Concept: A randomised multicentre trial of first line chemotherapy comparing three weekly cabazitaxel versus weekly paclitaxel in HER2 negative metastatic breast cancer.

BACKGROUND: Paclitaxel is commonly used as first-line chemotherapy for HER2-negative metastatic breast cancer (MBC) patients. However, with response rates of 21.5-53.7% and significant risk of peripheral neuropathy, there is need for better chemotherapy. PATIENTS AND METHODS: This open-label phase II/III trial randomised HER2-negative MBC patients 1:1 to either 6 cycles of three-weekly cabazitaxel (25 mg/m2), or, weekly paclitaxel (80 mg/m2) over 18 weeks. The primary endpoint was progression free survival (PFS). Secondary endpoints included objective response rate (ORR), time to response (TTR), overall survival (OS), safety and tolerability and quality of life (QoL). RESULTS: 158 patients were recruited. Comparing cabazitaxel to paclitaxel, median PFS was 6.7 vs 5.8 months (HR 0.87; 80%CI 0.70-1.08, P = 0.4). There was no difference in median OS (20.6 vs 18.2 months, HR 1.00; 95%CI 0.69-1.45, P = 0.99), ORR (41.8% vs 36.7%) or TTR (HR 1.09; 95%CI 0.68-1.75, P = 0.7). Grade ≥3 adverse events occurred in 41.8% on cabazitaxel and 46.8% on paclitaxel; the most common being neutropenia (16.5%) and febrile neutropenia (12.7%) cabazitaxel and neutropenia (8.9%) and lung infection (7.6%) paclitaxel. Peripheral neuropathy of any grade occurred in 54.5% paclitaxel vs 16.5% cabazitaxel. Mean EQ-5D-5L single index utility score (+0.05; 95%CI 0.004-0.09, P = 0.03) and visual analogue scale score (+7.7; 95%CI 3.1-12.3, P = 0.001) were higher in cabazitaxel vs paclitaxel. CONCLUSIONS: Three-weekly cabazitaxel in HER2-negative MBC does not significantly improve PFS compared to weekly paclitaxel, although it has a lower risk of peripheral neuropathy with better patient reported QoL outcomes. It is well tolerated and requires fewer hospital visits.

A Single-arm Phase II Trial of Neoadjuvant Cabazitaxel and Cisplatin Chemotherapy for Muscle-Invasive Transitional Cell Carcinoma of the Urinary Bladder.

INTRODUCTION: Neoadjuvant cisplatin-based combination chemotherapy improves survival in muscle-invasive bladder cancer. However, response rates and survival remain suboptimal. We evaluated the efficacy, safety, and tolerability of cisplatin plus cabazitaxel. METHODS: A phase II single-arm trial was designed to recruit at least 26 evaluable patients. This would give 80% power to detect the primary endpoint, an objective response rate defined as a pathologic complete response plus partial response (pathologic downstaging), measured by pathologic staging at cystectomy (p0 = 0.35 and p1 = 0.60, α = 0.05). RESULTS: Objective response was seen in 15 of 26 evaluable patients (57.7%) and more than one- third of patients achieved a pathologic complete response (9/26; 34.6%). Seventy-eight percent of the patients (21/27) completed all cycles of treatment, with only 6.7% of the reported adverse events being graded 3 or 4. There were 6 treatment-related serious adverse event reported, but no suspected unexpected serious adverse reactions. In the patients who achieved an objective response, the median progression-free survival and overall survival were not reached (median follow-up of 41.5 months). In contrast, the median progression-free survival (7.2 months) and overall survival (16.9 months) were significantly worse (P = .001, log-rank) in patients who did not achieve an objective response. CONCLUSION: Cabazitaxel plus cisplatin for neoadjuvant treatment of muscle-invasive bladder cancer can be considered a well-tolerated and effective regimen before definitive therapy with higher rates (57.7%) of objective response, comparing favorably to that with of cisplatin/gemcitabine (23%-26%). These results warrant further evaluation in a phase III study.

Intracellular cytarabine triphosphate in circulating blasts post-treatment predicts remission status in patients with acute myeloid leukemia.

