Intratumor morphologic and transcriptomic heterogeneity in V600EBRAF-mutated metastatic colorectal adenocarcinomas.

BACKGROUND: Intratumor heterogeneity (ITH) is described as the presence of various clones within one tumor, each with their own unique features in terms of morphology, inflammation, genetics or transcriptomics. Heterogeneity provides the fuel for drug resistance; therefore, an accurate assessment of tumor heterogeneity is essential for the development of effective therapies. The purpose of this study was to dissect morphologic and molecular ITH in colorectal adenocarcinoma. MATERIALS AND METHODS: A series of 120 V600EBRAF-mutated (V600EBRAFmt) consecutive metastatic colorectal adenocarcinomas was assessed for morphologic heterogeneity. The two heterogeneous components of each specimen underwent a histopathological, immunohistochemical and molecular characterization to evaluate: histologic variant, grading, tumor-infiltrating lymphocytes (TILs), mismatch repair proteins' expression, KRAS/BRAF/NRAS mutations, microsatellite instability (MSI) status and consensus molecular subtype (CMS). RESULTS: Thirty-one out of 120 (25.8%) V600EBRAFmt primary colorectal adenocarcinomas presented a heterogeneous morphology. Among these, eight cases had adequate material for molecular profiling. Five out of the eight (62.5%) cases resulted instable at MSI testing. The majority (62.5%) of the samples showed a CMS4 phenotype based on gene expression profiling. Heterogeneity in CMS classification was observed in four out of eight cases. One out of eight cases presented significant heterogeneity in the number of TILs between the two components of the tumor. CONCLUSIONS: Although the distribution of the immune infiltrate appears relatively conserved among heterogeneous areas of the same tumor, changes in gene expression profile and CMS occur in 50% of V600EBRAFmt adenocarcinoma cases in our small series and might contribute to variability in response to anticancer therapy and clinical outcomes. Assessment of morphological and molecular ITH is needed to improve colorectal cancer classification and to tailor anticancer treatments and should be included in the pathology report.

Isolated limb perfusion for the management limb threatening soft tissue sarcomas: The role of histological type on clinical outcomes.

BACKGROUND: Hyperthermic isolated limb perfusion (HILP) is an effective neoadjuvant treatment to avoid amputation in patients with locally advanced extremity soft tissue sarcomas (STS). We aimed to investigate whether STS histological type plays a role in predicting clinical outcomes. METHODS: This study reports a retrospective analysis of 125 patients with limb threatening STS (liposarcoma, n = 41; malignant peripheral nerve sheath tumor, n = 20; leiomyosarcoma, n = 20; miscellany, n = 44), who underwent HILP from 1990 through 2015 at our institution. The following endpoints were evaluated: tumor response (assessed by radiological imaging and histology), limb sparing rate, local progression-free survival (LPFS) and overall survival (OS). RESULTS: On average, overall (complete + partial) tumor response was significantly greater in patients affected with liposarcoma as compared to those with other histotypes (radiological response rate: 38/41, 92.7% vs 66/84, 78.6%, P-value: 0.048; mean histological necrosis: 83.6% vs 52.9%, P < 0.0001). Limb sparing rate was also higher among patients with liposarcoma as compared to other histotypes (39/41, 95.1% vs 62/84, 73.8%, P-value: 0.005). As regards survival, LPFS was similar across tumor types, whereas OS resulted significantly worse in patients with limb leiomyosarcoma (log-rank P-value: 0.009). CONCLUSIONS: HILP is a very effective treatment modality for limb threatening STS. In our series, liposarcoma appears to be the histological type most sensitive to HILP in terms of tumor response and thus limb sparing, which might help clinicians in the patient selection process.

Adherence to treatment guidelines for primary sarcomas affects patient survival: a side study of the European CONnective TIssue CAncer NETwork (CONTICANET).

