Late Local Recurrence after Neoadjuvant Therapy and Radical Resection for Locally Advanced Rectal Cancer.

Neoadjuvant radiochemotherapy (RCT) and lately total neoadjuvant therapy (TNT) improved local recurrence rates of rectal cancer significantly compared to total mesorectal excision (TME) alone. Yet the occurrence and impact of late local recurrences after many years appears to be a distinct biological problem. We included n = 188 patients with rectal cancer after RCT and radical resection in this study; n = 38 of which had recurrent disease (sites: local (8.0%), liver (6.4%), lung (3.7%)). We found that 68% of all recurrences developed within the first two years. Four patients, however, experience recurrence >8 years after surgery. Here, we report and characterize four cases of late local recurrence (10% of patients with recurrent disease), suggesting that neoadjuvant therapy in principle delays local recurrence.

Neither FDG-PET Nor CT can distinguish between a pathological complete response and an incomplete response after neoadjuvant chemoradiation in locally advanced rectal cancer: a prospective study.

OBJECTIVE: To prospectively compare the ability of flourodeoxyglucose-positron emission tomography (FDG-PET) and computed tomography (CT) to identify a pathological complete response (pCR) in patients with rectal cancer treated by chemoradiation. BACKGROUND: A major obstacle in pursuing nonoperative management in patients with rectal cancer after chemoradiation is the inability to identify a pCR preoperatively. METHODS: A total of 121 patients with rectal cancer were prospectively enrolled. FDG-PET scans and helical CT scans were obtained before and after neoadjuvant chemoradiation. Consensus readings of PET and CT scans were used to classify certainty of disease (5-point confidence rating scale). The ability of PET and CT scans to accurately distinguish a pCR (ypT0) from an incomplete response (ypT1-4) was estimated using the area under the receiver operating characteristic curve (AUC). RESULTS: Of the 121 patients, 26 (21%) had a pCR. PET and CT scans were equally inadequate at distinguishing a pCR from an incomplete response (AUC = 0.64 for both, P = 0.97). Among the 26 patients with a pCR, 14 (54%) and 5 (19%) were classified as complete responders on PET and CT scans, respectively. Among the 95 patients with an incomplete pathological response, 63 (66%) and 90 (95%) were classified as incomplete responders on PET and CT scans, respectively. None of the individual PET parameters, including visual response score, mean standard uptake value (SUVmean), maximum SUV (SUVmax), and total lesion glycolysis, accurately distinguished a pCR (AUCs = 0.57-0.73). CONCLUSIONS: Neither PET nor CT scans have adequate predictive value to be clinically useful in distinguishing a pCR from an incomplete response and, therefore, should not be obtained for the purpose of attempting to predict a pCR after neoadjuvant chemoradiation for rectal cancer.

Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model.

Oncolytic virotherapy constitutes a promising treatment option for many solid cancers, including peritoneal carcinomatosis (PC), which still represents a terminal stage of many types of tumors. To date, the in vitro efficacy of oncolytic viruses is mostly tested in 2D-cultured tumor cell lines due to the lack of realistic 3D in vitro tumor models. We have investigated the feasibility of virotherapy as a treatment option for PC in a human ex vivo peritoneum co-culture model. Human HT-29 cancer cells stably expressing marker genes GFP and firefly luciferase (GFP/luc) were cultured on human peritoneum and infected with two prototypic oncolytic viruses (GLV-0b347 and MeV-DsRed). Both viral constructs were able to infect HT-29 cells in patient-derived peritoneum with high tumor specificity. Over time, both GFP signal and luciferase activity decreased substantially, thereby indicating successful virus-induced oncolysis. Furthermore, immunohistochemistry stainings showed specific virotherapeutic infections of HT-29 cells and effective tumor cell lysis in infected co-cultures. Thus, the PC model established here provides a clinically relevant screening platform to evaluate the therapeutic efficacy of virotherapeutic compounds and also to investigate, in an autologous setting, the immunostimulatory potential of oncolytic viruses for PC in a unique human model system superior to standard 2D in vitro models.

