Paolo Bianchi

Reduced likelihood of metastases in patients with microsatellite-unstable colorectal cancer

Purpose: The outcome of patients with colorectal cancer is more favorable when the tumor exhibits high-frequency microsatellite instability (MSI). Although associated with earlier-stage tumors, MSI has been proposed as an independent predictor of survival. We tested the prognostic value of MSI in a large series of patients diagnosed with colorectal cancer in the last decade. Experimental Design: The survival of 893 consecutive patients with colorectal cancer characterized by microsatellite status was analyzed. The 89 (10%) patients with MSI cancer were classified according to tumor mismatch repair (MMR) defect, MMR germ-line mutation, hMLH1 and p16 promoter methylation, BRAF and K-ras mutations, and frameshifts of target genes. Results: The colorectal cancer–specific survival was significantly (P = 0.02) better in patients with MSI cancer than in those with stable tumor (MSS). MSI did not predict a significantly lower risk of cancer-related death if tumor stage was included in the multivariate analysis [hazard ratio, 0.72; 95% confidence interval (95% CI), 0.40-1.29; P = 0.27]. Instead, MSI was strongly associated with a decreased likelihood of lymph node (odds ratio, 0.31; 95% CI, 0.17-0.56; P < 0.001) and distant organ (odds ratio, 0.13; 95% CI, 0.05-0.33; P < 0.001) metastases at diagnosis, independently of tumor pathologic features. Molecular predictors of reduced metastatic risk, and then of more favorable prognosis, included TGFβRII mutation for all MSI tumors, hMSH2 deficiency for hereditary non-polyposis colorectal cancer, and absence of p16 methylation for sporadic hMLH1-deficient cancers. Conclusions: Tumor MSI is a stage-dependent predictor of survival in patients with colorectal cancer. The decreased likelihood of metastases in patients with MSI cancer is associated with specific genetic and epigenetic changes of the primary tumor.

Survival and recurrences after hepatic resection or radiofrequency for hepatocellular carcinoma in cirrhotic patients: a multivariate analysis

Hepatic resection is still considered the treatment of choice for hepatocellular carcinoma in patients with liver cirrhosis. Radiofrequency ablation is a new emerging modality. The aim of this study was to compare two homogeneous groups of patients who underwent either surgical resection or laparoscopic radiofrequency, analyzing the factors predicting survival and intrahepatic recurrences with use of a multivariate analysis. From February 1997 to April 2003, 98 patients were enrolled in this prospective study. Inclusion criteria were a single nodule of less than 5 cm, Child A-B class of liver function, and no previous treatment: 40 patients were in the surgical group and 58 patients were in the radiofrequency group. The two groups were homogeneous as far as preoperative characteristics were concerned. Operative mortality was zero, and the rates of operative morbidity were similar. Actuarial survival at 4 years was not significantly different (61% after resection and 45% after radiofrequency). There was a significant higher incidence of intrahepatic recurrences after radiofrequency than after resection (53% versus 30%; P = 0.018). This was mainly due to local recurrences, whereas those appearing in other liver segments were similar in both groups. A multivariate analysis showed that the significant factors predictive of an intrahepatic recurrence were the level of α-fetoprotein, the etiology of cirrhosis, and the type of the treatment. On the other hand, multivariate analysis of the survival showed that only the level of α-fetoprotein was an independent predictor of survival. The results of our study showed a significant lower incidence of intrahepatic recurrences after resection compared with after radiofrequency. This seems not to significantly influence the overall survival, probably because of a prompt and effective treatment of the recurrences themselves.

The Chemokine Receptor CX3CR1 Is Involved in the Neural Tropism and Malignant Behavior of Pancreatic Ductal Adenocarcinoma

Tumor perineural dissemination is a hallmark of human pancreatic ductal adenocarcinoma (PDAC) and represents a major source of local tumor recurrence after surgery. In this study, we provide in vitro and in vivo evidence that the chemokine receptor CX3CR1 may be involved in the neurotropism of PDAC cells to local peripheral nerves. Neoplastic cells from PDAC cell lines and surgical specimens express the chemokine receptor CX3CR1, absent in normal pancreatic ducts. Its unique ligand, the transmembrane chemokine CX3CL1, is expressed by neurons and nerve fibers. CX3CR1 + PDAC cell lines migrated in response to human recombinant CX3CL1 and specifically adhered to CX3CL1-expressing cells of neural origin via mechanisms involving activation of G proteins, beta1 integrins, and focal adhesion kinase. In vivo experiments with transplanted PDAC showed that only CX3CR1-transfected tumor cells infiltrated the local peripheral nerves. Immunohistochemistry of CX3CR1 in PDAC specimens revealed that 90% of the samples were positive with a heterogeneous pattern of expression. High receptor score was significantly associated with more prominent tumor perineural infiltration evaluated histologically (P = 0.026). Regression analyses (univariate and multivariate) showed that high CX3CR1 expression and perineural invasion were strongly associated with local and earlier tumor recurrence (P = 0.007). Collectively, this study shows that the CX3CR1 receptor may be involved in PDAC tumor neurotropism and is a relevant and independent risk factor to predict an early local tumor relapse in resected patients. Thus, the CX3CR1-CX3CL1 axis could represent a valuable therapeutic target to prevent tumor perineural dissemination in pancreatic cancer.

