Trastuzumab is effective in the treatment of HER2/neu over-expressing breast cancer, but not all patients benefit from it. In vitro data suggest a role for HER3 in the initiation of signaling activity involving the AKT–mTOR pathway leading to trastuzumab insensitivity. We sought to investigate the potential of HER3 alone and in the context of p95HER2 (p95), a trastuzumab resistance marker, as biomarkers of trastuzumab escape. Using the VeraTag® assay platform, we developed a dual antibody proximity-based assay for the precise quantitation of HER3 total protein (H3T) from formalin-fixed paraffin-embedded (FFPE) breast tumors. We then measured H3T in 89 patients with metastatic breast cancer treated with trastuzumab-based therapy, and correlated the results with progression-free survival and overall survival using Kaplan–Meier and decision tree analyses that also included HER2 total (H2T) and p95 expression levels. Within the sub-population of patients that over-expressed HER2, high levels of HER3 and/or p95 protein expression were significantly associated with poor clinical outcomes on trastuzumab-based therapy. Based on quantitative H3T, p95, and H2T measurements, multiple subtypes of HER2-positive breast cancer were identified that differ in their outcome following trastuzumab therapy. These data suggest that HER3 and p95 are informative biomarkers of clinical outcomes on trastuzumab therapy, and that multiple subtypes of HER2-positive breast cancer may be defined by quantitative measurements of H3T, p95, and H2T.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-013-2665-0) contains supplementary material, which is available to authorized users.
Breast cancer; HER2; HER3; p95HER2; Trastuzumab
The osteolytic nature of bone metastasis results from a tumor-driven increased bone resorption. Bone remodeling is orchestrated by the molecular triad RANK-RANKL-OPG. This process is dysregulated in bone metastases, mostly via induction of RANKL by tumor-derived factors. These factors increase expression of RANKL, which induce osteoclast formation, function, and survival, thereby increasing bone resorption. RANK is unexpectedly expressed by cancer cells, and the activation of RANKL-RANK pathway correlates with an increased invasive phenotype. To investigate the interaction between RANK expression in human breast and prostate cancer cells and their pro-metastatic phenotype we analyzed the activation of RANKL-RANK pathway and its effects on cell migration, invasion, gene expression in vitro, and osteolysis-inducing ability in vivo. RANKL activates kinase signaling pathways, stimulates cell migration, increases cell invasion, and up-regulates MMP-1 expression. In vivo, MMP-1 knockdown resulted in smaller x-ray osteolytic lesions and osteoclastogenesis, and decreased tumor burden. Therefore, RANKL inhibition in bone metastatic disease may decrease the levels of the osteoclastogenesis inducer MMP-1, contributing to a better clinical outcome.
The prognostic role, in terms of the overall survival outcome, of serum parathyroid hormone evaluated at baseline and after 3 months of zoledronic acid or placebo treatment in hormone-refractory prostate cancer patients with bone metastases enrolled in a randomized clinical trial was assessed.
Secondary hyperparathyroidism is frequent in prostate cancer patients with bone metastases, and this condition is worsened by the administration of potent bisphosphonates. Serum parathyroid hormone (PTH) elevation can impair the efficacy of these drugs in terms of survival.
The prognostic role of elevated serum PTH levels at baseline and after 3 months of zoledronic acid administration was assessed prospectively in 643 bone metastatic prostate cancer patients enrolled in a prospective randomized, placebo-controlled study.
On multivariate analysis, after adjusting for major prognostic factors and bone turnover markers, elevated baseline serum PTH level was negatively associated with overall survival (hazard ratio [HR], 1.448; 95% confidence interval [CI], 1.045–2.006; p < .03) in zoledronic acid–treated patients but not in placebo-treated patients. In patients with normal baseline PTH levels, there was a trend but insignificant association between zoledronic acid administration and a better survival outcome than with placebo (HR, 0.81; 95% CI, 0.65–1.01; p = .065), whereas a trend in the opposite direction was observed in patients with elevated PTH levels (HR, 1.45; 95% CI, 0.87–2.39; p = .151); interaction test, p = .040. Elevated serum PTH level after 3 months of zoledronic acid treatment was not significantly associated with survival outcome.