FDG-PET assessment of rectal cancer response to neoadjuvant chemoradiotherapy is not associated with long-term prognosis: a prospective evaluation.

BACKGROUND: At present there is no defined role for routine FDG-PET in the preoperative evaluation of nonmetastatic rectal cancer. OBJECTIVE: The primary objective of this study was to evaluate the ability of FDG-PET to predict long-term prognosis based on the response to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer. DESIGN: This was a prospective study. SETTINGS: This study was performed at an academic, tertiary care, comprehensive cancer center. PATIENTS: One hundred twenty-seven patients with locally advanced rectal cancer were enrolled between September 1999 and December 2005. INTERVENTIONS: All patients underwent FDG-PET scans before and after neoadjuvant chemoradiotherapy. MAIN OUTCOME MEASURES: FDG-PET parameters were evaluated by at least 2 study board-certified nuclear medicine physicians, and included mean standard uptake value, maximum standard uptake value, total lesion glycolysis, and visual response score. The main outcome measures were time to recurrence and disease-specific survival. RESULTS: Of 127 patients, 82 (65%) were men, the median age was 60 years (range, 27-82), 110 patients had stage II/III disease, and 17 patients had stage IV disease. Median follow-up among survivors was 77 months (range, 1-115 months). Nine patients had unresectable metastatic disease and were excluded from the time-to-recurrence analysis. At 5 years, 74% (95% CI = 66%-81%) of patients had not had recurrences (locally and/or distantly). The 5-year disease-specific survival was 89% (95% CI = 81%-93%). On univariate analysis, visual response score and time to recurrence came closest to having an association (HR = 0.83, 95% CI = 0.68-1.01, p = 0.06). On multivariate analysis, the visual response score was not significant (p = 0.85). No FDG-PET parameter was associated with disease-specific survival. CONCLUSIONS: Assessment of rectal cancer response to neoadjuvant chemoradiotherapy by FDG-PET provides no prognostic information. Therefore, serial FDG-PET before and after neoadjuvant chemoradiotherapy should not be performed for this purpose.

A new tool for bile duct tissue sampling: ex vivo clinical evaluation of intraductal cryobiopsy for cholangioscopy.

Background and study aims Indeterminate biliary strictures represent a major challenge in clinical diagnostics. Diagnostic yield of radiological, endoscopic imaging and histopathological diagnosis is insufficient. The cryobiopsy technique is a new method for tissue extraction already used in different clinical settings. The aim of this ex vivo clinical study was to investigate feasibility and tissue quality of cryobiopsy in the bile duct. Patients and methods We included 14 patients who underwent pancreaticoduodenectomy. Bile duct samples were taken with either a new prototype cryoprobe or one of two forceps types. Results were analyzed for general feasibility, specimen size, histological assessability as well as representativity of retrieved tissue. Results Feasibility of cholangioscopic forceps was poor compared to gastric biopsy forceps or cryobiopsy. Significantly larger tissue samples were obtained with cryobiopsy (5.6 ±â€Š4.5 mm 2 ) compared to gastric biopsy forceps (3.3 ±â€Š5.1 mm 2 , P  = 0.006). Furthermore, cryobiopsy was superior in histological assessment quality ( P  = 0.02) and concerning representativity ( P  = 0.03). Conclusions Cryobiopsy in the bile duct is feasible and the quality of the obtained tissue is high. Further investigation of bile duct cryobiopsy in vivo is warranted.

Pharmacologic Targeting of MMP2/9 Decreases Peritoneal Metastasis Formation of Colorectal Cancer in a Human Ex Vivo Peritoneum Culture Model.

