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Building upon the concept of schedule-specific biochemical modulation of 5-fluorouracil (FU), which alternates bolus and continuous infusion (CI) FU, we have incorporated oxaliplatin (l-OHP) following the bolus part of the regimen to explore the activity of this new combination. Patients with advanced, untreated, measurable colorectal cancer received sequential methotrexate (MTX) (days 1 and 15)-->l-OHP+FU (days 2 and 16) (200, 85 and 600 mg m(-2), respectively) followed by 3 weeks of CI FU (200 mg m(-2) day(-1)) given from day 29 to 50, modulated by weekly leucovorin (LV) (20 mg m(-2)). After 1 week of rest, the second cycle was started. The treatment was continued until progression or patient's refusal. According to the intention-to-treat analysis on all 46 patients accrued, the response rate was 42% (95% CL=28-55%), with three complete responses and 16 partial responses. The median overall survival was 15.9 months and the median progression-free survival 6.9 months. Toxicity was very mild, with the bolus part of the regimen more toxic than the infusional part (24 vs 7% of grade III-IV, respectively). This new combination of MTX -->l-OHP-FU followed by FU CI is well tolerated, feasible and produces activity results similar to other more simple, but more toxic, regimens. Pros and cons of the different fluoropyrimidines-l-OHP combinations are discussed.

Aim: Six months FOLFOX4 or XELOX is the standard adjuvant chemotherapy in radically resected stage III colon cancer, however a shorter duration of therapy could be equally efficacious. Methods: This is an Italian, multicenter, non-inferiority phase III trial randomizing patients (pts) with stage III and high risk stage II colon cancer to receive 3 (arm 3) vs 6 (arm 6) months of FOLFOX4 or XELOX. Primary end-point is relapse free survival (RFS), and the arm 3 will be considered non-inferior if the superior margin of the 95% CI of the HR is Results: From June 2007 to March 2013, 3,759 patients were accrued and 3,738 (1860 in arm 3 and 1878 in arm 6) included in the intent-to-treat (ITT) population, since 21 pts were excluded due to major violations. The proportion of FOLFOX4 vs XELOX was 64% vs 36% in both arms. Among the ITT population, demographic factors (including gender and age) were balanced in the two arms. As expected, compliance was higher in arm 3 than in arm 6: treatment completion rate without any modification of planned dose and timing was 35% in arm 3 and 12% in arm 6, while approximately 55% pts in both arms completed treatment with modifications of dose and/or timing, resulting in 90% vs 67% pts receiving all the planned cycles. Therapy was permanently interrupted due to toxicity in 5% of pts in arm 3 and 17% in arm 6. The overall incidence of grade 3-4 toxicity (including grade 2 oxaliplatin neurotoxicity) events was 37% in arm 3 and 60% in arm 6. Grade 2+ neuropathy was more frequent in arm 6 than in arm 3: 32 % vs 9%, respectively. With a median follow-up of 30 months, 476 pts relapsed, 195 died and 520 relapsed and/or died. Conclusions: TOSCA is the first trial completing the accrual among the international initiative of comparing 3 vs 6 months of adjuvant treatment in colon cancer (IDEA). In general, reported toxicity was low, but, as expected, significantly higher in the 6-month arm compared to that of the 3-month arm; accordingly, compliance was lower in the standard 6-month arm. Data for efficacy analysis are premature. Disclosure: All authors have declared no conflicts of interest.

In this study, we show that NKRP1A is expressed and functions on a subset of immature human thymocytes. We took advantage of the monoclonal antibody (mAb) 191B8 that was obtained by immunizing mice with cultured human thymocytes characterized by an immature surface phenotype [CD2- CD3- CD4- CD8- stem cell factor receptor (SCFR)+] and expressing cytoplasmic CD3 epsilon chain. The 191B8 antibody homogeneously reacted with the immunizing population but not with most unfractionated thymocytes. It stained a minor population of resting immature thymocytes co-expressing CD34, SCFR, or both. Following culture of the CD34+ or CD34- fractions of CD2- CD3- CD4- CD8- purified immature thymocytes with recombinant interleukin-2 (rIL-2), the 191B8-defined antigen was expressed on virtually all cells even when 191B8+ cells were removed from the starting population. On the other hand, no 191B8+ cells were detected in fresh or cultured thymocytes expressing a more mature phenotype. Biochemical analysis of 191B8 mAb-reactive molecules revealed, under non-reducing conditions, two bands displaying apparent molecular masses of 80 and 44 kDa and a single band of 44 kDa under reducing conditions. Digestion with proteases indicated that the 80-kDa form represented a homodimeric form of two 44-kDa molecules, while deglycosylation with N-glycanase suggested the existence of four N-glycosylation sites. Transfection of COS7 or NIH3T3 cells with hNKRP1A cDNA showed that the 191B8 mAb recognized NKRP1A as shown by both immunofluorescence analysis and immunoprecipitation experiments. Functional studies showed that the 191B8/NKRP1A molecule mediated strong inhibition of the cytolytic activity of cultured CD2- CD3- immature thymocytes against a panel of tumor target cells. More importantly, 191B8 mAb induced proliferation of CD2- CD3- fresh thymocytes which was not increased by rIL-2. Thus, we propose that NKRP1A molecules, which are expressed in highly immature thymocytes, may play a regulatory role in their growth and function.