BACKGROUND: The impact of adherence to clinical practice guidelines (CPGs) for loco-regional treatment (i.e. surgery and radiotherapy) and chemotherapy on local disease control and survival in sarcoma patients was investigated in a European study conducted in an Italian region (Veneto). PATIENTS AND METHODS: The completeness of the adherence to the Italian CPGs for sarcomas treatment was assessed by comparing the patient's charts and the CPGs. Propensity score-adjusted multivariate survival analysis was used to assess the impact of CPGs adherence on patient clinical outcomes. RESULTS: A total of 151 patients were included. Adherence to CPGs for loco-regional therapy and chemotherapy was observed in 106 out of 147 (70.2%) and 129 out of 139 (85.4%) patients, respectively. Non-adherence to CPGs for loco-regional treatment was independently associated with AJCC stage III disease [odds ratio (OR) 1.77, P = 0.011] and tumor-positive excision margin (OR 3.55, P = 0.003). Patients not treated according to the CPGs were at a higher risk of local recurrence [hazard ratio (HR) 5.4, P < 0.001] and had a shorter sarcoma-specific survival (HR 4.05, P < 0.001), independently of tumor stage. CONCLUSIONS: Incomplete adherence to CPGs for loco-regional treatment of sarcomas was associated with worse prognosis in patients with non-metastatic tumors.

Circumventing melanoma chemoresistance by targeting DNA repair.

Available evidence demonstrates that the DNA repair machinery is involved in melanoma resistance to chemotherapeutics. Furhtermore, preclinical findings suggest that interfering with DNA repair could increase chemosensitivity of melanoma cells. However, the clinical implementation of these principles is still in its infancy and no such strategy is currently proven to be effective in patients with advanced melanoma. Since the molecular mechanisms governing the relationship between chemoresistance and DNA repair are not fully elucidated, more basic and translational research is needed to understand the reasons for the failures and to identify novel targets. In this review we summarize the experimental and clinical findings that are fostering the research in this promising field of oncology.

Cancer resistance to type II topoisomerase inhibitors.

Type II topoisomerases (TOPO2) are ubiquitously expressed enzymes that overcome topological problems in genomic DNA, which can result from DNA replication, transcription and repair. The class of compounds targeting TOPO2 includes some of the most active chemotherapy agents currently available for the treatment of patients with different cancer types. Therefore, understanding of the molecular mechanisms underlying resistance to these drugs is of pivotal importance to improve their efficacy and ultimately increase the life expectancy of cancer patients. The first aim of this review is to summarize the molecular biology of TOPO2 inhibitors, which is the key to understand cancer resistance to them; the second part of this work is dedicated to overview and discuss the available evidence on the mechanisms of resistance to these drugs, with special attention to the strategies that might be useful to circumvent this phenomenon on the clinical ground.

The liver: another organ involved in Muir Torre syndrome?

Muir Torre syndrome is a rare autosomal dominant cancer-predisposing syndrome characterized by the occurrence of sebaceous gland neoplasms and/or keratoacanthomas associated with visceral malignancies that belong to the spectrum of hereditary non polyposis colorectal cancer (HNPCC), i.e., tumors of gastrointestinal and genitourinary tracts. Hepatobiliary malignancy in association with Muir Torre syndrome has rarely been reported. Here, we describe a case of Muir Torre syndrome associated with an hepatocellular-carcinoma in a patient with a non-cirrhotic liver and an HNPCC-family with multiple cases of hepatocellular carcinoma.

Prognostic factors and oncologic outcome in 146 patients with colorectal peritoneal carcinomatosis treated with cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy: Italian multicenter study S.I.T.I.L.O.

AIM: The present study was specifically designed to assess the major clinical and pathological variables of patients with colorectal peritoneal carcinomatosis in order to investigate whether currently used criteria appropriately select candidates for peritonectomy procedures (cytoreductive surgery) combined with hyperthermic intraperitoneal chemotherapy (HIPEC). PATIENTS AND METHODS: Preoperative, operative and follow-up data on 146 consecutive patients presenting with peritoneal carcinomatosis of colorectal origin and treated by surgical cytoreduction combined with HIPEC in 5 Italian Hospital and University Centers were prospectively entered in a common database. Univariate and multivariate analyses were used to assess the prognostic value of clinical and pathologic factors. RESULTS: Over a minimum 24-month follow-up, the overall morbidity rate was 27.4% (mortality rate: 2.7%) and was directly related to the extent of surgery. Peritoneal cancer index (PCI), unfavorable peritoneal sites, synchronous or previously resected liver metastasis and the completeness of cytoreduction, all emerged as independent prognostic factors correlated with survival. CONCLUSIONS: Until research provides more effective criteria for selecting patients based upon the biomolecular features of carcinomatosis, patients should be selected according to the existing independent prognostic variables.