BACKGROUND: Matrix metalloproteinases (MMPs) play a crucial role in tumour initiation, progression, and metastasis, including peritoneal carcinosis (PC) formation. MMPs serve as biomarkers for tumour progression in colorectal cancer (CRC), and MMP overexpression is associated with advanced-stage metastasis and poor survival. However, the molecular mechanisms of PC from CRC remain largely unclear. METHODS: We investigated the role of MMPs during peritoneal colonisation by CRC cell lines in a human ex vivo peritoneum model and in patient-derived CRC and corresponding PC samples. MMP2 and MMP9 were inhibited using the small-molecule inhibitors batimastat and the specific MMP2/9 inhibitor III. RESULTS: MMP2 and MMP9 were strongly upregulated in patient-derived samples and following peritoneal colonisation by CRC cells in the ex vivo model. MMP inhibition with batimastat reduced colonisation of HT29 and Colo205 cells by 36% and 68%, respectively (p = 0.0073 and p = 0.0002), while MMP2/9 inhibitor III reduced colonisation by 50% and 41%, respectively (p = 0.0003 and p = 0.0051). Fibronectin cleavage was enhanced in patient-derived samples of PC and during peritoneal colonisation in the ex vivo model, and this was inhibited by MMP2/9 inhibition. CONCLUSION: MMPs were upregulated in patient-derived samples and during peritoneal attachment of CRC cell lines in our ex vivo model. MMP2/9 inhibition prevented fibronectin cleavage and peritoneal colonisation by CRC cells. MMP inhibitors might thus offer a potential treatment strategy for patients with PC.

Evaluation of ¹8F-FDG-PET for early detection of suboptimal response of rectal cancer to preoperative chemoradiotherapy: a prospective analysis.

BACKGROUND: Early identification of inadequate response to preoperative chemoradiotherapy (CRT) may spare rectal cancer patients the toxicity of ineffective treatment. We prospectively evaluated tumor response with (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET) early in the course of preoperative CRT. METHODS: A total of 27 prospectively accrued patients with locally advanced rectal cancer (T(3-4)/N(1)) received preoperative CRT (5040 cGy + 5FU-based chemotherapy). Patients underwent PET scanning before and 8-14 days after commencement of CRT. Scans were interpreted using 3 standard parameters: SUV(max), SUV(avg), and total lesion glycolysis (TLG) as well as an investigational parameter: visual response score (VRS). Percent pathologic response was quantified as a continuous variable. All PET parameters were correlated with pathology. Pathologic complete/near-complete response was defined as ≥95% tumor destruction, suboptimal response as <95%. Statistical analysis was performed using the Wilcoxon rank sum test and receiver operating characteristic (ROC) curve analysis. RESULTS: Of the 27 patients, 11 (41%) had pathologic complete/near-complete response; 16 (59%) had suboptimal response. SUV(max), SUV(avg), and TLG did not discriminate between responders and nonresponders. Visual response score (VRS) was statistically significantly higher for complete/near-complete responders than for suboptimal responders (65 vs. 33%, P = 0.02). Suboptimal responders were identified with 94% sensitivity and 78% accuracy using a VRS cut-off of 50%. CONCLUSIONS: In this pilot study, FDG-PET at 8-14 days after the beginning of preoperative CRT was unsuccessful at predicting pathological response with enough accuracy to justify an early change in therapy.

Three dimensional cultivation increases chemo- and radioresistance of colorectal cancer cell lines.

Although 2D cell cultures are commonly used to predict therapy response, it has become clear that 3D cultures may better mimic the in vivo situation and offer the possibility of tailoring translational clinical approaches. Here, we compared the response of 2D and 3D colorectal cancer (CRC) cell lines to irradiation and chemotherapy. Classic 2D cultures and 3D spheroids of CRC cell lines (CaCo2, Colo205, HCT116, SW480) were thoroughly established, then irradiated with doses of 1, 4, or 10 Gy, using a clinical-grade linear accelerator. The response was assessed by immunohistochemistry, flow cytometry, and TUNEL assays. Upon irradiation, CRC 3D spheroids were morphologically altered. After irradiation with 10 Gy, annexin V/PI staining revealed a 1.8- to 4-fold increase in the apoptosis rate in the 2D cell cultures (95% CI 3.24±0.96), and a 1.5- to 2.4-fold increase in the 3D spheroids (95% CI 1.56±0.41). Irradiation with 1 Gy caused 3- and 4-fold increases in TUNEL positive cells in the CaCo2 and HCT116 (p = 0.01) 2D cultures, respectively, compared with a 2-fold increase in the 3D spheroids. Furthermore, the 2D and 3D cultures responded differently to chemotherapy; the 3D cultures were more resistant to 5-FU and cisplatin, but not to doxorubicin and mitomycin C, than the 2D cultures. Taken together, CRC cells cultured as 3D spheroids displayed markedly higher resistance to irradiation therapy and selected chemotherapeutic drugs than 2D cultures. This in vitro difference must be considered in future approaches for determining the ideal in vitro systems that mimic human disease.

