A colorectal cancer classification system that associates cellular phenotype and responses to therapy.

Colorectal cancer (CRC) is a major cause of cancer mortality. Whereas some patients respond well to therapy, others do not, and thus more precise, individualized treatment strategies are needed. To that end, we analyzed gene expression profiles from 1,290 CRC tumors using consensus-based unsupervised clustering. The resultant clusters were then associated with therapeutic response data to the epidermal growth factor receptor-targeted drug cetuximab in 80 patients. The results of these studies define six clinically relevant CRC subtypes. Each subtype shares similarities to distinct cell types within the normal colon crypt and shows differing degrees of 'stemness' and Wnt signaling. Subtype-specific gene signatures are proposed to identify these subtypes. Three subtypes have markedly better disease-free survival (DFS) after surgical resection, suggesting these patients might be spared from the adverse effects of chemotherapy when they have localized disease. One of these three subtypes, identified by filamin A expression, does not respond to cetuximab but may respond to cMET receptor tyrosine kinase inhibitors in the metastatic setting. Two other subtypes, with poor and intermediate DFS, associate with improved response to the chemotherapy regimen FOLFIRI in adjuvant or metastatic settings. Development of clinically deployable assays for these subtypes and of subtype-specific therapies may contribute to more effective management of this challenging disease.

In vivo evaluation of the anti-tumoral effect of sunitinib eluting beads in the rabbit VX2 tumor model

Purpose: To study the anti-tumoral effect of sunitinib eluting beads in the rabbit VX2 tumor modelMaterials: VX2 tumor were implanted in the left liver lobe of New-Zealand white rabbits. Seven animals received 0.2ml of DC Beads loaded with 6mg of sunitinb (group 1), 6 animals received 0.2ml of DC Beads (group 2) and 6 animals received NaCl 0.9% intra arterially in the left hepatic artery. One animal in each group was sacrificed at 24 hours and the others were left to survive. Liver enzyme were measured daily. In group 1 plasmatic sunitinib concentration were measured daily by LC MS/MS tandem mass spectroscopy. At day 15 all living animals were sacrficed. After sacrifice, or premature euthanasia the livers were harvested for determination of the VEGF receptor tyrosine kinase activity by western blot and histopathological examination.Results: In group 1, no animal died during follow-up. In group 2 and 3, respectively 2 and 3 animals died during follow-up. In group 1 plasmatic sunitinib level remained under therapeutic concentration during the whole experiment. There was an evident lack of phosphorylation of the RTK In group 1 and there was an augmentation of the RTK phosphorylation in group 2 at 24 hours. No difference in RTK activity was noticable at 15 days. From the histopathological point of view it was unpossible to differentiate treatment induced from spontaneous necrosis of tumors.Conclusions: Administration of sunitinib eluting Beads in VX2 carrying rabbits inhibits the activation of RTK's triggered by ischemia. It also seems to prolong survival of the treated animals.

Sorafenib fourth-line treatment in imatinib-, sunitinib-, and nilotinib-resistant metastatic GIST : a retrospective analysis : 10564

Background: Gastrointestinal stromal tumors (GISTs) are rare mesenchymal tumors usually caused by mutations in the KIT or PDGFRA gene. Advanced disease generally cannot be cured by surgery nor by tyrosine kinase inhibitors (TKI), but TKIs have considerably improved outcome for patients (pts) with advanced GIST. Patients failing TKI treatment with imatinib (IM), sunitinib (SU) or nilotinib (NI) have a poor prognosis. Sorafenib is a multi kinase inhibitor that blocks not only receptor tyrosine kinases such as KIT, VEGFR and PDGFR but also serine/threonine kinases along the RAS/RAF/MEK/ERK pathway. Recently, clinical activity of sorafenib in third-line treatment in patients with GIST after IM and SU failure has been shown (Wiebe et al. ASCO 2008, #10502). Methods: We report herein preliminary data of 32 pts treated with sorafenib in nine European centers. Centers were selected based on their previous and known experience in GIST and reported all pts treated. Pts received sorafenib after failure of IM, SU and NI in fourth-line treatment. Baseline characteristics and treatment details have been retrieved via questionary. Results: Median age at sorafenib treatment start was 62 years (range 33-81 y), and the majority of pts were male (63 %). Primary tumor site was gastric or small intestine in 25% and 41% of pts, respectively. All pts had failed IM, SU, NI. 19 % of pts achieved partial remission and 44% disease stabilization. Approximately half of the pts had an improvement of symptoms and/or performance. Half of the pts were on treatment longer than 4 months (actuarial data) and 41% of pts continue to receive sorafenib. Median progression-free survival is 20 weeks and median overall survival 42 weeks (Kaplan-Meier), at a median follow-up of 22 weeks (range 3-54). Conclusions: This is the largest series assessing efficacy of sorafenib fourth-line treatment for IM, SU and NI refractory GIST reported yet. Sorafenib displays significant clinical activity in this heavily pretreated group of patients.