The effect of the guanosine triphosphate-binding protein (G-protein) inhibitors cholera toxin (Ctx) and pertussis toxin (Ptx) has been analyzed on lymphocyte function-associated antigen 1 (LFA-1)-dependent adhesion and signal transduction in human natural killer (NK) cells. Ctx, but not Ptx, inhibited the LFA-1-dependent adhesion of NK cells to tumor target cells which constitutively express the intercellular cell adhesion molecule-1 (ICAM-1) and to NIH/3T3 mouse fibroblasts stably transfected with human ICAM-1. This effect was detectable only by the use of the entire Ctx but not of the Ctx B subunit. In addition, Ctx could inhibit both NK cell binding and spreading to purified ICAM-1 protein. NK cell treatment with Ctx modified neither the surface expression of LFA-1 nor its Mg2+ binding site. These findings, together with the absence of any detectable effect of Ctx on the constitutive phosphorylation of LFA-1 alpha, suggests that this toxin modifies the avidity of LFA-1 for ICAM-1 by acting on LFA-1-cytoskeletal protein association. Unlike Ctx, Ptx did not affect NK cell adhesion. The effects of Ctx and Ptx are unlikely to depend on intracellular levels of cyclic adenosine 3',5'-monophosphate (cAMP), since a strong increase of cAMP was induced by both toxins. Moreover, this was confirmed by the observation that the LFA-1-dependent adhesion was not inhibited by the adenylate cyclase activator forskolin (FSK), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX), or both, which increase intracellular cAMP levels. Unlike the differential effect on cell adhesion, both the intracellular calcium [Ca2+]i increase and phosphoinositide breakdown mediated via LFA-1 were consistently inhibited in a dose-dependent manner by both Ctx and Ptx. Also in this case, the inhibitory effect did not depend on an increase of intracellular cAMP as indicated by NK cell treatment with FSK, IBMX, or both. Further evidence of the involvement of G-proteins in LFA-1-mediated signal transduction was the inhibitory effect of the GDP analog guanosine-5'-O-2-thiodiphosphate (GDP beta S) on LFA-1-mediated calcium mobilization. Taken together, our data provide evidence that the LFA-1-mediated NK cell adhesion and signal transduction are partially independent phenomena which may be regulated by different G-proteins.

3612 Background: BRAF mutation is widely recognised as a strong negative prognostic factor in advanced CRC, while the prognostic value of KRAS mutations in codons 12-13 remains controversial. Exploring mutations in other downstream components of the EGFR pathway may have an impact on survival. Methods: A consecutive cohort of 201 metastatic CRC patients treated with systemic chemotherapy were analysed for KRAS (12-13-61-146), BRAF, PIK3CA and NRAS genotypes by pyrosequencing on PyroMarkTMQ96 ID instrument (Qiagen, Germany) with commercially available kits Anti-EGFR MoAb response (Diatech Pharmacogenetics, Italy). Accurate microdissection guaranteed more than 70% of cancer cells for each sample. For the purpose of the survival analysis 4 categories were created: (1) KRAS mutated (codons 12-13 only); (2) BRAF mutated; (3) any of KRAS codons 61-146, PIK3CA or NRAS mutations; (4) all-wild type. Log-rank and Cox proportional tests were applied for statistical analysis. Results: KRAS mutations were present in 96 (47.8%) patients: 86 (42.8%) were in codons 12-13. BRAF mutations were found in 11 (5.5%) samples while PIK3CA and NRAS in 33 (16.4%) and 7 (3.5%), respectively. All mutations were mutually exclusive except for 24 (11.9%) patients with concomitant KRAS/PIK3CA mutations. Median survivals for different categories are shown. Patients harbouring BRAF mutation had the worst outcome (p=0.0006). Mutations of any codon of KRAS (12-13-61-146) also negatively impacted on survival (p=0.026), while the all wild-type (KRAS/BRAF/PIK3CA/NRAS) patients had the longest survival (p=0.002). Conclusions: This study suggests the usefulness for early molecular profiling for advanced CRC patients. Mutational analysis of all EGFR pathway components may identify different prognostic subgroups. This information may drive treatment selection in clinical practice and stratification in clinical trials. [Table: see text]