Peptide-based anticancer vaccines: recent advances and future perspectives.

Anticancer active immunotherapy embodies the ideal antitumor therapy, as it theoretically combines target specificity with long-term disease control. Peptide-based cancer vaccines represent the most specific approach to polarize the immune system against malignant cells, since they are preparations made of single epitopes, the minimal immunogenic region of an antigen. Despite the strong rational, the promising preclinical results and the frequent induction of antigen-specific immune responses, peptide-based cancer vaccines have yielded relatively poor results in the clinical setting, a phenomenon likely due to the immunosuppressive properties of the tumor microenvironment that allow malignant cells to evade both naturally occurring and therapeutically induced immune surveillance. Nevertheless, advances in the engineering of peptides and in our understanding of the molecular mechanisms underlying an effective immune response against tumors have renewed the enthusiasm for peptide-based vaccination regimens in the setting of cancer, and a variety of clinical trials are being conducted based on the use of peptides. This review will describe the most recent insights in the rational design of peptide-based cancer vaccines, as well as the challenges to successful anticancer immunotherapy based on these short amino acid chains.

[Adjuvant chemotherapy after radical liver resection in the treatment of metastases from colorectal carcinoma]. / Ruolo della chemioterapia adiuvante dopo resezione epatica radicale nel trattamento delle metastasi da carcinoma del colon-retto.

Liver metastases are the leading cause of death in patients with colorectal carcinoma: approximately 25% present with metastases at diagnosis of the primary tumor and 30-50% will eventually develop metastases. Surgical therapy for metastases is the only curative treatment that will ensure five-year survival in 30-60% of patients; however, in 30-50% of these patients liver disease will recur. To improve these rates, various different studies have investigated the efficacy of postsurgical adjuvant therapy. The majority of randomized studies evaluated the efficacy of intra-arterial infusion associated or not with postsurgical systemic adjuvant treatment: this approach demonstrated benefit in terms of control of recurrent of liver disease but not in terms of overall survival. A reduction in the recurrence of liver disease was found in the two randomized studies published to date on the efficacy of systemic adjuvant therapy, and an improvement in survival in one trial. Given these data and the results obtained with the use of last generation chemotherapeutic agents (oxaliplatin and irinotecan) in the treatment of unresectable liver metastases from colorectal carcinoma, it can be conjectured that ongoing randomized clinical trials may confirm a significant advantage of adjuvant chemotherapy in the control of recurrence of liver disease and overall survival.

Subarachnoid anesthesia for loco-regional antiblastic perfusion with circulatory block (stop-flow perfusion).

AIM: Loco-regional antiblastic perfusion with circulatory block (stop-flow perfusion, SFP) is a procedure designed to treat solid tumors of the limb and pelvis in an advanced stage, like melanoma, sarcoma of the soft tissues and colon-rectal cancer. The aim of this study was to evaluate if subarachnoid anesthesia could represent a safe and suitable anesthetic technique for this procedure. METHODS: Thirty SFP procedures were performed in the angiographic room, 15 for the treatment of lower-limb neoplasias and 15 for pelvic neoplasias. The patients (ASA I-III) had a mean age of 59.1 years (range: 19-81 years). The patients were given different dosages of bupivacaine (range: 10-20 mg) in hyperbaric solution at the concentration of 0.5% and 1% by lumbar subarachnoid injection at different levels (from T12-L1 to L3-L4). Standard monitoring was set up (ECG, pulse-oximetry, and non-invasive artery pressure). The use of any anesthetic and analgesic drug, eventually used in the intra- or postoperative period, was recorded. RESULTS: The lumbar puncture was approached at L1-L2 and L2-L3 levels in 80% of the cases. Doses of bupivacaine between 12 mg and 14 mg were administered in 2/3 of the cases. Bupivacaine was formulated in hyperbaric solution and administered at a concentration of 0.5% (8 patients) or 1% (22 patients). Complica-tions related to the anesthetic technique were absent. Intraoperative pain control was almost complete with one exception, when the procedure lasted unusually long. Pain control was satisfying immediately after the procedure as well: only in 3 cases were non-opiod analgesics administered within the first 6 h. CONCLUSIONS: Spinal subarachnoid anesthesia has proven to be an effective, safe, and easy-to-manage technique for carrying out SFP procedure in a non-conventional environment such as an angiographic room. It was free of serious side effects and well tolerated even in patients in poor general conditions.