Secondary hyperparathyroidism has a negative prognostic impact in metastatic prostate cancer patients undergoing zoledronic acid administration. Counteracting elevated PTH levels by adequate doses of vitamin D may improve the efficacy of this drug.
Bone metastasis; Parathyroid hormone; Prostate cancer; Zoledronic acid
Triple-negative breast cancer (TNBC) is characterized by the lack of expression of ERα, PR and HER-2 receptors and the pathway(s) responsible for this downregulation and thus aggressiveness, remains unknown. Here we discovered that lactoferrin (Lf) efficiently downregulates the levels of ERα, PR and HER-2 receptors in a proteasome-dependent manner in breast cancer cells, and accounts for the loss of responsiveness to ER- or HER-2- targeted therapies. Further we found that Lf increases migration and invasiveness of both non-TNBC and TNBC cell lines. We discovered that Lf directly stimulates the transcription of endothelin-1 (ET-1), a secreted pro-invasive polypeptide that acts through a specific receptor ET(A)R, leading to secretion of bioactive ET-1 peptide. Interestingly, a therapeutic ET-1 receptor antagonist drug completely blocked Lf-dependent motility and invasiveness of breast cancer cells. Physiologic significance of this newly discovered Lf-ET-1 axis in the manifestation of TNBC phenotypes is revealed by elevated plasma and tissue Lf and ET-1 levels in TNBC patients as compared to those in ER+ cases. These findings describe the first physiologically relevant polypeptide as a functional determinant of downregulating all three therapeutic receptors in breast cancer which utilizes another secreted ET-1 system to confer invasiveness. Results presented here provide proof-of-principle evidence in support of therapeutic effectiveness of ET-1 receptor antagonist to completely block the Lf-induced motility and invasiveness of the TNBC as well as non-TBNC cells, and thus, opening a remarkable opportunity to treat TNBC by targeting the Lf-ET-1 axis using an approved developmental drug.
Approximately half of all HER2/neu-overexpressing breast cancer patients do not respond to trastuzumab-containing therapy. Therefore, there remains an urgent and unmet clinical need for the development of predictive biomarkers for trastuzumab response. Recently, several lines of evidence have demonstrated that the inflammatory tumor microenvironment is a major contributor to therapy resistance in breast cancer. In order to explore the predictive value of inflammation in breast cancer patients, we measured the inflammatory biomarkers serum ferritin and C-reactive protein (CRP) in 66 patients immediately before undergoing trastuzumab-containing therapy and evaluated their progression-free and overall survival. The elevation in pre-treatment serum ferritin (>250 ng/ml) or CRP (>7.25 mg/l) was a significant predictor of reduced progression-free survival and shorter overall survival. When patients were stratified based on their serum ferritin and CRP levels, patients with elevation in both inflammatory biomarkers had a markedly poorer response to trastuzumab-containing therapy. Therefore, the elevation in inflammatory serum biomarkers may reflect a pathological state that decreases the clinical efficacy of this therapy. Anti-inflammatory drugs and life-style changes to decrease inflammation in cancer patients should be explored as possible strategies to sensitize patients to anti-cancer therapeutics.
Hormone ablation therapy (HALT) for breast or prostate cancer accelerates the development of osteoporosis in both men and women by causing estrogen deficiency, which increases the risk for fracture by promoting bone resorption mediated by osteoclasts. Denosumab, a fully human monoclonal antibody that inhibits osteoclast formation and function, increases bone mass in patients undergoing hormone ablation therapy. In the HALT study of 1,468 men with prostate cancer on androgen-deprivation therapy, denosumab significantly reduced the risk of new vertebral fractures, increased bone mineral density (BMD), and reduced markers of bone turnover. In a study of 252 women with breast cancer undergoing adjuvant aromatase inhibitor (AI) therapy, denosumab increased BMD at 12 and 24 months, overall and in all patient subgroups. The overall rates of adverse events were similar to placebo. Clinicians should consider fracture risk assessment and therapies such as denosumab to increase bone mass in patients on hormone ablation therapy who are at high risk for fracture.