As shown recently, CD3+/TcR+ functional T lymphocytes can be derived in culture from embryonic liver cell precursors at a gestational age (6-8 weeks) preceding the colonization of the epithelial thymus. In this report, we analyzed the V beta repertoire of T lymphocytes derived from embryonic liver by applying a quantitative reverse transcriptase-polymerase chain reaction technique. To this end, oligonucleotide primers for C alpha or the various human V beta have been used to study both freshly derived embryonic liver cell suspensions and CD3+/TcR+ populations derived after approximately 6 weeks upon stimulation with 1% phytohemagglutinin and culture in 100 units/ml recombinant interleukin-2. In order to exclude possible contaminations with mother-derived T lymphocytes, only T cells displaying both X and Y chromosomal sequences (i.e. derived from male embryos) were further analyzed. While neither C alpha nor the various V beta could be detected in fresh liver cells, C alpha and the large majority of V beta were detected in in vitro cultured populations. The levels of the various V beta expressed by embryo-derived T cells was similar to that detected in adult peripheral blood-derived T lymphocytes. These experiments indicate that the immature liver precursors can potentially give rise in vitro to T cells which express a wide V beta repertoire and may provide a suitable in vitro system for the analysis of the selection processes mediated by either major histocompatibility complex antigen or superantigens.

The TA218 and T205 monoclonal antibodies (mAb) were selected on the basis of their ability to inhibit the non-major histocompatibility complex-restricted lysis of the murine mastocytoma P815 cell line mediated by CD3-CD16+ natural killer (NK) cells. Both mAb were found to react with CD45 molecules, as demonstrated by immunoprecipitation after surface iodination and western blot analysis. A panel of tumor target cells susceptible to lysis by polyclonal or clonal CD3-CD16+ NK cells was used to study the mAb-mediated inhibitory effect. The inhibition of cytolysis mediated by TA218 and T205 mAb was found to consistently parallel the inhibition mediated (with the same tumor target cells) by the anti-LFA1 alpha mAb TS.1.22 or by the anti-LFA1 beta mAb TS.1.18. However, different from the anti-LFA1 mAb, T205 or TA218 mAb did not inhibit the binding of activated CD3-CD16+ effector NK cells to the same tumor target cells. This finding supported the concept that the anti-CD45 mAb-mediated inhibition could occur at a post-binding stage. In polyclonal or clonal CD3-CD16+ NK cells T205 or TA218 mAb were found to reduce by 50-70% the intracellular Ca++ ([Ca++]i) mobilization induced by anti-LFA1 alpha or anti-LFA1 beta mAb. On the other hand, TA218 and T205 mAb did not inhibit the Ca++ mobilization induced by anti-CD16 mAb or phytohemagglutinin, thus suggesting that, in NK cells, CD45 molecules may exert a selective inhibitory effect on the signal transduction mediated by LFA1 molecules. In line with this hypothesis, the cytolytic activity of human NK clones was triggered in the presence of the hybridoma cells secreting either anti-CD16 or anti-LFA1 alpha mAb (as "triggering targets"). This effect of anti-LFA1 alpha, but not of anti-CD16 hybridoma was susceptible to inhibition by the anti-CD45 mAb T205 or TA218. Further, experiments on cloned NK cells indicated that T205 or TA218 mAb induced a strong decrease in the constitutive phosphorylation of the LFA1 alpha chain (but not of HLA class I antigens). Taken together, these studies suggest that in human NK lymphocytes, CD45 molecule may regulate both the activation state and the function of the LFA1 molecule.

