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Due to activation of fibroblast into cancer-associated fibroblasts, there is often an increased deposition of extracellular matrix and fibrillar collagens, e.g. type III collagen, in the tumor microenvironment (TME) that leads to tumor fibrosis (desmoplasia). Tumor fibrosis is closely associated with treatment response and poor prognosis for patients with solid tumors. To assure that the best possible treatment option is provided for patients, there is medical need for identifying patients with high (or low) fibrotic activity in the TME. Measuring unique collagen fragments such as the pro-peptides released into the bloodstream during fibrillar collagen deposition in the TME can provide a non-invasive measure of the fibrotic activity. Based on data from 8 previously published cohorts, this review provides insight into the prognostic value of quantifying tumor fibrosis by measuring the pro-peptide of type III collagen in serum of a total of 1692 patients with different solid tumor types and discusses the importance of tumor fibrosis for understanding prognosis and for potentially guiding future drug development efforts that aim at overcoming the poor outcome associated with a fibrotic TME., (© 2022. The Author(s).)

To define the growing significance of cellular targets and/or effectors of cancer drugs, we examined the fitness dependency of cellular targets and effectors of cancer drug targets across human cancer cells from 19 cancer types. We observed that the deletion of 35 out of 47 cellular effectors and/or targets of oncology drugs did not result in the expected loss of cell fitness in appropriate cancer types for which drugs targeting or utilizing these molecules for their actions were approved. Additionally, our analysis recognized 43 cellular molecules as fitness genes in several cancer types in which these drugs were not approved, and thus, providing clues for repurposing certain approved oncology drugs in such cancer types. For example, we found a widespread upregulation and fitness dependency of several components of the mevalonate and purine biosynthesis pathways (currently targeted by bisphosphonates, statins, and pemetrexed in certain cancers) and an association between the overexpression of these molecules and reduction in the overall survival duration of patients with breast and other hard-to-treat cancers, for which such drugs are not approved. In brief, the present analysis raised cautions about off-target and undesirable effects of certain oncology drugs in a subset of cancers where the intended cellular effectors of drug might not be good fitness genes and that this study offers a potential rationale for repurposing certain approved oncology drugs for targeted therapeutics in additional cancer types.

The therapeutic landscape of metastatic castration-resistant prostate cancer (mCRPC) has substantially evolved over the last decade. Nonetheless, a better understanding of bone-targeted agents (BTAs) action in mCRPC remains an unmet need. Theuse of BTAs aims to reduce the incidence of skeletal-related events (SREs) in patients with mCRPC. Less frequent BTA schedules are currently being studied to minimize adverse events. In this study, the impact of metastatic compartment (bone and extraskeletal metastases (BESM) vs. bone-only metastases (BOM)) on bone biomarker kinetics, time to first on-study SRE, and symptomatic skeletal events (SSEs) is evaluated. This is a retrospective analysis of the prospective, randomized, multicenter clinical trial of denosumab vs. zoledronic acid in patients with mCRPC and bone metastases. A total of 1901 patients were included, 1559 (82.0%) with BOM and 342 with BESM (18.0%). Bone metastases burden was balanced between groups. Baseline levels and normalization rates of corrected urinary N-terminal telopeptide and bone alkaline phosphatase did not differ between groups. However, BESM patients had a higher risk of SREs (adjusted HR 1.21; 95% CI 1.01-1.46; p = 0.043) and SSEs (adjusted HR 1.30; 95% CI 1.06-1.61; p = 0.014). This difference was more pronounced in the first 12 months of BTA treatment.In mCRPC, strategies of BTA schedule de-escalation may take into account presence of extraskeletal metastases.