Double seronegative myasthenia gravis with antiphospholipid syndrome: a case report.

INTRODUCTION: Myasthenia gravis is an autoimmune disease characterized by fluctuating muscle weakness. It is often associated with other autoimmune disorders, such as thyroid disease, rheumatoid arthritis, systemic lupus erythematosus, and antiphospholipid syndrome. Many aspects of autoimmune diseases are not completely understood, particularly when they occur in association, which suggests a common pathogenetic mechanism. CASE PRESENTATION: We report a case of a 42-year-old Caucasian woman with antiphospholipid syndrome, in whom myasthenia gravis developed years later. She tested negative for both antibodies against the acetylcholine receptor and against muscle-specific receptor tyrosine-kinase, but had typical decremental responses at the repetitive nerve stimulation testing, so that a generalized myasthenia gravis was diagnosed. Her thromboplastin time and activated partial thromboplastin time were high, anticardiolipin and anti-β2 glycoprotein-I antibodies were slightly elevated, as a manifestation of the antiphospholipid syndrome. She had a good clinical response when treated with a combination of pyridostigmine, prednisone and azathioprine. CONCLUSIONS: Many patients with myasthenia gravis test positive for a large variety of auto-antibodies, testifying of an immune dysregulation, and some display mild T-cell lymphopenia associated with hypergammaglobulinemia and B-cell hyper-reactivity. Both of these mechanisms could explain the occurrence of another autoimmune condition, such as antiphospholipid syndrome, but further studies are necessary to shed light on this matter.Clinicians should be aware that patients with an autoimmune diagnosis such as antiphospholipid syndrome who develop signs and neurological symptoms suggestive of myasthenia gravis are at risk and should prompt an emergent evaluation by a specialist.

Therapeutic drug monitoring in cancer - Are we missing a trick?

Therapeutic drug monitoring (TDM) can be defined as the measurement of drug in biological samples to individualise treatment by adapting drug dose to improve efficacy and/or reduce toxicity. The cytotoxic drugs are characterised by steep dose-response relationships and narrow therapeutic windows. Inter-individual pharmacokinetic (PK) variability is often substantial. There are, however, a multitude of reasons why TDM has never been fully implemented in daily oncology practice. These include difficulties in establishing appropriate concentration target, common use of combination chemotherapies and the paucity of published data from pharmacological trials. The situation is different with targeted therapies. The large interindividual PK variability is influenced by the pharmacogenetic background of the patient (e.g. cytochrome P450 and ABC transporters polymorphisms), patient characteristics such as adherence to treatment and environmental factors (drug-drug interactions). Retrospective studies have shown that targeted drug exposure correlates with treatment response in various cancers. Evidence for imatinib currently exists, others are emerging for compounds including nilotinib, dasatinib, erlotinib, sunitinib, sorafenib and mammalian target of rapamycin (mTOR) inhibitors. Applications for TDM during oral targeted therapies may best be reserved for particular situations including lack of therapeutic response, severe or unexpected toxicities, anticipated drug-drug interactions and concerns over adherence treatment. There are still few data with monoclonal antibodies (mAbs) in favour of TDM approaches, even if data showed encouraging results with rituximab and cetuximab. TDM of mAbs is not yet supported by scientific evidence. Considerable effort should be made for targeted therapies to better define concentration-effect relationships and to perform comparative randomised trials of classic dosing versus pharmacokinetically-guided adaptive dosing.

