Engineering CD276/B7-H3-targeted antibody-drug conjugates with enhanced cancer-eradicating capability.

CD276/B7-H3 represents a promising target for cancer therapy based on widespread overexpression in both cancer cells and tumor-associated stroma. In previous preclinical studies, CD276 antibody-drug conjugates (ADCs) exploiting a talirine-type pyrrolobenzodiazepine (PBD) payload showed potent activity against various solid tumors but with a narrow therapeutic index and dosing regimen higher than that tolerated in clinical trials using other antibody-talirine conjugates. Here, we describe the development of a modified talirine PBD-based fully human CD276 ADC, called m276-SL-PBD, that is cross-species (human/mouse) reactive and can eradicate large 500-1,000-mm3 triple-negative breast cancer xenografts at doses 10- to 40-fold lower than the maximum tolerated dose. By combining CD276 targeting with judicious genetic and chemical ADC engineering, improved ADC purification, and payload sensitivity screening, these studies demonstrate that the therapeutic index of ADCs can be substantially increased, providing an advanced ADC development platform for potent and selective targeting of multiple solid tumor types.

A Phase I Dose-escalation Study of AZD3965, an Oral Monocarboxylate Transporter 1 Inhibitor, in Patients with Advanced Cancer.

PURPOSE: Inhibition of monocarboxylate transporter (MCT) 1-mediated lactate transport may have cytostatic and/or cytotoxic effects on tumor cells. We report results from the dose-escalation part of a first-in-human trial of AZD3965, a first-in-class MCT1 inhibitor, in advanced cancer. PATIENTS AND METHODS: This multicentre, phase I, dose-escalation and dose-expansion trial enrolled patients with advanced solid tumors or lymphoma and no standard therapy options. Exclusion criteria included history of retinal and/or cardiac disease, due to MCT1 expression in the eye and heart. Patients received daily oral AZD3965 according to a 3+3 then rolling six design. Primary objectives were to assess safety and determine the MTD and/or recommended phase II dose (RP2D). Secondary objectives for dose escalation included measurement of pharmacokinetic and pharmacodynamic activity. Exploratory biomarkers included tumor expression of MCT1 and MCT4, functional imaging of biological impact, and metabolomics. RESULTS: During dose escalation, 40 patients received AZD3965 at 5-30 mg once daily or 10 or 15 mg twice daily. Treatment-emergent adverse events were primarily grade 1 and/or 2, most commonly electroretinogram changes (retinopathy), fatigue, anorexia, and constipation. Seven patients receiving ≥20 mg daily experienced dose-limiting toxicities (DLT): grade 3 cardiac troponin rise (n = 1), asymptomatic ocular DLTs (n = 5), and grade 3 acidosis (n = 1). Plasma pharmacokinetics demonstrated attainment of target concentrations; pharmacodynamic measurements indicated on-target activity. CONCLUSIONS: AZD3965 is tolerated at doses that produce target engagement. DLTs were on-target and primarily dose-dependent, asymptomatic, reversible ocular changes. An RP2D of 10 mg twice daily was established for use in dose expansion in cancers that generally express high MCT1/low MCT4).

Cancer cell survival depends on collagen uptake into tumor-associated stroma.

Collagen I, the most abundant protein in humans, is ubiquitous in solid tumors where it provides a rich source of exploitable metabolic fuel for cancer cells. While tumor cells were unable to exploit collagen directly, here we show they can usurp metabolic byproducts of collagen-consuming tumor-associated stroma. Using genetically engineered mouse models, we discovered that solid tumor growth depends upon collagen binding and uptake mediated by the TEM8/ANTXR1 cell surface protein in tumor-associated stroma. Tumor-associated stromal cells processed collagen into glutamine, which was then released and internalized by cancer cells. Under chronic nutrient starvation, a condition driven by the high metabolic demand of tumors, cancer cells exploited glutamine to survive, an effect that could be reversed by blocking collagen uptake with TEM8 neutralizing antibodies. These studies reveal that cancer cells exploit collagen-consuming stromal cells for survival, exposing an important vulnerability across solid tumors with implications for developing improved anticancer therapy.