Cytarabine remains the backbone of therapy in acute myeloid leukemia (AML). The ability to assess intracellular cytarabine triphosphate (ara-CTP) levels in patients receiving cytarabine represents a major goal in the prediction of treatment response. This study, conducted within a clinical setting, aimed to assess ara-CTP levels in circulating peripheral blasts from non-M3 AML patients receiving cytarabine at one of three dosing levels, using a novel biosensor assay. Results from the initial 72 hours post-commencement were correlated with day 28 remission status, with feasibility parameters concurrently assessed. Intracellular ara-CTP was detectable in ex vivo blasts post-treatment for standard-dose (SD) and high-dose (HD) patients (p < 0.05), and quantification revealed a 27-fold increase in intracellular steady-state concentration between the two dosing levels. For low-dose cytarabine, high rates of patient discharge and low intracellular concentrations limited analysis; however, assessment of intracellular ara-CTP concentration was achievable in a dwindling population of blasts for SD and HD treatment cohorts, with 4 hours post-treatment commencement potentially being most predictive of clinical response (r = -0.912, p = 0.0113). Concurrent assessment of peripheral leukemia-associated immunophenotype (LAIP)-positive cells revealed a decline in burden (0-72 hours), which correlated with remission status (p < 0.05). Unexpectedly high rates of night sampling led to challenges associated with sampling rates, but did not have an impact on patient compliance. Additional training of night staff improved feasibility substantially. Multiple peripheral sampling during the initial 72 hours of treatment is feasible in newly diagnosed patients, and ara-CTP is detectable over the initial 24 hours, facilitating prediction of chemosensitivity of leukemic blasts to cytarabine.

Insulin-like growth factor binding protein 2 (IGFBP-2) promotes growth and survival of breast epithelial cells: novel regulation of the estrogen receptor.

In breast tumors IGF binding protein-2 (IGFBP-2) is elevated, and the presence of IGFBP-2 has been shown to correlate with malignancy. However, how IGFBP-2 contributes to the malignant state is still unclear. Silencing IGFBP-2 blocked cell proliferation and in MCF-7 cells increased cell death, indicating that IGFBP-2 was acting in both a mitogenic and a survival capacity. Exogenous IGFBP-2 acting via integrin receptors to reduce phosphatase and tensin homolog deleted from chromosome 10 (PTEN) levels protected these cells against death induced by various chemotherapeutic agents. This was dependent on a functional estrogen receptor (ER)-α because silencing ER-α blocked the ability of IGFBP-2 to confer cell survival. Loss of IGFBP-2 increased levels of PTEN and improved chemosensitivity of the cells, confirming its role as a survival factor. Silencing IGFBP-2 had no effect on the response to IGF-II, but responses to estrogen and tamoxifen were no longer observed due to loss of ER-α, which could be prevented by the inhibition of PTEN. Conversely, exogenous IGFBP-2 increased ER-α mRNA and protein in both normal and cancer cells via its interaction with integrin receptors. These actions of IGFBP-2 on ER-α involved the IGF-I receptor and activation of phosphatidylinositol 3-kinase in the cancer cells but were independent of this in normal breast cells. The production of IGFBP-2 by breast cancer cells enhances their proliferative potential, increases their survival, and protects them against chemotherapy-induced death. IGFBP-2 not only modulates IGFs and directly regulates PTEN but also has a role in maintaining ER-α expression.

IGFBP-3 can either inhibit or enhance EGF-mediated growth of breast epithelial cells dependent upon the presence of fibronectin.

Progression of breast cancer is associated with remodeling of the extracellular matrix, often involving a switch from estrogen dependence to a dependence on EGF receptor (EGFR)/HER-2 and is accompanied by increased expression of the main binding protein for insulin-like growth factors (IGFBP-3). We have examined the effects of IGFBP-3 on EGF responses of breast epithelial cells in the context of changes in the extracellular matrix. On plastic and laminin with MCF-10A normal breast epithelial cells, EGF and IGFBP-3 each increased cell growth and together produced a synergistic response, whereas with T47D breast cancer cells IGFBP-3 alone had no effect, but the ability of EGF to increase cell proliferation was markedly inhibited in the presence of IGFBP-3. In contrast on fibronectin with MCF-10A cells, IGFBP-3 alone inhibited cell growth and blocked EGF-induced proliferation. With the cancer cells, IGFBP-3 alone had no effect but enhanced the EGF-induced increase in cell growth. The insulin-like growth factor-independent effects of IGFBP-3 alone on cell proliferation were completely abrogated in the presence of an EGFR, tyrosine kinase inhibitor, Iressa. Although IGFBP-3 did not affect EGFR phosphorylation [Tyr(1068)], it was found to modulate receptor internalization and was associated with activation of Rho and subsequent changes in MAPK phosphorylation. The levels of fibronectin and IGFBP-3 within breast tumors may determine their dependence on EGFR and their response to therapies targeting this receptor.

