decreasing ALCs were associated with increasing MMP-2 scores, the appearance of CTCs and relapse.

CD47 Expression in Circulating Tumor Cells and Circulating Tumor Microemboli from Non-Small Cell Lung Cancer Patients Is a Poor Prognosis Factor.

Circulating tumor cells (CTCs) and/or circulating tumor microemboli (CTM) from non-small cell lung cancer (NSCLC) patients may be a non-invasive tool for prognosis, acting as liquid biopsy. CTCs interact with platelets through the transforming growth factor-ß/transforming growth factor-ß receptor type 1 (TGF-ß/TGFßRI) forming clusters. CTCs also may express the Cluster of Differentiation 47 (CD47) protein, responsible for the inhibition of phagocytosis, the "don't eat me" signal to macrophages. OBJECTIVES: To isolate, quantify and analyze CTCs/CTMs from metastatic NSCLC patients, identify TGFßRI/CD47 expression in CTCs/CTMs, and correlate with progression-free survival (PFS). METHODS: Blood (10 mL) was collected at two time-points: T1 (before the beginning of any line of treatment; T2 (60 days after initial collection). CTCs were isolated using ISET®. Immunocytochemistry was conducted to evaluate TGFßRI/CD47 expression. RESULTS: 45 patients were evaluated. CTCs were observed in 82.2% of patients at T1 (median: 1 CTC/mL; range: 0.33-11.33 CTCs/mL) and 94.5% at T2 (median: 1.33 CTC/mL; 0.33-9.67). CTMs were observed in 24.5% of patients and significantly associated with poor PFS (10 months vs. 17 months for those without clusters; p = 0.05) and disease progression (p = 0.017). CTMs CD47+ resulted in poor PFS (p = 0.041). TGFßRI expression in CTCs/CTMs was not associated with PFS. CONCLUSION: In this study, we observed that CTC/CTM from NSCLC patients express the immune evasion markers TGFßRI/CD47. The presence of CTMs CD47+ is associated with poor PFS. This was the first study to investigate CD47 expression in CTCs/CTM of patients with NSCLC and its association with poor PFS.

Prognostic significance of circulating tumor cell measurement in the peripheral blood of patients with nasopharyngeal carcinoma.

OBJECTIVE: Nasopharyngeal Carcinoma (NPC) is lethal cancer. Typically, relapse and metastasis are the outcomes of most patients. Against this backdrop, this study aimed to investigate the correlation between Circulating Tumor Cell (CTC) profiles and clinicopathological features in patients with NPC. PATIENTS AND METHODS: A total of 119 blood samples from 79 patients were collected from patients with NPC during treatment. CanPatrolTM CTC enrichment and RNA In Situ Hybridization (RNA-ISH) were used to characterize CTCs, including epithelial, Mesenchymal (MCTCs), and epithelial/mesenchymal mixed types according to their surface markers. RESULTS: The number of CTCs and MCTCs in the pre-treatment group was significantly higher than that in the post-treatment group (p < 0.05). The total number of CTCs and MCTCs cell numbers was significant correlation with Tumor-Node-Metastasis (TNM) staging (p < 0.05), Progression-Free Survival (PFS), and Overall Survival (OS). The PFS of patients with > 7 CTCs or > 5 MCTCs per 5 mL blood was significantly shorter PFS than those patients with ≤ 7 CTCs or ≤ 5 MCTCs (p < 0.05). Patients treated with targeted therapy combined with chemoradiotherapy had poorer PFS and OS rates than those treated with chemoradiotherapy (p < 0.05). The Kaplan-Meier survival analysis also demonstrated that patients with changes in CTC > 4 were strongly associated with PFS and OS rates (p < 0.05). CONCLUSION: CTC and MCTC number detection in patients with NPC is a useful biomarker for predicting patient progress. Patients with more than 7 CTCs or 5 MCTCs in 5 mL of blood had shorter PFS and OS rates. CTC and MCTC count changes were also significantly associated with the patient's therapy.

HPV virus and biomarkers of resistance to chemoradiation in circulating tumor cells from patients with squamous cell carcinoma of the anus.