120 peritoneal carcinomatoses from colorectal cancer treated with peritonectomy and intra-abdominal chemohyperthermia: a S.I.T.I.L.O. multicentric study.

A multicentric study has been carried out on 120 patients affected by peritoneal carcinomatosis from colorectal cancer. Patients have been treated by cytoreductive surgery and intra-operative hyperthermic chemoperfusion (HIPEC) with cisplatin (CDDP) and mitomycin-c (MMC). A small group of patients were treated with oxaliplatin (LOHP) following the Elias et al. scheme [intravenous 5-fluorouracil (400 mg/m2) and leucovorin (20 mg/m2) followed by intraperitoneal perfusion with LOHP (460 mg/m2) in 2 l/m2, during 30 min at 43 degrees C]. CC-0 cytoreduction was achieved in 85.2% of the patients. Major morbidity and mortality was 22.5% and 3.3%, respectively. No G4 toxicity was registered. The three-year survival was 25.8%. The difference in survival evaluating complete cytoreduction (CC-0) vs. incomplete (CC1-2; residual tumor nodules greater than 2.5 mm) was statistically significant (p < 0.0001). Evaluating only the patients that could be cytoreduced to CC-0, the 3-year survival was raised to 33.5%. In our experience the peritoneal cancer index (PCI) has been demonstrated to be a weak prognostic factor reaching a statistical significance only after the exclusion of patients with resected hepatic metastases. The patients treated with oxaliplatin were alive and free-of-disease after a 16-month median follow-up.

IL-10 stimulatory effects on human NK cells explored by gene profile analysis.

The molecular mechanisms underlying the increase of natural killer (NK) cell anticancer activity mediated by interleukin (IL)-10 have not been elucidated. The aim of this study was to identify potential molecular mediators of IL-10 stimulatory effects by exploring the NK cell gene display induced by this cytokine. Gene profile was determined by high-throughput cDNA microarray and quantitative real-time PCR. In vitro, NK cells resting or conditioned with IL-10 were tested for cytotoxicity, migration and proliferation. IL-10 enhanced mRNA levels of cell activation/cytotoxicity-related genes (eg secretogranin, TIA-1, HMG-1, interferon-inducible genes) not upregulated by IL-2. In line with these findings, IL-10 increased NK cell in vitro cytotoxicity against Daudi cells. Unlike IL-2, IL-10 did not show any significant effect on NK cell in vitro proliferation and migration. However, gene profile analysis showed that IL-10 increased the expression of cell migration-related genes (eg L-selectin, vascular endothelium growth factor receptor-1, plasminogen activator, tissue; formyl peptide receptor, lipoxin A4 receptor), which might support a stimulatory effect not evident with the in vitro functional assay. Overall, gene profiling allowed us to formulate new hypotheses regarding the molecular pathways underlying the stimulatory effects of IL-10 on NK cells, supporting further investigation aimed at defining its role in cancer immune rejection.

Optimized procedure of real-time systemic leakage monitoring during isolated limb perfusion using a hand held gamma probe and 99mTc-HSA.