The safety and tolerability of pirfenidone for bronchiolitis obliterans syndrome after hematopoietic cell transplant (STOP-BOS) trial.

Bronchiolitis obliterans syndrome (BOS) is the most morbid form of chronic graft-versus-host disease (cGVHD) after hematopoietic cell transplantation (HCT). Progressive airway fibrosis leads to a 5-year survival of 40%. Treatment options for BOS are limited. A single arm, 52-week, Phase I study of pirfenidone was conducted. The primary outcome was tolerability defined as maintaining the recommended dose of pirfenidone (2403 mg/day) without a dose reduction totaling more than 21 days, due to adverse events (AEs) or severe AEs (SAEs). Secondary outcomes included pulmonary function tests (PFTs) and patient reported outcomes (PROs). Among 22 participants treated for 1 year, 13 (59%) tolerated the recommended dose, with an average daily tolerated dose of 2325.6 mg/day. Twenty-two SAEs were observed, with 90.9% related to infections, none were attributed to pirfenidone. There was an increase in the average percent predicted forced expiratory volume in 1 s (FEV1%) of 7 percentage points annually and improvements in PROs related to symptoms of cGVHD. In this Phase I study, treatment with pirfenidone was safe. The stabilization in PFTs and improvements in PROs suggest the potential of pirfenidone for BOS treatment and support the value of a randomized controlled trial to evaluate the efficacy of pirfenidone in BOS after HCT. The study is registered in ClinicalTrials.gov (NCT03315741).

Effect of oxaliplatin plus 5-fluorouracil or capecitabine on circulating and imaging biomarkers in patients with metastatic colorectal cancer: a prospective biomarker study.

BACKGROUND: Patients with metastatic colorectal cancer are treated with cytotoxic chemotherapy supplemented by molecularly targeted therapies. There is a critical need to define biomarkers that can optimise the use of these therapies to maximise efficacy and avoid unnecessary toxicity. However, it is important to first define the changes in potential biomarkers following cytotoxic chemotherapy alone. This study reports the impact of standard cytotoxic chemotherapy across a range of circulating and imaging biomarkers. METHODS: A single-centre, prospective, biomarker-driven study. Eligible patients included those diagnosed with colorectal cancer with liver metastases that were planned to receive first line oxaliplatin plus 5-fluorouracil or capecitabine. Patients underwent paired blood sampling and magnetic resonance imaging (MRI), and biomarkers were associated with progression-free survival (PFS) and overall survival (OS). RESULTS: Twenty patients were recruited to the study. Data showed that chemotherapy significantly reduced the number of circulating tumour cells as well as the circulating concentrations of Ang1, Ang2, VEGF-A, VEGF-C and VEGF-D from pre-treatment to cycle 2 day 2. The changes in circulating concentrations were not associated with PFS or OS. On average, the MRI perfusion/permeability parameter, Ktrans, increased in response to cytotoxic chemotherapy from pre-treatment to cycle 2 day 2 and this increase was associated with worse OS (HR 1.099, 95%CI 1.01-1.20, p = 0.025). CONCLUSIONS: In patients diagnosed with colorectal cancer with liver metastases, treatment with standard chemotherapy changes cell- and protein-based biomarkers, although these changes are not associated with survival outcomes. In contrast, the imaging biomarker, Ktrans, offers promise to direct molecularly targeted therapies such as anti-angiogenic agents.

Differential activity and expression of human 5ß-reductase (AKR1D1) splice variants.