denosumab; treatment-induced bone loss; hormone-ablation therapy; breast cancer; prostate cancer
In breast cancer cells with HER2 gene amplification, HER2 receptors exist on the cell surface as monomers, homodimers and heterodimers with EGFR/HER3. The therapeutic antibody trastuzumab, an approved therapy for HER2+ breast cancer, cannot block ligand-induced HER2 heterodimers, suggesting it cannot effectively inhibit HER2 signaling. Hence, HER2 oligomeric states may predict the odds of a clinical response to trastuzumab in HER2-driven tumors. To test this hypothesis, we generated non-transformed human MCF10A mammary epithelial cells stably expressing a chimeric HER2-FKBP molecule that could be conditionally induced to homodimerize by adding the FKBP ligand AP1510, or instead induced to heterodimerize with EGFR or HER3 by adding the heterodimer ligands EGF/TGFα or heregulin. AP1510, EGF, and heregulin each induced growth of MCF10A cells expressing HER2-FKBP. As expected, trastuzumab inhibited homodimer-mediated but not heterodimer-mediated cell growth. In contrast, the HER2 antibody pertuzumab, which blocks HER2 heterodimerization, inhibited growth induced by heregulin but not AP1510. Lastly, HER2/EGFR tyrosine kinase inhibitor lapatinib blocked both homodimer- and heterodimer-induced growth. AP1510 triggered phosphorylation of Erk1/2 but not AKT, whereas trastuzumab inhibited AP1510-induced Erk1/2 phosphorylation and Shc-HER2 homodimer binding, but not TGFα-induced AKT phosphorylation. Consistent with these observations, high levels of HER2 homodimers correlated with longer time to progression following trastuzumab therapy in a cohort of HER2-overexpressing patients. Together, our findings corroborate the hypothesis that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers reflects an inability to activate the PI3K/AKT pathway. One of the most important clinical implications of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab.
Trastuzumab; HER2; receptor dimerization; breast cancer; ErbB network
Bone metastases are prevalent among patients with advanced solid tumors. Metastatic bone disease alters bone homeostasis, resulting in reduced bone integrity and, consequently, increased skeletal complications. Biochemical markers of bone metabolism may meet an unmet need for useful, noninvasive, and sensitive surrogate information for following patients’ skeletal health.
Materials and methods
Data for this review were identified by searches of PubMed, and references from relevant articles using the search terms “bone markers” or individual bone marker nomenclature, “cancer,” and “metastases.” Abstracts and reports from meetings were included only when they related directly to previously published work. Only papers published in English between 1990 and 2007 were included.
Recent retrospective analyses with bisphosphonates, and particularly with zoledronic acid, have shown significant correlations between biochemical markers of bone metabolism levels and clinical outcomes, especially for bone resorption markers. Clinical results for biochemical markers of bone formation and resorption and other emerging markers of bone metabolism including bone sialoprotein, receptor–activator of nuclear factor-κB ligand, osteoprotegerin, and other markers are presented. However, biochemical markers of bone metabolism are not yet an established surrogate endpoint for treatment efficacy.
Biochemical markers of bone metabolism may allow physicians to identify which patients with metastatic bone disease are at high risk for skeletal-related events or death and who may be responding to therapy. Prospective randomized clinical trials are underway to further assess the utility of markers of bone metabolism in patients with bone metastases.
Bone alkaline phosphatase; Bone sialoprotein; C-telopeptide of type I collagen; N-telopeptide of type I collagen; Osteoprotegerin
Tumor metastasis to the skeleton affects over 400,000 individuals in the United States annually, more than any other site of metastasis, including significant proportions of patients with breast, prostate, lung and other solid tumors. Research on the bone microenvironment and its role in metastasis suggests a complex role in tumor growth. Parallel preclinical and clinical investigations into the role of adjuvant bone-targeted agents in preventing metastasis and avoiding cancer therapy-induced bone loss have recently reported exciting and intriguing results. A multidisciplinary consensus conference convened to review recent progress in basic and clinical research, assess gaps in current knowledge and prioritize recommendations to advance research over the next 5 years. The program addressed three topics: advancing understanding of metastasis prevention in the context of bone pathophysiology; developing therapeutic approaches to prevent metastasis and defining strategies to prevent cancer therapy-induced bone loss. Several priorities were identified: (1) further investigate the effects of bone-targeted therapies on tumor and immune cell interactions within the bone microenvironment; (2) utilize and further develop preclinical models to study combination therapies; (3) conduct clinical studies of bone-targeted therapies with radiation and chemotherapy across a range of solid tumors; (4) develop biomarkers to identify patients most likely to benefit from bone-targeted therapies; (5) educate physicians on bone loss and fracture risk; (6) define optimal endpoints and new measures of efficacy for future clinical trials; and (7) define the optimum type, dose and schedule of adjuvant bone-targeted therapy.
