The primary aim of this clinical trial was to determine the feasibility of delivering first-generation CAR T cell therapy to patients with advanced, CEACAM5+ malignancy. Secondary aims were to assess clinical efficacy, immune effector function and optimal dose of CAR T cells. Three cohorts of patients received increasing doses of CEACAM5+-specific CAR T cells after fludarabine pre-conditioning plus systemic IL2 support post T cell infusion. Patients in cohort 4 received increased intensity pre-conditioning (cyclophosphamide and fludarabine), systemic IL2 support and CAR T cells. No objective clinical responses were observed. CAR T cell engraftment in patients within cohort 4 was significantly higher. However, engraftment was short-lived with a rapid decline of systemic CAR T cells within 14 days. Patients in cohort 4 had transient, acute respiratory toxicity which, in combination with lack of prolonged CAR T cell persistence, resulted in the premature closure of the trial. Elevated levels of systemic IFNγ and IL-6 implied that the CEACAM5-specific T cells had undergone immune activation in vivo but only in patients receiving high-intensity pre-conditioning. Expression of CEACAM5 on lung epithelium may have resulted in this transient toxicity. Raised levels of serum cytokines including IL-6 in these patients implicate cytokine release as one of several potential factors exacerbating the observed respiratory toxicity. Whilst improved CAR designs and T cell production methods could improve the systemic persistence and activity, methods to control CAR T ‘on-target, off-tissue’ toxicity are required to enable a clinical impact of this approach in solid malignancies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-017-2034-7) contains supplementary material, which is available to authorized users.

Array comparative genomic hybridization, with a genome-wide resolution of ϳ1 Mb, has been used to investigate copy number changes in 48 colorectal cancer (CRC) cell lines and 37 primary CRCs. The samples were divided for analysis according to the type of genomic instability that they exhibit, microsatellite instability (MSI) or chromosomal instability (CIN). Consistent copy number changes were identified, including gain of chromosomes 20, 13, and 8q and smaller regions of amplification such as chromosome 17q11.2-q12. Loss of chromosome 18q was a recurrent finding along with deletion of discrete regions such as chromosome 4q34-q35. The overall pattern of copy number change was strikingly similar between cell lines and primary cancers with a few obvious exceptions such as loss of chromosome 6 and gain of chromosomes 15 and 12p in the former. A greater number of aberrations were detected in CIN؉ than MSI؉ samples as well as differences in the type and extent of change reported. For example, loss of chromosome 8p was a common event in CIN؉ cell lines and cancers but was often found to be gained in MSI؉ cancers. In addition, the target of amplification on chromosome 8q appeared to differ, with 8q24.21 amplified frequently in CIN؉ samples but 8q24.3 amplification a common finding in MSI؉ samples. A number of genes of interest are located within the frequently aberrated regions, which are likely to be of importance in the development and progression of CRC.