Steroid hormones, including glucocorticoids and androgens, exert a wide variety of effects in the body across almost all tissues. The steroid A-ring 5ß-reductase (AKR1D1) is expressed in human liver and testes, and three splice variants have been identified (AKR1D1-001, AKR1D1-002, AKR1D1-006). Amongst these, AKR1D1-002 is the best described; it modulates steroid hormone availability and catalyses an important step in bile acid biosynthesis. However, specific activity and expression of AKR1D1-001 and AKR1D1-006 are unknown. Expression of AKR1D1 variants were measured in human liver biopsies and hepatoma cell lines by qPCR. Their three-dimensional (3D) structures were predicted using in silico approaches. AKR1D1 variants were overexpressed in HEK293 cells, and successful overexpression confirmed by qPCR and Western blotting. Cells were treated with either cortisol, dexamethasone, prednisolone, testosterone or androstenedione, and steroid hormone clearance was measured by mass spectrometry. Glucocorticoid and androgen receptor activation were determined by luciferase reporter assays. AKR1D1-002 and AKR1D1-001 are expressed in human liver, and only AKR1D1-006 is expressed in human testes. Following overexpression, AKR1D1-001 and AKR1D1-006 protein levels were lower than AKR1D1-002, but significantly increased following treatment with the proteasomal inhibitor, MG-132. AKR1D1-002 efficiently metabolised glucocorticoids and androgens and decreased receptor activation. AKR1D1-001 and AKR1D1-006 poorly metabolised dexamethasone, but neither protein metabolised cortisol, prednisolone, testosterone or androstenedione. We have demonstrated the differential expression and role of AKR1D1 variants in steroid hormone clearance and receptor activation in vitro. AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues. In addition, AKR1D1-001 and AKR1D1-006 may have a limited, steroid-specific role in the regulation of dexamethasone action.

Brief report on the clinical characteristics of patients whose samples generate small cell lung cancer circulating tumour cell derived explants.

INTRODUCTION: Small cell lung cancer (SCLC) has a dismal prognosis. Circulating tumour cells (CTCs) can be used to generate CTC derived explants (CDX) for the study of SCLC biology and the development of novel therapeutics. We investigated whether there are demographic or clinical predictors of the success of CDX generation, and whether CDX models are representative of the SCLC patient population. METHODS: This was a single centre, retrospective analysis of SCLC patients who had participated in the CHEMORES Study. Paired blood samples were donated for CTC enumeration and CDX generation attempt at pre-treatment baseline, disease progression and intervening timepoints. Clinical and demographic data was collected from electronic records, and analysed for differences between patients whose samples did and did not generate a CDX. RESULTS: 231 paired blood samples were taken from 147 patients. 45 CDX were generated from 34 patients. CTC number was significantly higher in blood samples which successfully generated a CDX than those which didn't, at both baseline (p=<0.0001) and progression (p = 0.0001). The group with successful blood samples had a poorer performance status (p = 0.0067), and a higher proportion of patients with chemorefractory disease (p = 0.0077). Both progression free survival (PFS) (p = 0.0132) and overall survival (p=< 0.0001) were significantly shorter for patients with successful samples. CONCLUSIONS: Patients whose samples generate CDX models may have a higher disease burden and more aggressive disease. Thus, insights gained by study of SCLC CDX may have a significant impact, particularly in the SCLC subpopulation with the greatest clinical need.

Tumor stroma-targeted antibody-drug conjugate triggers localized anticancer drug release.

Although nonmalignant stromal cells facilitate tumor growth and can occupy up to 90% of a solid tumor mass, better strategies to exploit these cells for improved cancer therapy are needed. Here, we describe a potent MMAE-linked antibody-drug conjugate (ADC) targeting tumor endothelial marker 8 (TEM8, also known as ANTXR1), a highly conserved transmembrane receptor broadly overexpressed on cancer-associated fibroblasts, endothelium, and pericytes. Anti-TEM8 ADC elicited potent anticancer activity through an unexpected killing mechanism we term DAaRTS (drug activation and release through stroma), whereby the tumor microenvironment localizes active drug at the tumor site. Following capture of ADC prodrug from the circulation, tumor-associated stromal cells release active MMAE free drug, killing nearby proliferating tumor cells in a target-independent manner. In preclinical studies, ADC treatment was well tolerated and induced regression and often eradication of multiple solid tumor types, blocked metastatic growth, and prolonged overall survival. By exploiting TEM8+ tumor stroma for targeted drug activation, these studies reveal a drug delivery strategy with potential to augment therapies against multiple cancer types.