Structural insights into the interaction of insulin-like growth factor 2 with IGF2R domain 11.

The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.

Functional evaluation of novel soluble insulin-like growth factor (IGF)-II-specific ligand traps based on modified domain 11 of the human IGF2 receptor.

Ligands transported by the mannose 6-phosphate/insulin-like growth factor (IGF)-II receptor (IGF2R) include IGF-II- and mannose 6-phosphate-modified proteins. Increased extracellular supply of IGF-II, either secondary to loss of the clearance function of IGF2R, loss of IGF binding protein function, or increased IGF2 gene expression, can lead to embryonic overgrowth and cancer promotion. Reduced supply of IGF-II is detrimental to tumor growth, and this suggests that gain of function of IGF-II is a molecular target for human cancer therapy. Domain 11 of IGF2R binds IGF-II with high specificity and affinity. Mutagenesis studies have shown that substitution of glutamic acid for lysine at residue 1554 results in a 6-fold higher affinity for IGF-II (20.5 nmol/L) than native domain 11 (119 nmol/L). Here, we generate a novel high-affinity IGF-II ligand trap by fusion of mutated human 11(E1554K) to a COOH-terminal human IgG1 Fc domain (11(E1554K)-Fc). The resulting homodimer has a significantly increased affinity for IGF-II (1.79 nmol/L) when measured by surface plasmon resonance. IGF-II signaling via the IGF-I receptor and the proliferative effect of IGF-II were specifically inhibited by 11(E1554K)-Fc in both HaCaT and Igf2(-/-) mouse embryonic fibroblast cells. These data confirm that a novel engineered and soluble IGF2R-11(E1554K)-Fc protein functions as an IGF-II-specific and high-affinity ligand trap in vitro and that this protein has potential application as an IGF-II antagonist for cancer therapy following in vivo experimental evaluation.

How growth hormone may be linked to cancer: concerns and perspective.

Recent evidence from epidemiology indicates that inter-individual variations in the growth hormone (GH)/IGF-I pathway affect the risk of individuals developing common epithelial cancers. This is supported by associations between normal common variants within genes from the pathway and these cancers, which excludes many potential confounding issues, such as reverse causality. This raises concern for the increasing numbers of patients treated with GH; although replacement therapy for GH-deficiency should aim to restore normality, which should then only incur a normal risk. The links with cancer also offers promising new opportunities. Clinical trials treating cancer patients with pharmaceuticals targeting the IGF-I receptor are well advanced with promising initial findings. In the future, there has to be much more emphasis within oncology on prevention and the GH/IGF-I pathway is one of few identified risk factors that are modifiable, not just by pharmaceutical, but also nutritional, interventions that may, in the long term, be more appropriate. Assessing the status of the GH/IGF-I pathway in individuals may also provide a means for targeting screening programs and preventative measures.

Soluble IGF2 receptor rescues Apc(Min/+) intestinal adenoma progression induced by Igf2 loss of imprinting.

The potent growth-promoting activity of insulin-like growth factor-II (IGF-II) is highly regulated during development but frequently up-regulated in tumors. Increased expression of the normally monoallelic (paternally expressed) mouse (Igf2) and human (IGF2) genes modify progression of intestinal adenoma in the Apc(Min/+) mouse and correlate with a high relative risk of human colorectal cancer susceptibility, respectively. We examined the functional consequence of Igf2 allelic dosage (null, monoallelic, and biallelic) on intestinal adenoma development in the Apc(Min/+) by breeding with mice with either disruption of Igf2 paternal allele or H19 maternal allele and used these models to evaluate an IGF-II-specific therapeutic intervention. Increased allelic Igf2 expression led to elongation of intestinal crypts, increased adenoma growth independent of systemic growth, and increased adenoma nuclear beta-catenin staining. By introducing a transgene expressing a soluble form of the full-length IGF-II/mannose 6-phosphate receptor (sIGF2R) in the intestine, which acts as a specific inhibitor of IGF-II ligand bioavailability (ligand trap), we show rescue of the Igf2-dependent intestinal and adenoma phenotype. This evidence shows the functional potency of allelic dosage of an epigenetically regulated gene in cancer and supports the application of an IGF-II ligand-specific therapeutic intervention in colorectal cancer.