Metastasis; Bisphosphonate; RANK; Bone targeted; PTHrP; Bone loss; Denosumab; Zoledronic acid
Cancer and its treatment can compromise bone health, leading to fracture, pain, loss of mobility, and hypercalcemia of malignancy. Bone metastasis occurs frequently in advanced prostate and breast cancers, and bony manifestations are commonplace in multiple myeloma. Osteoporosis and osteopenia may be consequences of androgen-deprivation therapy for prostate cancer, aromatase inhibition for breast cancer, or chemotherapy-induced ovarian failure. Osteoporotic bone loss and bone metastasis ultimately share a pathophysiologic pathway that stimulates bone resorption by increasing the formation and activity of osteoclasts. Important mediators of pathologic bone metabolism include substances produced by osteoblasts, such as RANKL, the receptor activator of nuclear factor kappa B ligand, which spurs osteoclast differentiation from myeloid cells. Available therapies are targeted to various steps in cascade of bone metastasis.
Biochemical markers; bisphosphonates; bone health; bone metabolism; bone metastasis; bone pain; breast cancer; chemotherapy-induced bone loss; denosumab; metastatic cancer; multiple myeloma; osteoporosis; prostate cancer; RANKL
To evaluate the effects of timing and length of zoledronic acid (ZA) treatment on outcomes for patients with prostate cancer in clinical practice.
Materials and methods
Patients with prostate cancer and first bone metastasis diagnosed from January 2003 to October 2006 were included. Patients were considered ‘untreated’ if no ZA was given, ‘early ZA-treated’ if ZA was initiated before skeletal complication (SC) occurrence or ‘late ZA-treated’ if one or more SC was documented before or at ZA initiation. Patients were classified with short (≤90 days), medium (91–180 days) or long (>180 days) treatment persistence. Assessments included follow-up duration (FUP) and risk of developing one or more SC.
Among eligible patients, 847 were untreated, 243 were early ZA-treated and 218 were late ZA-treated. For untreated versus early ZA-treated groups, median FUP was 263 versus 357 days (p<0.0001), respectively, and time to first SC was 199 versus 273 days (p<0.0001), respectively. ZA treatment was associated with significantly longer FUP and lower SC risk. The early ZA-treated group had significantly longer FUP versus the late ZA-treated group (median days, 357 vs. 299.5); the late ZA-treated group experienced significantly higher SC risk vs. the early ZA-treated group (odds ratio, 1.51). Compared with the long-persistence group, FUP was 56% and 40% shorter in the short and medium groups, respectively (p<0.0001).
Treatment with and early initiation of ZA for patients with prostate cancer and bone metastasis significantly prolonged time to and reduced risk of developing SC, while extending FUP.
Bone metastasis; Prostate; Prostatic neoplasms; Skeletal complication; Zoledronic acid
Skeletal complications are a major cause of morbidity in men with hormone-refractory metastatic prostate cancer. These analyses were designed to identify the variables associated with a greater risk of skeletal complications.
The 643 subjects in this report were participants in a randomized placebo-controlled trial to evaluate the effects of zoledronic acid on the incidence of skeletal-related events. All subjects had bone metastases and disease progression despite medical or surgical castration. The relationships between the baseline covariates and the time to the first skeletal-related event were assessed by Cox proportional hazard analyses. The serum bone-specific alkaline phosphatase (BAP) and urinary N-telopeptide level was assessed as a representative specific marker of osteoblastic and osteoclastic activity, respectively. The other covariates included in the model were age, cancer duration, Eastern Cooperative Oncology Group performance status, analgesic use, and prostate-specific antigen, hemoglobin, and lactate dehydrogenase levels.