Eradication of Tumors through Simultaneous Ablation of CD276/B7-H3-Positive Tumor Cells and Tumor Vasculature.

Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.

Challenges in enumeration of CTCs in breast cancer using techniques independent of cytokeratin expression.

INTRODUCTION: Given the current postulated plasticity between epithelial and mesenchymal states of migratory cancer cells the detection of non-epithelial CTCs is an important scientific and clinical goal. METHODS: We used the filtration-based ISET technology to enrich circulating tumour cells (CTCs) in early breast cancer blood samples and identify them using a morphology-based immunocytochemistry (ICC) approach. RESULTS: We found greater numbers of putative CTCs by this approach than by the cytokeratin-based CellSearch technology, but a high number of CTC false positives were identified in healthy volunteer samples which were not reduced in successive blood draws. Preliminary work using an oestrogen receptor (ER)-based multiplex ICC method in metastatic breast cancer ISET samples indicated a low number of ER+ CTCs even at this advanced stage. CONCLUSIONS: This work highlights the challenges in enumerating CTCs without conventional epithelial markers.

Molecular analysis of circulating tumor cells identifies distinct copy-number profiles in patients with chemosensitive and chemorefractory small-cell lung cancer.

In most patients with small-cell lung cancer (SCLC)-a metastatic, aggressive disease-the condition is initially chemosensitive but then relapses with acquired chemoresistance. In a minority of patients, however, relapse occurs within 3 months of initial treatment; in these cases, disease is defined as chemorefractory. The molecular mechanisms that differentiate chemosensitive from chemorefractory disease are currently unknown. To identify genetic features that distinguish chemosensitive from chemorefractory disease, we examined copy-number aberrations (CNAs) in circulating tumor cells (CTCs) from pretreatment SCLC blood samples. After analysis of 88 CTCs isolated from 13 patients (training set), we generated a CNA-based classifier that we validated in 18 additional patients (testing set, 112 CTC samples) and in six SCLC patient-derived CTC explant tumors. The classifier correctly assigned 83.3% of the cases as chemorefractory or chemosensitive. Furthermore, a significant difference was observed in progression-free survival (PFS) (Kaplan-Meier P value = 0.0166) between patients designated as chemorefractory or chemosensitive by using the baseline CNA classifier. Notably, CTC CNA profiles obtained at relapse from five patients with initially chemosensitive disease did not switch to a chemorefractory CNA profile, which suggests that the genetic basis for initial chemoresistance differs from that underlying acquired chemoresistance.

Circulating Tumor Cells Detected in the Tumor-Draining Pulmonary Vein Are Associated with Disease Recurrence after Surgical Resection of NSCLC.

Tumor recurrence after surgical resection of NSCLC obstructs long-term disease-free survival in approximately 50% of cases. Our data suggest that combining circulating tumor cell enumeration (as single cells or clusters) in tumor-draining pulmonary vein and peripheral blood (assessed by CellSearch) at the time of NSCLC surgery better identifies those patients at higher risk for lung cancer recurrence than does peripheral circulating tumor cell number alone.

Identification and Targeting of Long-Term Tumor-Propagating Cells in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a neuroendocrine lung cancer characterized by fast growth, early dissemination, and rapid resistance to chemotherapy. We identified a population of long-term tumor-propagating cells (TPCs) in a mouse model of SCLC. This population, marked by high levels of EpCAM and CD24, is also prevalent in human primary SCLC tumors. Murine SCLC TPCs are numerous and highly proliferative but not intrinsically chemoresistant, indicating that not all clinical features of SCLC are linked to TPCs. SCLC TPCs possess a distinct transcriptional profile compared to non-TPCs, including elevated MYC activity. Genetic and pharmacological inhibition of MYC in SCLC cells to non-TPC levels inhibits long-term propagation but not short-term growth. These studies identify a highly tumorigenic population of SCLC cells in mouse models, cell lines, and patient tumors and a means to target them in this most fatal form of lung cancer.