Intraoperative ultrasound during thoracoscopic procedures for solitary pulmonary nodules

BACKGROUND Traditional nonoperative diagnostic approaches to the solitary pulmonary nodule (bronchoscopy and percutaneous needle biopsy) can be inconclusive. Video-assisted thoracic surgery (VATS) provides a minimally invasive way to diagnose and treat these nodules. We evaluated the use of a dedicated intraoperative ultrasound probe as an aid in localization of small pulmonary nodules during VATS. METHODS An intraoperative ultrasound examination during a thoracoscopic procedure was performed on 18 patients to localize deep pulmonary nodules less than 20 mm in diameter without a definitive diagnosis by preoperative imaging techniques. RESULTS In the 18 patients, all nodules were successfully identified by intraoperative ultrasound. A definitive pathologic diagnosis was obtained from thoracoscopic biopsy or resection. The final diagnoses were primary lung cancer in 5 patients, metastatic lesions in 4 patients, hamartoma or chondroma in 4, granuloma in 3, and interstitial fibrosis in 2 patients. CONCLUSIONS In our experience, intraoperative ultrasound can safely and effectively localize invisible or nonpalpable pulmonary nodules at the time of thoracoscopy. This may help surgeons perform minimally invasive lung resections with clear surgical margins.

Differences and evolution of the methods for the assessment of microsatellite instability

Microsatellite instability (MSI) originates from the systematic accumulation of uncorrected deletion/insertion in repetitive DNA tracts in cancer cells with a deficient mismatch repair system. Among colorectal cancers, the MSI signature identifies hereditary cases arising in patients with germline mutations in hMLH1, hMSH2, PMS2 and a fraction of those with hMSH6 mutations, as well as sporadic cancers with epigenetic hMLH1 promoter hypermethylation. Considering the specific pathogenesis, pathological features, natural history and response to 5-fluoro-uracil-based chemotherapy of the MSI cancers, confusion about the genetic markers for MSI recognition seems surprising. In this clinically relevant field, an agreement has not been reached concerning the use of di- or mononucleotide markers for MSI assessment. The Revised Bethesda Guidelines still recommend a panel of markers consisting of mono- and dinucleotides, despite being questioned whether it is congruous to continue to use dinucleotide markers for MSI identification. In any event, no single marker is accurate enough for MSI testing, and an awareness of their pros and cons is required for proper interpretation of results. In recent years, several papers have reported different prevalence of MSI in unrelated series, largely depending on the detection and classification method, suggesting that MSI test interpretation also requires the understanding of the phenomenon rather than simply the crude satisfaction of panel recommendations. Inaccuracies can otherwise lead to under- or overdiagnosis and inaccurate disease classification, which always have a negative impact on the clinical practice of medicine.

Contrast-enhanced intraoperative ultrasonography during hepatectomies for colorectal cancer liver metastases

Preliminary reports showed that contrast-enhanced intraoperative ultrasonography (CEIOUS) provides information on primary or metastatic tumors of the liver that is not obtainable with conventional intraoperative ultrasonography (IOUS). This study validates the impact of CEIOUS, focusing on resective surgery for colorectal cancer (CRC) liver metastases. Twenty-four consecutive patients underwent liver resection using IOUS and CEIOUS for CRC liver metastases. CEIOUS was accomplished with intravenous injection of 4.8 mL of sulphur-hexafluoride microbubbles. CEIOUS found lesions missed at preoperative imaging and at IOUS in four patients and confirmed all of the new findings of IOUS in four patients. In addition, CEIOUS helped to define the tumor margins of the main lesion in 29% of patients with CRC liver metastases. No adverse effects were observed in relation with CEIOUS. In conclusion, CEIOUS improves IOUS accuracy with a significant impact on surgical strategy and radicality in patients who undergo surgery for CRC liver metastases.

Occurrence of Tertiary Lymphoid Tissue Is Associated with T-Cell Infiltration and Predicts Better Prognosis in Early-Stage Colorectal Cancers

Purpose: Tumor-infiltrating T lymphocytes (TIL) play a key role in the clinical outcome of human colorectal cancer; however, the dynamics of their recruitment along colorectal cancer clinical progression have not been fully elucidated. Tertiary lymphoid tissue (TLT) is an ectopic organized lymph node–like structure that typically forms at sites of chronic inflammation and is involved in adaptive immune responses. Its occurrence in cancer is sporadically documented and its role and clinical relevance is largely unknown. Experimental Design: The occurrence of TLT, the correlation with TILs, and the clinical relevance were evaluated retrospectively, in a cohort study involving a consecutive series of 351 patients with stage II and III colorectal cancer. The role of TLT in lymphocyte recruitment was assessed in a preclinical model of colorectal cancer. Results: In both human colorectal cancer and in a murine model of colorectal cancer, we identified organized TLT, highly vascularized (including high endothelial venules), and correlated with the density of CD3+ TILs. Intravenous injection in mice of GFP splenocytes resulted in homing of lymphocytes to TLT, suggesting an active role of TLT in the recruitment of lymphocytes to tumor areas. Accordingly, TLT density and TIL infiltration correlated and were coordinated in predicting better patients outcome among patients with stage II colorectal cancer. Conclusions: We provide evidence that TLT is associated with lymphocyte infiltration in colorectal cancer, providing a pathway of recruitment for TILs. TLT cooperates with TILs in a coordinated antitumor immune response, when identifying patients with low-risk early-stage colorectal cancer, thus, representing a novel prognostic biomarker for colorectal cancer. Clin Cancer Res; 20(8); 2147–58. ©2014 AACR.