A human ex vivo coculture model to investigate peritoneal metastasis and innovative treatment options.

OBJECTIVES: Peritoneal metastasis (PM) is commonly observed in patients with colorectal cancer (CRC). The outcome of these patients is poor, with an average survival of only six months without therapy, which requires a better understanding of PM biology and new treatment strategies. METHODS: We established and characterized a human ex vivo peritoneal model to investigate the mechanisms of peritoneal seeding and possible treatment options. For this, CRC cell lines and patient-derived tumor organoids were cultured together with human peritoneum to investigate the invasion of malignant cells and the effects of local chemotherapy. RESULTS: Fresh human peritoneum was cultured for up to three weeks in a stainless steel ring system, allowing for survival of all peritoneal structures. Peritoneal cell survival was documented by light microscopy and immunohistochemical staining. Further, immunohistological characterization of the tissue revealed CD3-positive T-lymphocytes and vimentin-positive fibroblasts within the peritoneum. In addition, extracellular matrix components (collagens, matrix metalloproteinases) were localized within the tissue. Coculture with CRC cell lines and patient-derived CRC organoids revealed that cancer cells grew on the peritoneum and migrated into the tissue. Coculture with CRC cells confirmed that hyperthermal treatment at 41 °C for 90 min significantly enhanced the intracellular entry of doxorubicin. Moreover, treatment with mitomycin C under hyperthermic conditions significantly reduced the amount of cancer cells within the peritoneum. CONCLUSIONS: This human ex vivo peritoneal model provides a stringent and clinically relevant platform for the investigation of PM and for further elucidation of possible treatment options.

The role of neoadjuvant therapy in sphincter-saving surgery for mid and distal rectal cancer.

Current treatment of mid and lower rectal cancer includes neoadjuvant therapy, such as radiotherapy or radiochemotherapy. Randomized trials have demonstrated that neoadjuvant therapy improves local control. However, with the exception of one study, it does not improve overall and cancer-specific survival. In addition, the role of neoadjuvant therapy in enhancing the surgeon's ability to perform sphincter-saving surgery is still controversial. We provide an overview of reported randomized trials using neoadjuvant therapy in patients with rectal cancer. We have examined the most significant factors influencing sphincter preservation and the manner in which these factors are affected by neoadjuvant treatment.

Positron emission tomography detection of distant metastatic or synchronous disease in patients with locally advanced rectal cancer receiving preoperative chemoradiation.

BACKGROUND: Patients with locally advanced rectal cancer may present with synchronous distant metastases. Choice of optimal treatment--neoadjuvant chemoradiation versus systemic chemotherapy alone--depends on accurate assessment of distant disease. We prospectively evaluated the ability of [18F]fluorodeoxyglucose ([18F]FDG) positron emission tomography (PET) to detect distant disease in patients with locally advanced rectal cancer who were otherwise eligible for combined modality therapy (CMT). METHODS: Ninety-three patients with locally advanced rectal cancer underwent whole-body [18F]FDG PET scanning 2-3 weeks before starting CMT. Sites other than the rectum, mesorectum, or the area along the inferior mesenteric artery were considered distant and were divided into nine groups: neck, lung, mediastinal lymph node (LN), abdomen, liver, colon, pelvis, peripheral LN, and soft tissue. Two nuclear medicine physicians blinded to clinical information used PET images and a five-point scale (0-4) to determine certainty of disease. A score greater than 3 was considered malignant. Confirmation was based on tissue diagnosis, surgical exploration, and subsequent imaging. RESULTS: At a median follow-up of 34 months, the overall accuracy, sensitivity, and specificity of PET in detecting distant disease were 93.7%, 77.8%, and 98.7% respectively. Greatest accuracy was demonstrated in detection of liver (accuracy = 99.9%, sensitivity = 100%, specificity = 98.8%) and lung (accuracy = 99.9%, sensitivity = 80%, specificity = 100%) disease; PET detected 11/12 confirmed malignant sites in liver and lung. A total of 10 patients were confirmed to have M1 stage disease. All 10 were correctly staged by pre-CMT PET; abdominopelvic computed tomography (CT) scans accurately detected nine of them. CONCLUSION: Baseline PET in patients with locally advanced rectal cancer reliably detects metastatic disease in liver and lung. PET may play a significant role in defining extent of distant disease in selected cases, thus impacting the choice of neoadjuvant therapy.