Cediranib or placebo in combination with cisplatin and gemcitabine chemotherapy for patients with advanced biliary tract cancer (ABC-03): a randomised phase 2 trial.

BACKGROUND: Cisplatin and gemcitabine is the standard first-line chemotherapy regimen for patients with advanced biliary tract cancer; expression of VEGF and its receptors is associated with adverse outcomes. We aimed to assess the effect of the addition of cediranib (an oral inhibitor of VEGF receptor 1, 2, and 3) to cisplatin and gemcitabine on progression-free survival. METHODS: In this multicentre, placebo-controlled, randomised phase 2 study, we recruited patients aged 18 years or older with histologically confirmed or cytologically confirmed advanced biliary tract cancer from hepatobiliary oncology referral centres in the UK. Patients were eligible if they had an ECOG performance status of 0-1 and an estimated life expectancy of longer than 3 months. Patients were given first-line cisplatin and gemcitabine chemotherapy (25 mg/m(2) cisplatin and 1000 mg/m(2) gemcitabine [on days 1 and 8 every 21 days, for up to eight cycles]) with either 20 mg oral cediranib or placebo once a day until disease progression. We randomly assigned patients (1:1) with a minimisation algorithm, incorporating the stratification factors: extent of disease, primary disease site, previous treatment, ECOG performance status, and centre. The primary endpoint was progression-free survival in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT00939848, and was closed on Sept 30, 2014; results of the final analysis for the primary endpoint are presented. FINDINGS: Between April 5, 2011, and Sept 28, 2012, we enrolled 124 patients (62 in each group). With a median follow-up of 12·2 months (IQR 7·3-18·5), median progression-free survival was 8·0 months (95% CI 6·5-9·3) in the cediranib group and 7·4 months (5·7-8·5) in the placebo group (HR 0·93, 80% CI 0·74-1·19, 95% CI 0·65-1·35; p=0·72). Patients who received cediranib had more grade 3-4 toxic effects than did patients who received placebo: hypertension (23 [37%] vs 13 [21%]; p=0·05), diarrhoea (eight [13%] vs two [3%]; p=0·05); platelet count decreased (ten [16%] vs four [6%]; p=0·09), white blood cell decreased (15 [24%] vs seven [11%]; p=0·06) and fatigue (16 [24%] vs seven [11%]; p=0·04). INTERPRETATION: Cediranib did not improve the progression-free survival of patients with advanced biliary tract cancer in combination with cisplatin and gemcitabine, which remains the standard of care. Although patients in the cediranib group had more adverse events, we recorded no unexpected toxic effects. The role of VEGF inhibition in addition to chemotherapy for patients with advanced biliary tract cancer remains investigational. FUNDING: Cancer Research UK and AstraZeneca Pharmaceuticals.

Comparison of biosimilar filgrastim with originator filgrastim for peripheral blood stem cell mobilization and engraftment in patients with multiple myeloma undergoing autologous stem cell transplantation.