Isolation and validation of human prepubertal skeletal muscle cells: maturation and metabolic effects of IGF-I, IGFBP-3 and TNFalpha.

We have developed a primary skeletal muscle cell culture model derived from normal prepubertal children to investigate the effects of insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-3 (IGFBP-3) and tumour necrosis factor alpha (TNFalpha) on growth, differentiation and metabolism. Cells of myoblast lineage were characterized morphologically by desmin staining and differentiated successfully into multinucleated myotubes. Differentiation was confirmed biochemically by an increase in creatine kinase (CK) activity and IGFBP-3 secretion over time. IGF-I promoted whilst TNFalpha inhibited myoblast proliferation, differentiation and IGFBP-3 secretion. IGF-I partially rescued the cells from the inhibiting effects of TNFalpha. Compared to adult myoblast cultures, children's skeletal muscle cells demonstrated higher basal and day 7 CK activities, increased levels of IGFBP-3 secretion, diminished IGF-I/TNFalpha action and absence of the inhibitory effect of exogenous IGFBP-3 on differentiation. Additional studies demonstrated that TNFalpha increased basal glucose transport via GLUT1, nitric oxide synthase and p38MAPK-dependent mechanisms. These studies provide baseline data to study the interactivity effects of growth factors and cytokines on differentiation and metabolism in muscle in relation to important metabolic disorders such as obesity, type II diabetes or chronic wasting diseases.

Differential signalling mechanisms predisposing primary human skeletal muscle cells to altered proliferation and differentiation: roles of IGF-I and TNFalpha.

To gain a clearer insight into the mechanisms of skeletal muscle cell growth, differentiation and maintenance, we have developed a primary adult human skeletal muscle cell model. Cells were cultured from biopsies of rectus muscle from the anterior abdominal wall of patients undergoing elective surgery. Under differentiating conditions, all cultures formed myotubes, irrespective of initial myoblast number. Stimulation with both IGF-I and tumour necrosis factor alpha (TNFalpha) increased cellular proliferation but while IGF-I subsequently increased myoblast differentiation, via both hyperplasia and hypertrophy, TNFalpha inhibited the initiation of differentiation, but did not induce apoptosis. Addition of IGF-I stimulated both the MAP kinase and the phosphatidylinositide 3-kinase (PI 3-kinase) signalling pathways while treatment with TNFalpha preferentially led to MAP kinase activation although with a very different profile of activation compared to IGF-I. Data using the MEK inhibitor UO126 showed MAP kinase activity is not only needed for cellular proliferation but is also necessary for both the initiation and the progression of primary human myoblast differentiation. The PI 3-kinase pathway is also involved in differentiation, but activation of this pathway could not relieve inhibition of differentiation by TNFalpha or UO126. Our results show that the controlled temporal and amplitude of activation of multiple signalling pathways is needed for successful myoblast differentiation.

Role of insulin-like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human adult skeletal myoblasts.

Although muscle satellite cells were identified almost 40 years ago, little is known about the induction of their proliferation and differentiation in response to physiological/pathological stimuli or to growth factors/cytokines. In order to investigate the role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in adult human myoblast differentiation we have developed a primary human skeletal muscle cell model. We show that under low serum media (LSM) differentiating conditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5 was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis. IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially by proteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer of cells into LSM but then increased with myotube formation. Treatment with 20 ng/ml tumour necrosis factor-alpha (TNFalpha), which inhibits myoblast differentiation, blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, which stimulates myoblast differentiation, increased IGFBP-3 secretion. The TNFalpha-induced decrease in IGFBP-3 production and inhibition of differentiation could not be rescued by addition of IGF-I. LongR(3)IGF-I, which does not bind to the IGFBPs, had a similar effect on differentiation and IGFBP-3 secretion as IGF-I, both with and without TNFalpha, confirming that increased IGFBP-3 is not purely due to increased stability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretion using antisense oligonucleotides led to an inhibition of differentiation. Taken together these data indicate that IGFBP-3 supports myoblast differentiation.