p27 expression in post-treatment rectal cancer: a potential novel approach for predicting residual nodal disease.

BACKGROUND: Expression profiles of p21, p27, p53, Ki-67, and thymidylate synthase may be associated with response to neoadjuvant chemoradiation. The relationship between post-treatment protein expression and regional lymph node involvement has not been fully explored. METHODS: Tumor cores from 126 rectal cancer patients underwent immunohistochemical analysis for the aforementioned proteins. Staining indices (SIs) using percentage of stained cells and staining intensity were calculated for 10 tumor cores per patient. SI for each marker was compared between node negative and node positive patients. RESULTS: Twenty-six (20.6%) cancer patients had a pathologic complete response and 37 had inadequate tissue or cancer cells, leaving 63 for analysis. Thirty-seven (58.7%) cancer patients were node negative and 26 (41.3%) were node positive. There was an association between increased p27 SI and nodal positivity (P = .04). CONCLUSION: Increased p27 expression in post-treatment rectal cancer is associated with nodal positivity and may determine which patients are suitable for local excision.

Pilot study of PET imaging of 124I-iodoazomycin galactopyranoside (IAZGP), a putative hypoxia imaging agent, in patients with colorectal cancer and head and neck cancer.

BACKGROUND: Hypoxia within solid tumors confers radiation resistance and a poorer prognosis. 124I-iodoazomycin galactopyranoside (124I-IAZGP) has shown promise as a hypoxia radiotracer in animal models. We performed a clinical study to evaluate the safety, biodistribution, and imaging characteristics of 124I-IAZGP in patients with advanced colorectal cancer and head and neck cancer using serial positron emission tomography (PET) imaging. METHODS: Ten patients underwent serial whole-torso (head/neck to pelvis) PET imaging together with multiple whole-body counts and blood sampling. These data were used to generate absorbed dose estimates to normal tissues for 124I-IAZGP. Tumors were scored as either positive or negative for 124I-IAZGP uptake. RESULTS: There were no clinical toxicities or adverse effects associated with 124I-IAZGP administration. Clearance from the whole body and blood was rapid, primarily via the urinary tract, with no focal uptake in any parenchymal organ. The tissues receiving the highest absorbed doses were the mucosal walls of the urinary bladder and the intestinal tract, in particular the lower large intestine. All 124I-IAZGP PET scans were interpreted as negative for tumor uptake. CONCLUSIONS: It is safe to administer 124I-IAZGP to human subjects. However, there was insufficient tumor uptake to support a clinical role for 124I-IAZGP PET in colorectal cancer and head and neck cancer patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT00588276.

Hantavirus and acute appendicitis--the diagnosis behind the diagnosis?

A 33-year-old man with a history of acute lower abdominal pain was admitted to the emergency room. After laparoscopic appendectomy and pathological confirmed acute appendicitis the patient developed thrombocytopenia and acute renal failure. Serological testing for hantaviruses revealed a positive result for PUUV IgG and IgM. Immunohistochemical work-up detected PUUV antigen in endothelial cells of capillaries and larger vessels. The high percentage of patients with hantavirus infection and severe abdominal pain is remarkable and, up to now, unexplained. To our knowledge this is the first report demonstrating PUUV antigen in the human intestine. Further studies are warranted whether hantaviruses are setting the stage for a secondary bacterial infection or cause an inflammation itself.

Significance of acellular mucin pools in rectal carcinoma after neoadjuvant chemoradiotherapy.