BACKGROUND: Nivestim is a biosimilar approved for the same indications as Neupogen including the mobilization of autologous peripheral blood stem cells (PBSCs). The clinical efficacy and safety of Nivestim for this use have not been formally assessed in clinical trials. STUDY DESIGN AND METHODS: In our retrospective single-center study we compared variables of PBSC mobilization and engraftment of 60 patients mobilized with Nivestim to that of 38 patients mobilized with Neupogen. RESULTS: We found no difference between Nivestim and Neupogen in peripheral blood CD34+ at first leukapheresis (47 × 10(6) cells/L vs. 60 × 10(6) cells/L, p = 0.48) nor the total CD34+ collected (5.37 × 10(6)/kg vs. 4.59 × 10(6) /kg, p = 0.22). However, a difference in the median number of leukapheresis procedures (one vs. two, p = 0.0007) was observed. Eighty-one patients (51 Nivestim and 30 Neupogen mobilized) went on to transplantation. Median time to neutrophil engraftment (15 days vs. 13.5 days, p = 0.09) and platelet (PLT) engraftment (20 days vs. 18 days, p = 0.01) was longer in the Nivestim group. The significant delay in PLT engraftment did not, however, translate to increased PLT transfusions (two vs. three, p = 0.2) or impact significantly on hospitalization time for admissions within 30 days posttransplant (20 days vs. 18 days, p = .17). CONCLUSION: Nivestim is as effective as Neupogen for PBSC mobilization; however, its use was associated with a delay in PLT recovery. A prospective study should be conducted to confirm our findings.

Optimisation of immunofluorescence methods to determine MCT1 and MCT4 expression in circulating tumour cells.

BACKGROUND: The monocarboxylate transporter-1 (MCT1) represents a novel target in rational anticancer drug design while AZD3965 was developed as an inhibitor of this transporter and is undergoing Phase I clinical trials ( http://www.clinicaltrials.gov/show/NCT01791595 ). We describe the optimisation of an immunofluorescence (IF) method for determination of MCT1 and MCT4 in circulating tumour cells (CTC) as potential prognostic and predictive biomarkers of AZD3965 in cancer patients. METHODS: Antibody selectivity was investigated by western blotting (WB) in K562 and MDAMB231 cell lines acting as positive controls for MCT1 and MCT4 respectively and by flow cytometry also employing the control cell lines. Ability to detect MCT1 and MCT4 in CTC as a 4(th) channel marker utilising the Veridex™ CellSearch system was conducted in both human volunteer blood spiked with control cells and in samples collected from small cell lung cancer (SCLC) patients. RESULTS: Experimental conditions were established which yielded a 10-fold dynamic range (DR) for detection of MCT1 over MCT4 (antibody concentration 6.25 µg/mL; integration time 0.4 seconds) and a 5-fold DR of MCT4 over MCT 1 (8 µg/100 µL and 0.8 seconds). The IF method was sufficiently sensitive to detect both MCT1 and MCT4 in CTCs harvested from cancer patients. CONCLUSIONS: The first IF method has been developed and optimised for detection of MCT 1 and MCT4 in cancer patient CTC.

Circulating Tumor Cell Enumeration in a Phase II Trial of a Four-Drug Regimen in Advanced Colorectal Cancer.

BACKGROUND: Multidrug regimens are active against advanced colorectal cancer (ACRC). However, the increased toxicity requires the use of biomarkers to select the patients who will derive the most benefit. We assessed circulating tumor cells (CTCs) as a prognostic biomarker in patients treated with a 4-drug regimen. PATIENTS AND METHODS: A single-arm phase II trial (Erbitux Study of CPT11, Oxaliplatin, UFToral Targeted-therapy [eSCOUT]) was undertaken in patients with previously untreated KRAS wild-type ACRC using a regimen of irinotecan, oxaliplatin, and tegafur-uracil with leucovorin and cetuximab. Baseline CTCs were enumerated using CellSearch. The endpoints were an objective response rate (ORR) and overall survival (OS). We modeled our results and compared them with those modeled for the capecitabine, oxaliplatin, bevacizumab +/- cetuximab (CAIRO2) trial, stratifying patients a priori into low (< 3) and high (≥ 3) CTC groups. RESULTS: For 48 eligible patients, the best ORR from the 4-drug regimen was 71%, with a disease control rate of 98%. The median OS for patients with a high and low CTC count was 18.7 and 22.3 months (log-rank test, P = .038), respectively. In our modeled data, for patients with a low CTC count, no differences were found between the median OS in the eSCOUT trial and that in the CAIRO2 trial (22.2 vs. 22.0 months). However, for the high CTC group, a clinically relevant improvement was seen in median OS (eSCOUT vs. CAIRO2, 18.7 vs. 13.7 months; P = .001). CONCLUSION: These data are hypothesis generating-for patients with ACRC, stratification by CTC count can identify those who might benefit the most from an intensive 4-drug regimen, avoiding high-toxicity regimens in low CTC groups. This hypothesis warrants validation in a phase III biomarker-driven trial.