Continuous sunitinib treatment in patients with unresectable hepatocellular carcinoma (HCC): A multicenter phase II trial (SAKK 77/06 and SASL 23)

Background: Sunitinib (SU) is a multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenetic activity. Evidence for clinical activity in HCC was reported in 2 phase II trials [Zhu et al and Faivre et al, ASCO 2007] using either a 37.5 or a 50 mg daily dose in a 4 weeks on, 2 weeks off regimen. The objective of this trial was to demonstrate antitumor activity of continuous SU treatment in patients (pts) with HCC. Methods: Key eligibility criteria included unresectable or metastatic HCC, no prior systemic anticancer treatment, measurable disease and Child- Pugh A or B liver dysfunction. Pts received 37.5 mg SU daily until progression or unacceptable toxicity. The primary endpoint was progression free survival at 12 weeks (PFS12) defined as 'success' if the patient was alive and without tumor progression assessed by 12 weeks (±7 days) after registration. A PFS12 of _20% was considered uninteresting and promising if _40%. Using the Simon-two minimax stage design with 90% power and 5% significance the sample size was 45 pts. Secondary endpoints included safety assessments, measurement of serum cobalamin levels and tumor density. Results: From September 2007 to August 2008 45 pts, mostly male (87%), were enrolled in 10 centers. Median age was 63 years, 89% had Child-Pugh A and 47% had distant metastases. Median largest lesion diameter was 84mm (range: 18-280) and 18% had prior TACE. Reasons for stopping therapy were: PD 60%, symptomatic deterioration 16%, toxicity 11%, death 2% (due to tumor), and other reasons 4%; 7% remain on therapy. PFS12 was rated as success in 15 pts (33%) (95% CI: 20%, 49%) and failure in 27 (60%); 3 were not evaluable (due to refusal). Over the whole trial period 1 CR and 40% SD as best response were achieved. Median PFS, duration of disease stabilization, TTP and OS were 2.8, 3.2, 2.8 and 9.3 months, respectively. Grade 3 and 4 adverse events were infrequent and all deaths due to the tumor. Conclusions: Continuous SU treatment with 37.5 mg/d daily is feasible and demonstrates moderate activity in pts with advanced HCC and mild to moderately impaired liver dysfunction. Under this trial design the therapy is considered promising (>13 PFS12 successes).

Nilotinib in the treatment of advanced gastrointestinal stromal tumours resistant to both imatinib and sunitinib.

Patients diagnosed with advanced gastrointestinal stromal tumours (GISTs) who are resistant or intolerant to both imatinib and second-line sunitinib have a poor prognosis and few therapeutic options. We evaluated the efficacy of nilotinib, a novel tyrosine kinase inhibitor (TKI) in patients pretreated with imatinib and sunitinib. Fifty-two consecutive patients treated with oral nilotinib, 400mg twice daily, within the nilotinib compassionate use programme in 12 European cancer centres, were included in this retrospective analysis. Median age was 59 years (range 24-80), and all patients had WHO performance score better than 3. All patients had failed both imatinib and sunitinib pretreatment, either due to progressing GIST (96%) or intolerance (4%). Five patients (10%; 95% confidence interval (CI) 2-18) responded to nilotinib and 19 patients (37%; 95% CI 24-50) achieved a disease stabilisation. Nilotinib was generally well tolerated, but six patients (12%) discontinued treatment due to intolerance. Median progression-free survival of nilotinib treatment was 12 weeks (95% CI 9-15; range 0-104) and median overall survival was 34 weeks (95% CI 3-65; range 2-135). Nilotinib is active in GIST resistant to both imatinib and sunitinib. These results warrant further investigation of nilotinib in GIST.