Localized anal cancer is mostly represented by squamous cell carcinoma of the anus (SCCA) and is cured in ≥80 % of cases by chemoradiation (CRT). Development of techniques for detection/evaluating circulating tumor cells (CTCs) for diagnosis/ prognosis/response to therapy can change the manner we treat/follow SCCA patients. OBJECTIVE: to detect CTCs from patients with SCCA and evaluate the presence of HPV virus, p16 expression and markers related to resistance to CRT (RAD23B/ ERCC1/ TYMS) in CTCs at baseline and after CRT. METHODS: CTCs were isolated/quantified by ISET®, protein expressions were analyzed by immunocytochemistry and HPV DNA was detected by chromogenic in situ hybridization. RESULTS: We enrolled 15 patients: median age was 61 (43-73) years, the majority was women (10/15). CTCs were detected in all patients at baseline (median= 0.4 (0.4-3.33) CTCs/mL) and in 8/9 patients, after CRT (median= 2.33 (0-7.0) CTCs/mL). DNA from HPV was found in CTCs in 14/15 patients (93.33 %) at baseline and in 7/9 (77.7 %) after treatment. At a median follow-up of 22.20 (1.45-38.55) months, three patients expressed ERCC1 in CTCs after treatment, with one of them having disease recurrence. CONCLUSION: We showed that detection of HPV in CTCs from patients with non-metastatic SCCA is feasible and appears to be a sensitive diagnostic method. These results may be clinically useful for better monitoring these patients. However, future larger cohorts may demonstrate whether there is any correlation between the presence of HPV and the expression of screening markers for CRT in SCCA.

Fusion Cell Markers in Circulating Tumor Cells from Patients with High-Grade Ovarian Serous Carcinoma.

Cancer is primarily a disease in which late diagnosis is linked to poor prognosis, and unfortunately, detection and management are still challenging. Circulating tumor cells (CTCs) are a potential resource to address this disease. Cell fusion, an event discovered recently in CTCs expressing carcinoma and leukocyte markers, occurs when ≥2 cells become a single entity (hybrid cell) after the merging of their plasma membranes. Cell fusion is still poorly understood despite continuous evaluations in in vitro/in vivo studies. Blood samples from 14 patients with high-grade serous ovarian cancer (A.C. Camargo Cancer Center, São Paulo, Brazil) were collected with the aim to analyze the CTCs/hybrid cells and their correlation to clinical outcome. The EDTA collected blood (6 mL) from patients was used to isolate/identify CTCs/hybrid cells by ISET. We used markers with possible correlation with the phenomenon of cell fusion, such as MC1-R, EpCAM and CD45, as well as CEN8 expression by CISH analysis. Samples were collected at three timepoints: baseline, after one month (first follow-up) and after three months (second follow-up) of treatment with olaparib (total sample = 38). Fourteen patients were included and in baseline and first follow-up all patients showed at least one CTC. We found expression of MC1-R, EpCAM and CD45 in cells (hybrid) in at least one of the collection moments. Membrane staining with CD45 was found in CTCs from the other cohort, from the other center, evaluated by the CellSearch® system. The presence of circulating tumor microemboli (CTM) in the first follow-up was associated with a poor recurrence-free survival (RFS) (5.2 vs. 12.2 months; p = 0.005). The MC1-R expression in CTM in the first and second follow-ups was associated with a shorter RFS (p = 0.005). CEN8 expression in CTCs was also related to shorter RFS (p = 0.035). Our study identified a high prevalence of CTCs in ovarian cancer patients, as well as hybrid cells. Both cell subtypes demonstrate utility in prognosis and in the assessment of response to treatment. In addition, the expression of MC1-R and EpCAM in hybrid cells brings new perspectives as a possible marker for this phenomenon in ovarian cancer.

A Novel Detection Method of Breast Cancer through a Simple Panel of Biomarkers.