Elevated BAP levels were consistently associated with a greater risk of adverse skeletal outcomes. Elevated BAP was significantly associated with a shorter time to the first skeletal-related event on multivariate analyses of the entire study population (relative risk 1.84, 95% confidence interval 1.40 to 2.43; P <0.001) and in subset analyses of the placebo and zoledronic acid groups. Elevated BAP levels were also consistently associated with adverse skeletal outcomes on multivariate analyses of the time to radiotherapy and pathologic fracture, the most common types of skeletal-related events in the study population. No other baseline variable was consistently associated with the risk of adverse skeletal outcomes.
The results of our study have shown that elevated serum BAP levels are associated with a greater risk of adverse skeletal outcomes in men with hormone-refractory prostate cancer and bone metastases.
This phase 1 study was conducted to determine the recommended phase 2 dose of the selective insulin-like growth factor type 1 receptor (IGF-IR) inhibitor figitumumab (F, CP-751,871) given in combination with paclitaxel and carboplatin in patients with advanced solid tumors.
Patients received paclitaxel 200 mg/m2, carboplatin (area under the curve of 6), and F (0.05–20 mg/kg) q3 weeks for up to six cycles. Patients with objective response or stable disease were eligible to receive additional cycles of single agent F until disease progression. Safety, efficacy, pharmacokinetic, and pharmacodynamic endpoints were investigated.
Forty-two patients, including 35 with stages IIIB and IV non-small cell lung cancer (NSCLC), were enrolled in eight dose escalation cohorts. A maximum tolerated dose was not identified. Severe adverse events possibly related to F included fatigue, diarrhea, hyperglycemia, gamma glutamyl transpeptidase elevation, and thrombocytopenia (one case each). F plasma exposure parameters increased with dose. Fifteen objective responses (RECIST) were reported, including two complete responses in NSCLC and ovarian carcinoma. Notably, levels of bioactive IGF-1 seemed to influence response to treatment with objective responses in patients with a high baseline-free IGF-1 to IGF binding protein-3 ratio seen only in the 10 and 20 mg/kg dosing cohorts.
F was well tolerated in combination with paclitaxel and carboplatin. Based on its favorable safety, pharmacokinetic, and pharmacodynamic properties, the maximal feasible dose of 20 mg/kg has been selected for further investigation.
IGF-1R; Figitumumab; CP-751,871; NSCLC
Bone is among the most common sites of metastasis in patients with advanced cancer, and the development of bone metastases places patients at increased risk for skeletal complications.
This retrospective claims analysis included only patients with a diagnosis of bone metastasis who had a single type of solid tumor of the breast (women), prostate, or lung and experienced ≥1 skeletal complication between January 2002 and October 2005.
The mean follow-up (±standard deviation) for zoledronic acid (ZA)-treated patients versus untreated patients was 12.2 ± 9.05 months versus 8.7 ± 9.28 months, respectively (P <.001). The monthly rate of skeletal complications in ZA-treated patients versus untreated patients was 0.29 ± 0.3 per month versus 0.43 ± 0.4 per month, respectively (P <.001). Persistent ZA use was associated with longer follow-up duration (P <.05) and a greater probability of continuing follow-up. Greater persistency was associated with lower monthly rates of skeletal complications (P <.05). The length of follow-up for ZA use according to the recommended dosing schedule was 17.11 months compared with 9.93 months for nonrecommended schedules and 8.68 months for no treatment (analysis of variance; P <.001). The rate of skeletal complications with ZA use on the recommended schedule was 0.16 events per month versus 0.31 events per month for nonrecommended schedules and 0.43 events per month for no treatment. In the subgroup analysis, the mean time to first complication was 185 ± 210 days in the ZA-treated group versus 98 ± 161 days in the untreated group (P <.0001). The mean time from the first complication to the second complication was 111 ± 124 days in the ZA-treated group versus 86 ± 114 days in the untreated group (P <.05).
Real-world evidence indicated that ZA reduced the skeletal morbidity rate and delayed the time to skeletal complications.
zoledronic acid; skeletal complications; retrospective claims analysis; persistency; bone; cancer
Summarize current knowledge, critical gaps in knowledge, and recommendations to advance the field of metastatic bone cancer.