Inhibition of the Kit Ligand/c-Kit Axis Attenuates Metastasis in a Mouse Model Mimicking Local Breast Cancer Relapse after Radiotherapy.

PURPOSE: Local breast cancer relapse after breast-saving surgery and radiotherapy is associated with increased risk of distant metastasis formation. The mechanisms involved remain largely elusive. We used the well-characterized 4T1 syngeneic, orthotopic breast cancer model to identify novel mechanisms of postradiation metastasis. EXPERIMENTAL DESIGN: 4T1 cells were injected in 20 Gy preirradiated mammary tissue to mimic postradiation relapses, or in nonirradiated mammary tissue, as control, of immunocompetent BALB/c mice. Molecular, biochemical, cellular, histologic analyses, adoptive cell transfer, genetic, and pharmacologic interventions were carried out. RESULTS: Tumors growing in preirradiated mammary tissue had reduced angiogenesis and were more hypoxic, invasive, and metastatic to lung and lymph nodes compared with control tumors. Increased metastasis involved the mobilization of CD11b(+)c-Kit(+)Ly6G(high)Ly6C(low)(Gr1(+)) myeloid cells through the HIF1-dependent expression of Kit ligand (KitL) by hypoxic tumor cells. KitL-mobilized myeloid cells homed to primary tumors and premetastatic lungs, to give rise to CD11b(+)c-Kit(-) cells. Pharmacologic inhibition of HIF1, silencing of KitL expression in tumor cells, and inhibition of c-Kit with an anti-c-Kit-blocking antibody or with a tyrosine kinase inhibitor prevented the mobilization of CD11b(+)c-Kit(+) cells and attenuated metastasis. C-Kit inhibition was also effective in reducing mobilization of CD11b(+)c-Kit(+) cells and inhibiting lung metastasis after irradiation of established tumors. CONCLUSIONS: Our work defines KitL/c-Kit as a previously unidentified axis critically involved in promoting metastasis of 4T1 tumors growing in preirradiated mammary tissue. Pharmacologic inhibition of this axis represents a potential therapeutic strategy to prevent metastasis in breast cancer patients with local relapses after radiotherapy. Clin Cancer Res; 18(16); 4365-74. ©2012 AACR.

Rôle de l'EGFR dans le cancer pulmonaire non à petites cellules [The role of EGFR in non-small cell lung carcinoma]

EGFR receptor is expressed on most of the non small cell lung carcinoma (NSCLC) cells. Its relative importance in oncogenesis and tumour progression seems to greatly vary among NSCLC. Two molecules targeting differently EGFR are currently used for the treatment of metastatic NSCLC. cetuximab, a monoclonal antibody directed against the extracellular domain of the receptor, leads to a moderate survival benefit when associated with standard first-line chemotherapy. Erlotinib, a small EGFR tyrosine-kinase inhibitor molecule is used in 2nd or 3rd treatment line. Predictive factors for efficiency of these new treatments are subjects of intense research, in order to allow a better selection of the patients who could benefit from such a strategy.

Sorafenib as third- or fourth-line treatment of advanced gastrointestinal stromal tumour and pretreatment including both imatinib and sunitinib, and nilotinib: A retrospective analysis.

BACKGROUND: Tyrosine kinase inhibitors (TKI) improve the outcome of patients with advanced gastrointestinal stromal tumour (GIST), but treatment failure is frequent, and prognosis then bleak. Smaller trials in this setting suggested activity for sorafenib, a multikinase inhibitor of receptor tyrosine kinases and RAF serine/threonine kinases. PATIENTS AND METHODS: We retrospectively evaluated the efficacy of sorafenib, starting dose 400mg twice daily, in a large community-based cohort of 124 patients treated in 12 European and one United States (U.S.) cancer centre. All but one patient had a WHO performance score 0-2. All had failed both imatinib and sunitinib, 68 patients nilotinib and 26 had failed investigational therapy, too. RESULTS: Twelve (10%) patients responded to sorafenib and 70 (57%) patients achieved disease stabilisation. Sorafenib was moderately tolerated, and toxicity reported in 56% of the patients. Rash, hand-foot-syndrome and diarrhea occurred frequently. Sorafenib dosage was reduced in a third of patients, but this did not have an impact on progression-free survival (PFS) (p=0.15). Median PFS was 6.4months (95% confidence interval [CI], 4.6-8.0months) and median overall survival (OS) 13.5months (95% CI, 10.0-21.0months). Patients with a good performance status and those who responded to sorafenib had a significant better PFS. CONCLUSION: We conclude that sorafenib is active in GIST resistant to imatinib, sunitinib and nilotinib. These results warrant further investigation of sorafenib or similar molecules in GIST.