Circulating tumor cells (CTCs) have been identified as responsible for the spread of tumors to other organs of the body. In this sense, the development of sensitive and specific assays for their detection is important to reduce the number of deaths due to metastases. Here, we assessed whether the detection of CTCs in peripheral blood can serve in the construction of a panel of diagnosis and monitoring treatments of breast cancer (BC), focusing on the expression of markers of epithelial-mesenchymal transition. Through analyzing the blood from women without breast alterations (control), women with benign alterations, women with breast cancer without chemotherapy, and women with breast cancer with chemotherapy, we identified the best markers by transcriptional levels and determined three profiles of CTCs (mesenchymal, intermediate, and epithelial) by flow cytometry which, combined, can be used for diagnosis and therapy monitoring with sensitivity and specificity between 80% and 100%. Therefore, we have developed a method for detecting breast cancer based on the analysis of CTC profiles by epithelial-mesenchymal transition markers which, combined, can be used for the diagnosis and monitoring of therapy.

Liquid Biopsy as a Tool for the Diagnosis, Treatment, and Monitoring of Breast Cancer.

Breast cancer (BC) is a highly heterogeneous disease. The treatment of BC is complicated owing to intratumoral complexity. Tissue biopsy and immunohistochemistry are the current gold standard techniques to guide breast cancer therapy; however, these techniques do not assess tumoral molecular heterogeneity. Personalized medicine aims to overcome these biological and clinical complexities. Advances in techniques and computational analyses have enabled increasingly sensitive, specific, and accurate application of liquid biopsy. Such progress has ushered in a new era in precision medicine, where the objective is personalized treatment of breast cancer, early screening, accurate diagnosis and prognosis, relapse detection, longitudinal monitoring, and drug selection. Liquid biopsy can be defined as the sampling of components of tumor cells that are released from a tumor and/or metastatic deposits into the blood, urine, feces, saliva, and other biological substances. Such components include circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) or circulating tumor RNA (ctRNA), platelets, and exosomes. This review aims to highlight the role of liquid biopsy in breast cancer and precision medicine.

Clinical significance of circulating tumor cells in predicating the outcomes of patients with colorectal cancer.

BACKGROUND: Relapse and metastasis of patients with Colorectal Cancer (CRC) is the major obstacle to the long-term life of patients. Its mechanisms remain defined. METHODS: A total of 48 CRC patients were enrolled and 68 samples were obtained from the peripheral blood of patients before or after treatments in this study. Twenty non-cancer patients were also detected as a negative control. Circulating Tumor Cells (CTCs), including Epithelial CTCs (eCTCs), Mesenchymal (MCTCs), and epithelial/mesenchymal mixed phenotypes (mixed CTCs), were identified by CanPatrolTM CTC enrichment and RNA in situ hybridization. The relationship between CTCs number and Progression-Free Survival (PFS) or Overall Survival (OS) was evaluated. RESULTS: Thirty-four of 48 patients (70.8%) were found to have positive CTCs. Total CTCs and MCTCs in the post-treatment had a significant correlation PFS and OS. When total CTCs or MCTCs in 5 mL blood of patients were more than 6 CTCs or 5 MCTCs, PFS of the patients was significantly shorter (p < 0.05) than that in patients with less than 6 CTCs or 5 MCTCs. The patients with > 5 CTCs count changes were found to exhibit poor PFS and OS rates (p < 0.05). CONCLUSION: Total CTCs and MCTCs number detection in patients with colorectal cancer was very useful biomarker for predicting the prognosis of patients. Higher CTCs or MCTCs had poorer PFS and OS rates.