Isolated limb perfusion (ILP) therapy using a combination of tumour necrosis factor alpha (TNF) and cytostatic agents in hyperthermic conditions has proven to be effective in treating cancers limited to limbs or to a single organ such as the liver. A critical step for ILP is the accurate and real-time monitoring of that TNF toxic effects become relevant when overcoming the 10% limit of the 'effective' therapeutic dose administered during ILP. The most diffuse procedure for systemic leakage monitoring is based on the utilization of human soluble serum albumin (HSA) labelled with 131I and an external scintillation detector. In order to overcome some drawbacks connected with the properties of 131I, we developed a new procedure based on the utilization of HSA labelled with 99mTc in combination with a hand held gamma probe used as a detector. Our procedure consists of the following steps: (1) a 99mTc-HSA dose standardized as 0.5 MBq x kg(-1) body weight is injected into the ILP circuit before TNF administration; (2) a hand held gamma probe is placed over the pre-cordial area in a zone pre-marked on the patient's skin during a simulation test; (3) 48-72 h before ILP, a simulation test is obtained using a 99mTc-HSA dose corresponding to 10% of the dose calculated for ILP (i.e., 0.05 MBq x kg(-1) body weight); (4) during the simulation test the maximum count-rate zone detected on the pre-cordial area is marked on patient's skin; (5) a 60 min time-activity curve corresponding to the circulating 99mTc-HSA radioactivity effective decay is calculated and fitted; and (6) this time-activity curve is used to compensate for the leakage systemic counting observed during ILP. In order to compare the external, probe counting with the circulating radioactivity, in the first 10 patients from a total series of 43 treated patients, the results of external, probe monitoring were compared with the results of patient blood and perfusion circuit samples taken simultaneously every 5 min and measured by a laboratory gamma counter placed in the operating theatre. A good correlation was found between the two methods (R2 = 0.965, P < 0.01). It is concluded that the proposed procedure, based on the combination of 99mTc-HSA as the radiotracer and a hand held gamma probe as the detector, appears to be technically simple and accurate enough in the real-time monitoring of perfusion leakage in ILP cancer therapy. Moreover, using 99mTc-HSA as the radiotracer, the risk of radioactive contamination is significantly lower in comparison with 131I-HSA.

Hyperthermic isolated liver perfusion for unresectable liver cancers: pilot study.

Isolated hepatic perfusion (IHP) is a recently reconsidered locoregional approach for unresectable primary or metastatic cancer and encouraging results have been achieved from its clinical application. Ten patients underwent hyperthermic IHP with melphalan. There was no intraoperative mortality. In the postoperative period two patients died due to multi-organ failure. Four patients had significant but transient hepatic toxicity. In 8 assessable patients, the overall response rate was 63%. We observed objective tumor regression in a significant percentage of patients refractory to standard treatments. Locoregional toxicity was significant, which underscores the need for a more accurate preoperative evaluation of hepatic function.

Hyperthermic isolated perfusion with low-dose TNFalpha and doxorubicin in patients with locally advanced soft tissue limb sarcomas.

We report here the results of 27 patients who underwent hyperthermic isolated limb perfusion with low-dose TNFalpha (1 mg) and doxorubicin (8.5 mg/l of limb volume) for locally advanced soft tissue sarcomas. A tumor response was observed in 85% of cases. After a median follow-up of 30 months, limb salvage and local disease control were achieved in 82 and 85% of patients, respectively. Locoregional toxicity was low or mild in 14 patients, while 2 patients had severe limb toxicity. Systemic side effects were negligible. The perfusate/plasma area under the curve (AUC) ratio for TNFalpha was 56. HILP with low-dose TNFalpha and DXR proved to be an active neoadjuvant drug regimen against limb-threatening STS.

[Indications for integrated surgical treatment of peritoneal carcinomatosis of colorectal origin: experience of the Italian Society of Locoregional Integrated Therapy in Oncology]. / Indicazioni al trattamento chirurgico integrato delle carcinosi peritoneali di origine colorettale: esperienza della società Italiana di terapie integrate locoregionali in oncologia.

A multicentric prospective study has been carried on 69 patients affected by peritoneal carcinomatosis from colorectal cancer. Patients have been treated by cytoreductive surgery and intraoperative hyperthermic chemoperfusion. CC 0-1 has been achieved in 82%. Major morbidity and mortality was respectively 21.7% and 2.9%. Three years overall survival was 26.7% for all series. Difference in survival evaluating CC 0-1 vs CC 2 patients and PCI < or = 10 vs > 10 was statistically significant. Evaluating only patients CC 0-1 and PCI < or = 10 overall survival rised up to 44.7% at 4 years. A smaller subgroup of patients with a disease-free interval to peritoneal carcinomatosis > or = 2-year showed a 50% disease-free survival at 5 years. In conclusion PCI < or = 10, complete or optimal cytoreduction feasibility and disease-free interval have to be considered for the patients selection to the integrate treatment.

Phase I-II study on isolation antiblastic fotemustine perfusion after dacarbazine chemosensitization for advanced melanoma of the extremities.