Functional education of invariant NKT cells by dendritic cell tuning of SHP-1.

Invariant NKT (iNKT) cells play key roles in host defense by recognizing lipid Ags presented by CD1d. iNKT cells are activated by bacterial-derived lipids and are also strongly autoreactive toward self-lipids. iNKT cell responsiveness must be regulated to maintain effective host defense while preventing uncontrolled stimulation and potential autoimmunity. CD1d-expressing thymocytes support iNKT cell development, but thymocyte-restricted expression of CD1d gives rise to Ag hyperresponsive iNKT cells. We hypothesized that iNKT cells require functional education by CD1d(+) cells other than thymocytes to set their correct responsiveness. In mice that expressed CD1d only on thymocytes, hyperresponsive iNKT cells in the periphery expressed significantly reduced levels of tyrosine phosphatase SHP-1, a negative regulator of TCR signaling. Accordingly, heterozygous SHP-1 mutant mice displaying reduced SHP-1 expression developed a comparable population of Ag hyperresponsive iNKT cells. Restoring nonthymocyte CD1d expression in transgenic mice normalized SHP-1 expression and iNKT cell reactivity. Radiation chimeras revealed that CD1d(+) dendritic cells supported iNKT cell upregulation of SHP-1 and decreased responsiveness after thymic emigration. Hence, dendritic cells functionally educate iNKT cells by tuning SHP-1 expression to limit reactivity.

Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease.

The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity, mitochondrial antiviral signaling protein (MAVS) and TRIF, a phosphatase involved in growth factor signaling, T-cell protein tyrosine phosphatase (TC-PTP), and the E3 ubiquitin ligase component UV-damaged DNA-binding protein 1 (DDB1). Here we explored quantitative proteomics to identify novel cellular substrates of the NS3-4A protease. Cell lines inducibly expressing the NS3-4A protease were analyzed by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. This approach identified the membrane-associated peroxidase GPx8 as a bona fide cellular substrate of the HCV NS3-4A protease. Cleavage by NS3-4A occurs at Cys 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic HCV. Overexpression and RNA silencing studies revealed that GPx8 is involved in viral particle production but not in HCV entry or RNA replication. Conclusion: We provide proof-of-concept for the use of quantitative proteomics to identify cellular substrates of a viral protease and describe GPx8 as a novel proviral host factor targeted by the HCV NS3-4A protease. (Hepatology 2014;59:423-433).

Identification of HMX1 target genes: a predictive promoter model approach.

PURPOSE: A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway's function, we sought to identify the target genes. METHODS: We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. RESULTS: The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. CONCLUSIONS: Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1.

Combinations of vascular endothelial growth factor pathway inhibitors with metronomic chemotherapy: Rational and current status.

Chemotherapy given in a metronomic manner can be administered with less adverse effects which are common with conventional schedules such as myelotoxicity and gastrointestinal toxicity and thus may be appropriate for older patients and patients with decreased performance status. Efficacy has been observed in several settings. An opportunity to improve the efficacy of metronomic schedules without significantly increasing toxicity presents with the addition of anti-angiogenic targeted treatments. These combinations rational stems from the understanding of the importance of angiogenesis in the mechanism of action of metronomic chemotherapy which may be augmented by specific targeting of the vascular endothelial growth factor (VEGF) pathway by antibodies or small tyrosine kinase inhibitors. Combinations of metronomic chemotherapy schedules with VEGF pathway targeting drugs will be discussed in this paper.