e17555 Background: Costs of cancer care account for a growing proportion of European health care spending. Despite the rising price of new cancer drugs draws the attention of medical oncologists and decision makers, personnel spending takes the largest share of health care costs. Nonetheless, few data exist about the relationship between workload and human resources demand in a cancer unit. Here we describe an empirical model to determine staffing requirements according to the annual number of patients. Methods: The Department of Oncology at the University Hospital of Udine, Italy, is a computerized Unit within the regional cancer network taking care of about 1,500 pts/year. Each clinical episode is recorded on a centralized database. Standard time limits are set for different types of consultation. We queried the database to obtain the total number of consultations for every pt taken charge of by the Unit during 2006. Considering both the time scheduled for each type of visit and the number of yearly working hours per employee, we could reach a reasonable estimate of the annual physician and nurse hours per pt and of the number of needed personnel. Results: In 2006, each case generated an average of 16 clinical evaluations, with small differences among breast, lung and colorectal cancer. Corresponding physician- and nurse-time per pt was of 8 and 16 hours, respectively. For the first year - in Units with a mean number of 1,500 pts/year - this translates into a global amount of about 12,000 physician- and 24,000 nurse-hours and a needed number of 7 physicians and 13 nurses, respectively. In the second year, the same cases induced a mean of 4.5 consultations; using a similar approach, the demand for additional time and resources could be estimated as well. Conclusions: A preliminary assessment of the time required for different types of consultation, together with a centralized recording of each clinical episode, allows a reasoned estimate of needed time and personnel resources, thus providing a simple tool to determine the total staff expenditure of a cancer unit. No significant financial relationships to disclose.

The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells.

Purified CD3-4- thymocyte populations were cultured in the presence of interleukin-2 (IL-2) and peripheral blood lymphocytes (PBL) and/or tumor cell lines as a source of irradiated feeder cells. Maximal cell proliferation was obtained in the presence of a mixture of H9 leukemic cells and normal PBL. More importantly, under these culture conditions, 30%-50% of these cells were found to express CD16 surface antigen after 1-2 weeks of culture. Similar proportions of CD16+ cells could be detected in CD3-4- thymocyte populations that had been further depleted of CD16+ cells. Cloning of CD3-4-16- thymocytes under limiting dilution conditions resulted, in the presence of H9 cells, in more than 50% of CD16+ clones (cloning efficiency 3%-8%). Since some of the surface CD3- clones expressed cytoplasmic CD3 antigen, it has been possible to identify four distinct phenotypic groups of clones (CD16+cyCD3+, CD16+cyCD3-, CD16-cyCD3+, CD16-cyCD3-). Independently of their phenotype, all thymus-derived CD3- clones expressed a strong cytolytic activity against natural killer (NK)-sensitive and NK-resistant tumour target cells. In addition, following stimulation with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (or PHA alone) all clones released interferon-gamma and tumour necrosis factor-alpha, but not IL-2. Taken together, our data provide evidence that cells which share their phenotypic and functional properties with CD3-CD16+ NK cells can be derived from thymic precursors.

Highly purified CD1-3-4-8- human thymocytes were obtained by panning techniques combined with cell depletion with antibody-coated magnetic beads. Most of these cells expressed cytoplasmic CD3 antigen, as assessed by mAbs known to react with the CD3 epsilon chain. After culture with low doses of PMA (0.5 ng/ml) and subsequent addition (at 24 h) of recombinant interleukin 2 (rIL-2; 100 U/ml) cells underwent extensive proliferation (40-60-fold of the initial cell input after 2 wk). The majority of the proliferating cells were CD3-TCR-. The remaining cells (5-40%) were represented by CD3+ TCR gamma/delta+ (BB3- A13+) cells. Further removal of CD3+ TCR-gamma/delta+ cells resulted in highly purified CD3- populations that further proliferated in culture with no substantial phenotypic changes. When CD3+ thymocytes were cultured under the same experimental conditions, only CD3+ TCR-alpha/beta+ cells could be detected, thus indicating that PMA did not affect the surface expression of the CD3/TCR complex, but rather induced preferential growth of CD3- thymocytes. Surface marker analysis of cultured CD3- thymocytes showed that they were homogeneously CD7+, whereas low proportions of cells expressed CD2 and CD8 antigens. Among the natural killer (NK) cell markers, CD56 was highly expressed by all cells, whereas CD16, CD57, CD11b, NKH2, and GL183 were absent. Importantly, these cells were different from peripheral NK cells, as 80-95% of them expressed cytoplasmic CD3 antigen. Functional analysis revealed a strong cytolytic activity against both NK-sensitive (K562) and NK-resistant (M14, Daudi) human target cells. In a redirected killing assay against the Fc gamma R+ P815 cells, mAbs specific for triggering molecules including CD3, CD2, and CD16 failed to augment target cell lysis, while a strong cytolytic effect was induced by PHA. In addition, PHA alone or in combination with PMA induced tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) (but not IL-2) production by CD3- thymocytes. Cloning of fresh CD1-3-4-8-thymocytes in the presence of PMA and rIL-2 resulted in CD3-CD56+ clones that displayed a pattern of cytolytic activity and lymphokine production similar to that of the polyclonal populations. Northern blot analysis of transcripts coding for CD3/TCR molecules revealed the presence of CD3 zeta, epsilon, and gamma transcripts, while CD3 delta was undetectable. Mature transcripts for both gamma and delta TCR chains could be detected, whereas no TCR-alpha mRNA and only a truncated (1.0 kb) form of TCR-beta mRNA were revealed.(ABSTRACT TRUNCATED AT 400 WORDS)