Isolation limb perfusion (ILP) is the treatment of choice for locally advanced limb melanoma. With melphalan, the referral drug, complete response (CR) is achieved in about 50% of patients, but significant local toxicity occurs in up to 30%. The aim of the present phase I-II study was to challenge fotemustine (F) in ILP after systemic chemosensitization with dacarbazine (DTIC), given its lower toxicity and greater efficacy, as reported in a previous pilot study. Eleven patients with locally advanced limb melanoma were subdivided into triplets, and given F ILP at escalating doses (starting from 25 mg/l) after intravenous administration of 500 mg/m2 DTIC. Acute and chronic locoregional and systemic toxicity, tumour response and clinical outcome were evaluated. Two patients in the first triplet had G3-G4 local toxicity, so that the scheduled F dosage was halved. At drug levels of 12.5, 15.6 and 18.2 mg/l, local toxicity decreased, but only one of eight patients showed CR. The trial was then interrupted due to the low tolerability and poor efficacy of this perfusion regimen. At present, F ILP after DTIC chemosensitization should not be recommended for the treatment of locally advanced limb melanoma.

Sitilo experience on peritoneal carcinomatosis from colorectal cancer: clinical prognostic features.

UNLABELLED: A multicentric prospective study has been carried on 69 patients affected by peritoneal carcinomatosis from colorectal cancer. Patients have been treated by cytoreductive surgery and intraoperative hyperthermic chemoperfusion. CC 0-1 has been achieved in 82%. Major morbidity and mortality was 21.7% and 2.9% respectively. Three-yrs overall survival was 26.7% for all series. Difference in survival evaluating CC 0-1 vs. CC 2 pts and PCI < or = 10 vs. >10 was statistically significant. Evaluating only patients CC 0-1 and PCI < or = 10 4-yrs overall survival rised up to 44.7%. A smaller subgroup of patients with a disease-free interval to peritoneal carcinomatosis > or = 2-yrs showed a 5-yrs disease-free survival of 50%. CONCLUSIONS: PCI < or = 10, complete or optimal cytoreduction feasibility have to be considered for the patients selection to the integrate treatment. Disease-free interval seems to be a powerful prognostic indicator and deserve to be better outlined in further studies.

TNF-based limb perfusion for cutaneous melanoma in transit metastases: suggestions for modification of the perfusional schedule.

Isolated limb perfusion (ILP) is currently considered the standard treatment for melanoma patients with extensive in-transit disease, and L-PAM, combined or not with TNF, represents the most active drug. We here report on our clinical experience with TNF-based limb perfusion. Thirty-seven stage III patients underwent TNF-based limb perfusion, 22 with bulky disease, 15 with recurrences after perfusion with L-PAM. Ten patients were enrolled in a phase I-II study and treated with escalating doses of TNF (0.5-3 mg). The impact of disease burden, temperature, perfusion duration was assessed on tumor response. No postoperative death was observed. No significant systemic toxicity was recorded. Locoregional toxicity was G5 in one patient, G3 in 2, G2 in 9 and G1 in 25. Twenty-four (66%) patients had complete response, 11 (31%) partial and 1 (3%) no change. After a median follow-up of 20 months 14 (38%) patients are NED, 10 (27%) are AWD and 13 (35%) DOD. No significant statistical difference for tumor response were seen for disease burden, ILP temperatures and duration. Our results showed that it is possible to modify the perfusion schedule, without compromising the response rate but with lower cost and toxicity.

Treatment options for liver metastases from colorectal cancer.

The liver is the most common site of distant metastasis from colorectal cancer. About one-fourth of patients with liver metastases from colorectal cancer have no other sites of metastasis. Surgical resection of colorectal cancer liver metastases can result in a 24%-38% five-year survival. However, only a minority of patients are candidates for resection and no adjuvant treatment has been demonstrated to be effective in increasing the survival rate following radical surgery. For unresectable disease, several treatments have been tested in the clinical setting, although none of them can be currently considered a standard approach. Systemic chemotherapy is not generally considered a treatment for cure, although newer regimens appear to improve median survival. Locoregional therapies such hepatic intra-arterial chemotherapy and isolated hepatic perfusion may be offered to patients with unresectable liver metastases in the absence of extra-hepatic disease. However, the efficacy of these treatments is still being determined. Both systemic and locoregional chemotherapy might be useful in the neoadjuvant setting to increase the resectability of liver metastases initially not amenable to surgical resection.