ORIGINAL TECHNIQUE FOR SMALL COLORECTAL TUMOR LOCALIZATION DURING LAPAROSCOPIC SURGERY

INTRODUCTION: Small colonic tumor localization and correct extension of colonic resection is critical in laparoscopic surgery. Currently used techniques are sometimes inconclusive and may carry some morbidity. We describe an original method of small tumor localization during laparoscopic colorectal operations through the use of preoperative clip applications by colonoscopy and intraoperative ultrasound of the colon. METHODS: Eight patients with small colonic lesions necessitating preoperative marking were included into this study. A two-step technique was used. Before the operation two metal clips were endoscopically applied proximally and distally to the lesion site. At surgery an intraoperative ultrasound examination of the colon or rectum surface was performed to localize the clips. Subsequent laparoscopic colon resection was performed. RESULTS: Endoscopic metallic clips were easily applied around the lesion in all cases without complications. No dislodgement of clips was documented. At surgery laparoscopic ultrasound visualized the clips in all cases. The examination took between 5 and 17 minutes with no specific morbidity. The lesions with the surrounding clips were always found in the resected specimen. CONCLUSIONS: Endoscopic metal clipping and intraoperative laparoscopic ultrasound proved to be an easy, safe, and accurate technique in locating small colonic tumors.

Contrast-Enhanced Intraoperative Ultrasonography During Surgery for Hepatocellular Carcinoma in Liver Cirrhosis: Is It Useful or Useless? A Prospective Cohort Study of Our Experience

BackgroundPreliminary results showed that contrast-enhanced intraoperative ultrasonography (CEIOUS) could provide information not obtainable with conventional IOUS during surgery for hepatocellular carcinoma (HCC). The aim of the study was to prospectively validate the role of CEIOUS on the basis of a larger experience and to establish a new classification that takes into account its findings.MethodsEighty-seven consecutive patients underwent hepatecomies for HCC. Those patients with new lesions at IOUS underwent CEIOUS: for that patients received intravenously 4.8 mL sulphurhexafluoride microbubbles. Pattern of enhancement was classified in 4 categories: A1 (full enhancement in the arterial phase and wash-out in the delayed phases), A2 (intralesional signs of neovascularization during all phases), A3 (no nodular enhancement but detectability during the liver enhancement), and B (undetectability during the liver enhancement). Resection was recommended for A1-3 nodules and no treatment for B nodules.ResultsTwenty-nine patients (33%) had 59 new lesions at IOUS and underwent CEIOUS. Twenty-seven nodules showed a B pattern at CEIOUS and were not removed; 32 nodules were classified as A1 in 5 patients, A2 in 11 patients, and A3 in 16 patients. The nodules were removed, and by histology, five A1, nine A2, and six A3 nodules were confirmed to be HCC. CEIOUS modified the operative decision making in 79% of these patients.ConclusionsCEIOUS is useful during surgery for HCC; it complements the accuracy of IOUS and affects the radicalness of the surgical. Specificity of CEIOUS has to be further improved, although intrinsic drawbacks exist in the diagnostic criterion of tumor vascularity.

Genetic and epigenetic changes in primary metastatic and nonmetastatic colorectal cancer

Colorectal cancer (CRC) develops as multistep process, which involves genetic and epigenetic alterations. K-Ras, p53 and B-Raf mutations and RASSF1A, E-Cadherin and p16INK4A promoter methylation were investigated in 202 CRCs with and without lymph node and/or liver metastasis, to assess whether gene abnormalities are related to a metastogenic phenotype. K-Ras, B-Raf and p53 mutations were detected in 27, 3 and 32% of the cases, with K-Ras mutations significantly associated with metastatic tumour (P=0.019). RASSF1A, E-Cadherin and p16INK4A methylation was documented in 20, 44 and 33% of the cases with p16INK4A significantly associated with metastatic tumours (P=0.001). Overall, out of 202 tumours, 34 (17%) did not show any molecular change, 125 (62%) had one or two and 43 (21%) three or more. Primary but yet metastatic CRCs were prevalent in the latter group (P=0.023) where the most frequent combination was one genetic (K-Ras in particular) and two epigenetic alterations. In conclusion, this analysis provided to detect some molecular differences between primary metastatic and nonmetastatic CRCs, with K-Ras and p16INK4A statistically altered in metastatic tumours; particular gene combinations, such as coincidental K-Ras mutation with two methylated genes are associated to a metastogenic phenotype.