In this study we analyzed the effect of CK226 monoclonal antibody (mAb) on human B cell activation and proliferation. This mAb was shown to recognize a 75-kDa surface molecule expressed on both T and B lymphocytes and to mediate T lymphocyte activation and proliferation. Flow cytometry analysis of B cell populations isolated from peripheral blood, tonsil and spleen showed that CK226 surface antigen is highly expressed on 40-80% of surface Ig+ cells. When purified B cells were cultured in the presence of CK226 mAb, up-regulation of major histocompatibility complex class II and CD23 surface structures and the de novo expression of CD25 antigen could be detected within 48 h. In addition, B cells underwent proliferation ([3H] thymidine uptake) in the absence of either T cells or exogenous lymphokines. Proliferation was potentiated by the addition of suboptimal concentrations (0.5 ng/ml) of phorbol 12-myristate 13-acetate (PMA). Cells recovered at day 5 were surface Ig+ and no CD3+ cells could be detected. CK226-induced proliferation (either in the presence or in the absence of PMA) was not inhibited by anti-CD25 mAb. Addition of exogenous interleukin 2 to CK226-stimulated B cells resulted in further increase of B cell proliferation. On the other hand, CK226 mAb did not display a co-stimulatory effect with submitogenic concentrations of either anti-Ig antibody or Staphylococcus aureus Cowan strain I bacteria. In addition proliferation induced by mitogenic concentrations of the above stimuli was inhibited in a dose-dependent fashion by CK226 mAb.

14530 Background: Advances in systemic treatment for metastatic CRC confer survival advantage, allowing patients to reach a median survival of almost 2 years. As a consequence of this remarkable life extension, the incidence of BM from CRC, though still low, is increasing over time. The presence of BM is considered an index of poor prognosis, even when surgical resection is possible. Methods: To determine if an aggressive approach to BM from CRC could provide patients with clinical benefit or survival advantage, we retrospectively collected data from 29 CRC patients who underwent neurosurgical resection of BM between March 1998 and July 2006. Results: The median age at the time of surgical procedure was 65 years, median ECOG PS was 1, and the majority of patients (26 out of 29) had concomitant metastases at other sites. Lung and liver metastases were both common. Median number of previous chemotherapy regimens was two, with almost all of the patients being exposed both to oxaliplatinum and irinotecan-based regimens. After resection, 30 Gy of external whole-brain radiotherapy were administered to the majority of the patients. At the time of the analysis, 26 out of 29 patients had died, with a median survival time after brain metastasectomy of 210 days (8–682). Only 2 patients died within a month from surgery. We did observe prolonged survival for patients who received post-surgical radiotherapy (Kaplan Maier, p=0.033). Although death due to encephalic progression was reported for the patients with brain as the sole metastatic site, intracranial disease progression was not a clear predictor for poor survival in the whole cohort (p>0.05). Conclusions: Surgical resection of BM from CRC, whether followed by radiotherapy or not, is a feasible and safe technique, offering a chance of prolonged survival. Patients who received radiotherapy after complete neurosurgical resection have a better outcome. No significant financial relationships to disclose.