The presence of mucin pools lacking neoplastic epithelium ("acellular" mucin) in resection specimens of rectal carcinoma after neoadjuvant chemoradiotherapy (CRT) is a well-recognized phenomenon. The current recommendation by the College of American Pathologists is to regard acellular mucin as a type of treatment response and not as residual tumor. However, data-based evidence for or against such an approach is incomplete. In this study, we systematically analyzed the pattern and significance of mucin pools in 108 consecutive, prospectively collected resection specimens from patients who had uT3-4 and/or uN1 rectal cancer and were treated with preoperative long-course CRT. The 108 patients, 39 female and 69 male, had a median age of 58.5 years. With every tumor entirely examined in whole-mount sections, mucin pools were identified in 33 cases (33 of 108, 31%); in 16 (15%) they were all acellular. The mucin pools were focal (10% to 50% of the entire lesion) in 25 cases and extensive (>50%) in 8 cases. Mucin pools were also noted in the lymph nodes in 6 cases (6%); 3 of these were entirely acellular. Five cases had mucin pools in both the primary site and the lymph nodes. When acellular mucin was considered as "no residual tumor," the complete pathologic response rate for the entire cohort was 22% (24 of 108). The pathologic stage of the residual tumor (ypT) was 0 or 1 for 27 cases (25%) and 2 to 4 for 81 cases. The pathologic stage of nodal disease (ypN) was 0 for 83 (77%) and 1 or 2 for 25 cases. When acellular mucin was considered as "residual tumor," the complete pathologic response rate dropped to 17%; ypT was upstaged in 10 tumors and ypN was upstaged in 2 tumors. With a median follow-up of 31 months, the 3-year recurrence-free survival (RFS) was 73% for the entire group. Advanced pathologic response and low pathologic stage of the residual tumor (determined based on the depth of only viable tumor cells) correlated significantly with better RFS. However, the correlation between pathologic response and RFS became insignificant when acellular mucin pools were considered as residual tumor. Neither the presence of mucin pools nor their extent or cellularity had an impact on RFS. Furthermore, none of the 12 patients whose ypT or ypN was upstaged by acellular mucin had recurrent disease (3-y RFS of 100%). Thus, our results suggest that mucin pools in rectal carcinoma after neoadjuvant CRT do not have a significant impact on patient outcome, supporting the College of American Pathologists recommendation that only viable tumor cells, not acellular mucin, are to be interpreted as residual disease in the tumor pathologic staging.

Prognostic implications of the distribution of lymph node metastases in rectal cancer after neoadjuvant chemoradiotherapy.

PURPOSE: After preoperative chemoradiotherapy of rectal cancer, the number of retrievable and metastatic lymph nodes is decreased. The current TNM classification is based on number and not location of lymph node metastases and may understage disease after chemoradiotherapy. The aim of this study was to examine the prognostic significance of location of involved lymph nodes in rectal cancer patients after preoperative chemoradiotherapy. PATIENTS AND METHODS: We prospectively examined whole-mount specimens from 121 patients with uT3-4 and/or N+ rectal cancer who received preoperative chemoradiotherapy followed by resection. Location of involved lymph nodes was compared with median number of lymph nodes involved as well as presence of distant metastasis at presentation. RESULTS: Lymph node metastases were detected in 37 patients (31%). Thirteen patients with lymph node involvement along major supplying vessels (proximal lymph node metastases) had a significantly higher rate of distant metastatic disease at time of surgery than patients without proximal lymph node involvement (P < .001); median number of lymph nodes involved was two for patients with proximal lymph node metastases and 1.5 for patients with mesorectal lymph node involvement alone. CONCLUSION: Our data suggest that, after preoperative chemoradiotherapy, proximal lymph node involvement is associated with a high incidence of metastatic disease at time of surgery. Because the median number of involved lymph nodes is low after preoperative chemoradiotherapy, the TNM staging system may not provide an accurate assessment of metastatic disease. Therefore, the ypTNM staging system should incorporate distribution as well as number of lymph node metastases after preoperative chemoradiotherapy for rectal cancer.