Multianalyte Prognostic Signature Including Circulating Tumor DNA and Circulating Tumor Cells in Patients With Advanced Pancreatic Adenocarcinoma.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is associated with a poor prognosis. Multianalyte signatures, including liquid biopsy and traditional clinical variables, have shown promise for improving prognostication in other solid tumors but have not yet been rigorously assessed for PDAC. MATERIALS AND METHODS: We performed a prospective cohort study of patients with newly diagnosed locally advanced pancreatic cancer (LAPC) or metastatic PDAC (mPDAC) who were planned to undergo systemic therapy. We collected peripheral blood before systemic therapy and assessed circulating tumor cells (CTCs), cell-free DNA concentration (cfDNA), and circulating tumor KRAS (ctKRAS)-variant allele fraction (VAF). Association of variables with overall survival (OS) was assessed in univariate and multivariate survival analysis, and comparisons were made between models containing liquid biopsy variables combined with traditional clinical prognostic variables versus models containing traditional clinical prognostic variables alone. RESULTS: One hundred four patients, 40 with LAPC and 64 with mPDAC, were enrolled. CTCs, cfDNA concentration, and ctKRAS VAF were all significantly higher in patients with mPDAC than patients with LAPC. ctKRAS VAF (cube root; 0.05 unit increments; hazard ratio, 1.11; 95% CI, 1.03 to 1.21; P = .01), and CTCs ≥ 1/mL (hazard ratio, 2.22; 95% CI, 1.34 to 3.69; P = .002) were significantly associated with worse OS in multivariate analysis while cfDNA concentration was not. A model selected by backward selection containing traditional clinical variables plus liquid biopsy variables had better discrimination of OS compared with a model containing traditional clinical variables alone (optimism-corrected Harrell's C-statistic 0.725 v 0.681). CONCLUSION: A multianalyte prognostic signature containing CTCs, ctKRAS, and cfDNA concentration outperformed a model containing traditional clinical variables alone suggesting that CTCs, ctKRAS, and cfDNA provide prognostic information complementary to traditional clinical variables in advanced PDAC.

Increasing Immune Dysfunction is Associated with Increasing Matrix-Metalloproteinase-2 Expression and Predicts Biochemical Failure in Men with Bone Marrow Micro-Metastasis Positive Localized Prostate Cancer.

INTRODUCTION: To determine if there was an association of the ALC (absolute lymphocyte count) and LCP (lymphocytopenia) with the expression of MMP-2 in bone marrow micro-metastasis, the changes occurring during follow-up and association with biochemical failure. METHODS AND PATIENTS: One month after surgery blood and bone marrow samples were taken to determine the presence of micro-metastasis, the presence of circulating prostate cells (CPCs) and ALC. CPCs and micro-metastasis were detected using immunocytochemistry and MMP-2 expression determined in micro-metastasis. Only men positive for micro-metastasis participated in the study. At end follow blood was taken for serum PSA, ALC and CPCs, if the ALC decreased by more than 10% bone marrow sampling was repeated and MMP-2 expression determined, similarly for men with BF. Men who had stable ALCs had an end of study evaluation of the bone marrow. RESULTS: 402 men underwent radical prostatectomy, one month post surgery 79 men were positive for only bone marrow micro-metastasis and formed the study group; of whom 36/79 (45%) underwent BF. Clinical pathological findings were not significantly different between men with or without BF. In men with BF the ALC was significantly lower one-month post surgery. The 5 and 10 year Kaplan-Meier survival was 100% at 5-years and 65% at 10-years for the whole cohort. Men without BF had stable ALCs. A decrease of >10% in the ALC was associated with increasing MMP-2 expression in the micro-metastasis and surrounding stromal tissue, the appearance of CPCs 6-12 months later and BF. CONCLUSIONS: the immune host-tumour cell interaction in the microenvironment is dynamic and changes with time. A decreasing ALC may be a valuable marker in identifying men with high risk of BF and changes in immune mediated dormancy before the PSA rises.

Cell-free DNA analysis in current cancer clinical trials: a review.

Cell-free DNA (cfDNA) analysis represents a promising method for the diagnosis, treatment selection and clinical follow-up of cancer patients. Although its general methodological feasibility and usefulness has been demonstrated, several issues related to standardisation and technical validation must be addressed for its routine clinical application in cancer. In this regard, most cfDNA clinical applications are still limited to clinical trials, proving its value in several settings. In this paper, we review the current clinical trials involving cfDNA/ctDNA analysis and highlight those where it has been useful for patient stratification, treatment follow-up or development of novel approaches for early diagnosis. Our query included clinical trials, including the terms 'cfDNA', 'ctDNA', 'liquid biopsy' AND 'cancer OR neoplasm' in the FDA and EMA public databases. We identified 1370 clinical trials (FDA = 1129, EMA = 241) involving liquid-biopsy analysis in cancer. These clinical trials show promising results for the early detection of cancer and confirm cfDNA as a tool for real-time monitoring of acquired therapy resistance, accurate disease-progression surveillance and improvement of treatment, situations that result in a better quality of life and extended overall survival for cancer patients.