13556 Background: whenever surgical resection of liver metastases from colorectal cancer is not possible, thermal ablation is a mini-invasive local treatment that can be considered as an alternative approach. Complete thermal ablation can be achieved by laser treatment. When thermoablation is obtained by radiofrequency, patients treated for a small dominant lesion (less than 3 cm) experience a better outcome. This evidence is less clear when pLIT is used. Patients and Methods: 30 patients (22 with a single lesion, 8 with up to three metastases) were consecutively treated. Maximum diameter of the dominant lesion was less than 3 cm in 20 pts and more than 3 cm in ten. With mild sedation and local anaesthesia, optical fibers were inserted directly into the tumor with echo-guided percutaneous needle placement. Each optical fiber was connected to a neodymium:yttrium-aluminium-garnet (ND:YAG) laser, which delivers concentrated light at a wave-length of 1064 nm, with a 5-Watt power and a 1800-Joule energy per single fiber. A minimum of two and a maximum of four needles were used, with a 5 to 8 mm distance from one needle to another. Results: all patients tolerated LIT procedure well, without major complications. Post-treatment CT-scan demonstrated complete thermonecrosis in 39 out of 44 (87%) of the treated lesions, and almost complete in the remaining 5, 4 of which of diameter larger than 3 cm. Local failure was reported in 12% of the lesions at six-month follow-up. The median Kaplan-Meier survival for all patients was 607 days, with survival rate of 82% at one year and 55% at three years. Patients treated for a smaller dominant lesion had a significantly better survival than the others (850 vs 420 days, p=0.04, logrank). Conclusions: pLIT permitted in most cases a complete ablation of liver metastases with a high local tumor control rate and a low complication rate. Patients with a smaller dominant lesion do best after pLIT procedure. Local treatment coupled with systemic chemotherapy offers a chance of prolonged survival in patients not amenable to hepatic surgery. No significant financial relationships to disclose.

Background Emerging data suggest that sex-related immune system composition affect both immune response and efficacy of immunotherapy in cancer patients (pts). This study aimed to investigate the sex-related prognostic role of MLR in metastatic colorectal cancer (mCRC) pts. Methods We analyzed a retrospective consecutive cohort of 490 mCRC pts treated in 2004-2017 at the Oncology Departments of Aviano and Pordenone (training set) and Udine (validation set), Italy. Association analysis was explored by Chi-squared or Kruskal-Wallis test, as appropriate. The prognostic impact of MLR on overall survival (OS) was evaluated with uni- and multivariable Cox regression models. The best cut-off value to predict survival was defined through ROC analyses. Results Overall, we identified 288 males and 202 females; 161 pts (33%) had a right cancer and 324 (67%) a left one. Interestingly, sex was associated with MLR (p = 0.004). The obtained cut-off value for MLR in females and males was 0.27 and 0.49, respectively. At univariate analysis of training set, MLR >0.27 in females (HR 1.95, p = 0.003) and MLR >0.49 in males (HR 2.65, p = 0.010) were associated with poorer OS. Even in the validation set, MLR >0.27 in females (HR 2.21, p = 0.010) and MLR >0.49 in males (HR 2.99, p = 0.002) were associated with shorter OS. In the overall cohort, at univariate analysis MLR >0.27 in females (HR 2.07, p ≤ 0.001), MLR >0.49 in males (HR 2.87, p ≤ 0.001), KRAS mutation (HR 1.37 p = 0.008), BRAF mutation (HR 1.69 p = 0.009), sidedness (right vs left HR 1.59, p ≤ 0.001) and peritoneal metastases (HR 2.32, p ≤ 0.001) were associated with shorter OS. Instead, primary tumor resection (HR 0.37 p ≤ 0.001) was associated with prolonged OS. At multivariate analysis, MLR >0.27 in females (HR 2.77, p = 0.002), MLR >0.49 in males (HR 5.39, p ≤ 0.001), BRAF mutation (HR 3.38, p ≤ 0.001) and peritoneal metastases (HR 2.50, p = 0.003) were still independently associated with worse OS. Noteworthy, high MLR was more frequently found in females than in males (41% vs 9%). Conclusions Males and females have a different immune response. Our study showed that high MLR, both in males and females, is an unfavorable independent prognostic factor. Further prospective studies are needed to confirm these data. Legal entity responsible for the study IRCCS CRO National Cancer Institute, Aviano, Italy. Funding Has not received any funding. Disclosure F. Puglisi: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: Hoffmann-La Roche; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Amgen; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Celgene; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Eisai; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Eli Lilly; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Ipsen; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Pierre-Fabre; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Takeda; Advisory / Consultancy: Merck Sharp & Dohme. All other authors have declared no conflicts of interest.