A multidisciplinary consensus conference was convened to review recent progress in basic and clinical research, assess critical gaps in current knowledge, and prioritize recommendations to advance research in the next 5 years. The program addressed three principal topics: biology of metastasis, preserving normal bone health, and optimizing bone-targeted therapies.
A variety of specific recommendations were identified as important to advance research and clinical care over the next 5 years.
Priorities for research in bone biology include characterizing components of the stem cell niche in bone, developing oncogenic immunocompetent animal models of bone metastasis, and investigating the unique contribution of the bone microenvironment to tumor growth and dormancy. Priorities for research in preserving normal bone health include developing methods to measure and characterize disseminating tumor cells, assessing outcomes from the major prevention trials currently in progress, and improving methodologies to assess risks and benefits of treatment. Priorities for optimizing bone-targeted therapies include advancing studies of serum proteomics and genomics to reliably identify patients who will develop bone metastases, enhancing imaging for early detection of bone metastases and early response evaluation, and developing new tests to evaluate response to bone-directed treatments.
Bone metastases are a common cause of skeletal morbidity in patients with advanced cancer. The pattern of skeletal morbidity is complex, and the number of skeletal complications is influenced by the duration of survival. Because many patients with cancer die before trial completion, there is a need for survival-adjusted methods to accurately assess the effects of treatment on skeletal morbidity.
Recently, a survival-adjusted cumulative mean function model has been generated that can provide an intuitive graphic representation of skeletal morbidity throughout a study. This model was applied to the placebo-control arm of a pamidronate study in patients with malignant bone disease from breast cancer.
Analysis by bone lesion location showed that spinal metastases were associated with the highest cumulative mean incidence of skeletal-related events (SREs), followed by chest and pelvic metastases. Metastases located in the extremities were associated with an intermediate incidence of SREs, and those in the skull were associated with the lowest incidence of SREs.
Application of this model to data from the placebo arm of this trial revealed important insight into the natural history of skeletal morbidity in patients with bone metastases. Based on these observations, treatment for the prevention of SREs is warranted regardless of lesion location except for metastases on the skull.
The First Cambridge Conference on Advances in Treating Metastatic Bone Cancer, a symposium held in Cambridge, Massachusetts, October 28 to 29, 2005, was convened to discuss recent advances and research related to the natural history of bone metastases and skeletal complications, bone cancer biology, treatment of myeloma and other solid tumors, and treatment-induced bone loss. The conference format combined brief presentations with extended periods of discussion. The conclusions reached during the 2-day meeting are summarized in this article and presented in more detail in the individual articles and accompanying discussion sessions that comprise the conference proceedings.
HER-2/neu status of the primary breast cancer (PBC) is determined by immunohistochemistry and fluorescent in situ hybridization. Because of a variety of technical factors, however, the PBC may not accurately reflect the metastatic tumor in terms of HER-2/neu status. Recently published guidelines recommend that tumors be defined as HER-2/neu positive if 30% or more of the cells are 3+. Circulating levels of the HER-2 extracellular domain can be measured in serum using a test cleared by the US Food and Drug Administration, and increased serum HER-2/neu levels to above 15 ng/ml can reflect tumor progression. Studies comparing tissue HER-2/neu status of the PBC and HER-2/neu levels above 15 ng/ml in metastatic breast cancer patients are also reviewed.
Weissbach, Arthur (National Institutes of Health, Bethesda, Md.), Allan Lipton, and Arnold Lisio. Intracellular forms of λ deoxyribonucleic acid in Escherichia coli infected with clear or virulent mutants of bacteriophage λ. J. Bacteriol. 91:1489–1493. 1966.—Infection of either the sensitive or lysogenic strain of Escherichia coli K-112S by λ+ leads to the formation of a new phage deoxyribonucleic acid (DNA) species having the properties of a twisted circular DNA duplex. This new phage DNA species is also seen in cells infected with clear or virulent mutants of λ which cannot lysogenize, or do so at a low frequency. The sedimentation rate of circular λ DNA duplex at various pH values and its lability were examined.