Immune Dysfunction as Measured by the Systemic Immune-Inflammation Index is Associated with the Sub-Type of Minimal Residual Disease and Outcome in Stage II Colon Cancer Treated with Surgery alone.

OBJECTIVE: Within 5 years after curative surgery for stage II colon cancer 25% of patients will relapse due to minimal residual disease (MRD). MRD is the net result of the biological properties of subpopulations of primary tumour cells which enable them to disseminate, implant in distant tissues and survive and the immune system's ability to eliminate them. We hypothesize that markers of immune dysfunction such as the systemic inflammation index (SII) are associated with the sub-type of MRD defined by bone marrow micro-metastasis (mM) and circulating tumour cells (CTCs). A higher immune dysfunction being associated with a more aggressive MRD and worse prognosis. METHODS AND PATIENTS: Blood and bone marrow samples were taken to detect CTCs and mM using immunocytochemistry with anti-CEA one month after surgery. The SII, absolute neutrophil, platelet and lymphocyte counts (ANC, APC, ALC) were determined immediately pre-surgery and one month post-surgery. These were compared with the sub-types of MRD; Group I MRD (-); Group II mM positive and Group III CTC positive; cut-off values of SII of >700 and >900 were used. Follow-up was for up to 5 years or relapse and survival curves using Kaplan-Meier (KM) were calculated. RESULTS: One hundred and eighty one patients (99 women) participated, mean age 68 years, median follow up 4.04 years; I: = 105 patients, II: N= 36 patients, III: N=40 patients. The SII significantly decreased post-surgery only in Group I patients. The frequency of SII >700 and >900 was significantly higher in Group III, between Groups I and II there was no significant difference.  The SII was significantly associated with the number of CTCs detected. The 5-year KM was 98% Group I, 68% Group II and 7% Group III. CONCLUSIONS: The results of the study suggest that the severity of immune dysfunction as determined by the SII is associated with differing sub-types of MRD and a worse prognosis; increasing immune dysfunction is associated with a more aggressive CTC positive MRD sub-type; a more severe immune dysfunction is associated with a higher number of CTCs detected.
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Molecular and Dynamic Evaluation of Proteins Related to Resistance to Neoadjuvant Treatment with Chemoradiotherapy in Circulating Tumor Cells of Patients with Locally Advanced Rectal Cancer.

The heterogeneity of response to neoadjuvant chemoradiotherapy (NCRT) is still a challenge in locally advanced rectal cancer (LARC). The evaluation of thymidylate synthase (TYMS) and RAD23 homolog B (RAD23B) expression in circulating tumor cells (CTCs) provides complementary clinical information. CTCs were prospectively evaluated in 166 blood samples (63 patients) with LARC undergoing NCRT. The primary objective was to verify if the absence of RAD23B/TYMS in CTCs would correlate with pathological complete response (pCR). Secondary objectives were to correlate CTC kinetics before (C1)/after NCRT (C2), in addition to the expression of transforming growth factor-ß receptor I (TGF-ßRI) with survival rates. CTCs were isolated by ISET and evaluated by immunocytochemistry (protein expression). At C1, RAD23B was detected in 54.1% of patients with no pCR and its absence in 91.7% of patients with pCR (p = 0.014); TYMS- was observed in 90% of patients with pCR and TYMS+ in 51.7% without pCR (p = 0.057). Patients with CTC2 > CTC1 had worse disease-free survival (DFS) (p = 0.00025) and overall survival (OS) (p = 0.0036) compared with those with CTC2 ≤ CTC1. TGF-ßRI expression in any time correlated with worse DFS (p = 0.059). To conclude, RAD23B/TYMS and CTC kinetics may facilitate the personalized treatment of LARC.