Loss of chromosome 18q21 is well documented in colorectal cancer, and it has been suggested that this loss targets the DCC, DPC4͞ SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-␤ signaling pathway, in colorectal cancer has been highlighted, although the frequency of SMAD4 mutations appears much lower than that of 18q21 loss. We set out to investigate allele loss, mutations, protein expression, and cytogenetics of chromosome 18 copy number in a collection of 44 colorectal cancer cell lines of known status with respect to microsatellite instability (MSI). Fourteen of thirty-two MSI ؊ lines showed loss of SMAD4 protein expression; usually, one allele was lost and the other was mutated in one of a number of ways, including deletions of various sizes, splice site changes, and missense and nonsense point mutations (although no frameshifts). Of the 18 MSI ؊ cancers with retained SMAD4 expression, four harbored missense mutations in the 3 part of the gene and showed allele loss. The remaining 14 MSI ؊ lines had no detectable SMAD4 mutation, but all showed allele loss at SMAD4 and͞or DCC. SMAD4 mutations can therefore account for about 50 -60% of the 18q21 allele loss in colorectal cancer. No MSI ؉ cancer showed loss of SMAD4 protein or SMAD4 mutation, and very few had allelic loss at SMAD4 or DCC, although many of these MSI ؉ lines did carry TGFBIIR changes. Although SMAD4 mutations have been associated with late-stage or metastatic disease, our combined molecular and cytogenetic data best fit a model in which SMAD4 mutations occur before colorectal cancers become aneuploid͞polyploid, but after the MSI ؉ and MSI ؊ pathways diverge. Thus, MSI ؉ cancers may diverge first, followed by CIN ؉ (chromosomal instability) cancers, leaving other cancers to follow a CIN ؊ MSI ؊ pathway. For many years the allele loss observed around 18q21.1 in colorectal cancer was believed to be targeting DCC (2, 3). It has more recently been shown, however, that mutations of DCC and SMAD2 are very rare in colorectal cancer (4). By contrast, changes in SMAD4 are much more common in the pathogenesis and evolution of colorectal cancer (5, 6), as has also been shown in pancreatic cancer (7). In particular, inactivation of SMAD4 has been associated with late stage or metastatic colorectal cancer (6,8,9). Additionally, it has recently been shown that constitutional mutations in SMAD4 can cause Juvenile Polyposis Syndrome (JPS; refs. 10 and 11), a disorder characterized by the presence of hamartomatous polyps in the gastrointestinal tract and a markedly increased risk of developing a gastrointestinal malignancy.The SMAD4 gene codes for a protein involved as a downstream regulator in the transforming growth factor ␤ (TGF-␤) signal transduction pathway. The SMAD4 protein acts as a trimer and forms complexes with the receptor-phosphorylated SMAD2 and SMAD3; these heteromeric complexes then translocate from the cytoplasm to the nucleus where association with DNA binding factors facilitates the transcription of ...

Background:Rucaparib is an orally available potent selective small-molecule inhibitor of poly(ADP-ribose) polymerase (PARP) 1 and 2. Rucaparib induces synthetic lethality in cancer cells defective in the homologous recombination repair pathway including BRCA-1/2. We investigated the efficacy and safety of single-agent rucaparib in germline (g) BRCA mutation carriers with advanced breast and ovarian cancers.Methods:Phase II, open-label, multicentre trial of rucaparib in proven BRCA-1/2 mutation carriers with advanced breast and or ovarian cancer, WHO PS 0–1 and normal organ function. Intravenous (i.v.) and subsequently oral rucaparib were assessed, using a range of dosing schedules, to determine the safety, tolerability, dose-limiting toxic effects and pharmacodynamic (PD) and pharmacokinetic (PK) profiles.Results:Rucaparib was well tolerated in patients up to doses of 480 mg per day and is a potent inhibitor of PARP, with sustained inhibition ⩾24 h after single doses. The i.v. rucaparib (intermittent dosing schedule) resulted in an objective response rate (ORR) of only 2% but with 41% (18 out of 44) patients achieved stable disease for ⩾12 weeks and 3 patients maintaining disease stabilisation for >52 weeks. The ORR for oral rucaparib (across all six dose levels) was 15%. In the oral cohorts, 81% (22 out of 27) of the patients had ovarian cancer and 12 out of 13, who were dosed continuously, achieved RECIST complete response/partial response (CR/PR) or stable disease (SD) ⩾12 weeks, with a median duration of response of 179 days (range 84–567 days).Conclusions:Rucaparib is well tolerated and results in high levels of PARP inhibition in surrogate tissues even at the lowest dose levels. Rucaparib is active in gBRCA-mutant ovarian cancer and this activity correlates with platinum-free interval. The key lessons learned from this study is that continuous rucaparib dosing is required for optimal response, the recommended phase 2 dose (RP2D) for continuous oral scheduling has not been established and requires further exploration and, thirdly, the use of a PD biomarker to evaluate dose–response has its limitations.