Circulating biomarkers in hepatocellular carcinoma.

PURPOSE: Our aims are to determine levels of circulating cellular and protein biomarkers in hepatocellular carcinoma (HCC) patients and to analyse any relationships with clinical parameters. METHODS: Fifty-four consenting patients were recruited. Circulating tumour cells (CTCs) were enumerated (by CellSearch) and characterised via filtration [by isolation by size of epithelial tumour cells (ISET)] with downstream immunohistochemistry (IHC). Glypican-3 (GPC3) expression in tumour biopsies and CTCs (by IHC) was compared, and levels of circulating caspase-cleaved and full-length cytokeratin 18 (CK18, measured using M30 and M65 ELISAs) were examined as a putative prognostic factor and marker of tumour burden. RESULTS: CTCs were identified in 14 out of 50 (28%) patients by CellSearch and in 19 out of 19 (100%) patients by ISET. The presence of GPC3-positive CTCs by ISET was 100% concordant with the presence of GPC3-positive cells in the original tumour (n = 5). No statistically significant correlations were observed between CTC number and clinical characteristics, although trends were noted between CTC subtypes, Child-Pugh score and tumour node metastasis stage. Serum M30 and M65 levels (as continuous variables) significantly correlated with overall survival (OS) in a univariate analysis (p = 0.003 and p < 0.001, respectively); M65 levels remained statistically significant in a multivariate analysis (p = 0.029). CONCLUSIONS: This is the first study to detect GPC3-positive CTCs in HCC, important for drug development with this target. The significant association of circulating CK18 with OS in HCC further exemplifies the utility of circulating biomarkers in cancer.

Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer.

Small-cell lung cancer (SCLC), an aggressive neuroendocrine tumor with early dissemination and dismal prognosis, accounts for 15-20% of lung cancer cases and ∼200,000 deaths each year. Most cases are inoperable, and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs), which are prevalent in SCLC, present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice, and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged, and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms.

Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells.

BACKGROUND: AZD3514 inhibits and down regulates the androgen receptor (AR) and has undergone clinical trials in prostate cancer. To provide proof-of-mechanism (POM) in patients, an immunohistochemistry (IHC) method for determination of AR in circulating tumour cells (CTC) was developed and validated. METHODS: After an assessment of specificity validation focused on intra- and inter-operator reproducibility utilising a novel modification of incurred sample reanalysis (ISR). ß-Content γ-confidence tolerance intervals (BCTI) and Cohen's Kappa (κ) were employed in statistical analysis of results. RESULTS: In a first set of IHC reproducibility experiments, almost perfect agreement was recorded (κ=0.94) when two different operators scored CTC as overall positive or negative for AR. However, BCTI analysis identified a specific bias in scoring staining intensity, where one operator favoured moderate over strong assignments, whereas the reverse was the case with the second operator. After a period of additional training involving deployment of a panel of standardised images, a second set of validation experiments were conducted. These showed correction of the inter-operator bias by BCTI with κ for scoring intensity increasing from 0.59 to 0.81, indicative of almost perfect agreement. CONCLUSIONS: By application of BCTI to the validation of IHC, operator bias and therefore poor reproducibility can be identified, characterised and corrected to achieve a level of error normally associated with a quantitative biomarker assay, such as an ELISA. The methodological approach described herein can be applied to any generic IHC technique.