Pancreatic ductal adenocarcinoma (PDAC) patients have poor prognosis and poor response to treatment. This is largely due to PDAC being associated with a dense and active stroma and tumor fibrosis (desmoplasia). Desmoplasia is characterized by excessive degradation and formation of the extracellular matrix (ECM) generating collagen fragments that are released into circulation. We evaluated the association of specific collagen fragments measured in pre-treatment serum with outcome in patients with PDAC. Matrix metalloprotease (MMP)-degraded type I collagen (C1M), type III collagen (C3M), type IV collagen (C4M) and a pro-peptide of type III collagen (PRO-C3) were measured by ELISA in pre-treatment serum from a randomized phase 3 clinical trial of patients with stage III/IV PDAC treated with 5-fluorouracil based therapy (n = 176). The collagen fragments were evaluated for their correlation (r, Spearman) with serum CA19-9 and for their association with overall survival (OS) based on Cox-regression analyses. In this phase 3 PDAC trial, pre-treatment serum collagen fragment levels were above the reference range for 67%-98% of patients, with median values in PDAC approximately two-fold higher than reference levels. Collagen fragment levels did not correlate with CA19-9 (r = 0.049-0.141, p = ns). On a continuous basis, higher levels of all collagen fragments were associated with significantly shorter OS. When evaluating degradation (C3M) and formation (PRO-C3) of type III collagen further, higher PRO-C3 was associated with poor OS (>25 th percentile cut-point, HR = 2.01, 95%CI = 1.33-3.05) and higher C3M/PRO-C3 ratio was associated with improved OS (>25 th percentile cut-point, HR = 0.53, 95%CI = 0.34-0.80). When adjusting for CA19-9 and clinical covariates, PRO-C3 remained significant (HR = 1.65, 95%CI = 1.09-2.48). In conclusion, collagen remodeling quantified in pre-treatment serum as a surrogate measure of desmoplasia was significantly associated with OS in a phase 3 clinical PDAC trial, supporting the link between desmoplasia, tumorigenesis, and response to treatment. If validated, these biomarkers may have prognostic and/or predictive potential in future PDAC trials.

Exemestane (EXE) is an aromatase inhibitor used for the prevention and treatment of breast cancer. The major metabolic pathway for EXE is reduction to form the active 17β-dihydro-EXE (17β-DHE) and subsequent glucuronidation to 17β-hydroxy-EXE-17-O-β-D-glucuronide (17β-DHE-Gluc) by UGT2B17. The aim of the present study was to determine the effects of UGT2B17 copy number variation on the levels of urinary and plasma 17β-DHE-Gluc and 17β-DHE in patients taking EXE. Ninety-six post-menopausal Caucasian breast cancer patients with ER+ breast tumors taking 25 mg EXE daily were recruited into this study. UGT2B17 copy number was determined by a real-time PCR copy number variant assay and the levels of EXE, 17β-DHE and 17β-DHE-Gluc were quantified by UPLC/MS in patients' urine and plasma. A 39-fold decrease (P<0.0001) in the levels of creatinine-adjusted urinary 17β-DHE-Gluc was observed among UGT2B17 (*2/*2) subjects vs subjects with the UGT2B17 (*1/*1) genotype. The plasma levels of 17β-DHE-Gluc was decreased 29-fold (P<0.0001) in subjects with the UGT2B17 (*2/*2) genotype vs subjects with UGT2B17 (*1/*1) genotype. The levels of plasma EXE-adjusted 17β-DHE was 28% higher (P=0.04) in subjects with the UGT2B17 (*2/*2) genotype vs subjects with the UGT2B17 (*1/*1) genotype. These data indicate that UGT2B17 is the major enzyme responsible for 17β-DHE-Gluc formation in vivo and that the UGT2B17 copy number variant may play a role in inter-individual variability in 17β-DHE levels in vivo.

Bone is a common site for metastasis in breast cancer patients and is associated with a series of complications that significantly compromise patient survival, partially due to the advanced stage of disease at the time of detection. Currently, no clinically-approved biomarkers can identify or predict the development of bone metastasis. We recently identified a unique peptide fragment of parathyroid hormone-related protein (PTHrP), PTHrP(12-48), as a validated serum biomarker in breast cancer patients that correlates with and predicts the presence of bone metastases. In this study, the biological activity and mode of action of PTHrP(12-48) was investigated. Sequence-based and structure-based bioinformatics techniques predicted that the PTHrP(12-48) fragment formed an alpha helical core followed by an unstructured region after residue 40 or 42. Thereafter, detailed structure alignment and molecular docking simulations predicted a lack of interaction between PTHrP(12-48) and the cognate PTH1 receptor (PTHR1). The in silico prediction was confirmed by the lack of PTHrP(12-48)-stimulated cAMP accumulation in PTHR1-expressing human SaOS2 cells. Using a specific human PTHrP(12-48) antibody that we developed, PTHrP(12-48) was immunolocalized in primary and bone metastatic human breast cancer cells, as well as within human osteoclasts (OCLs) in bone metastasis biopsies, with little or no localization in other resident bone or bone marrow cells. In vitro, PTHrP(12-48) was internalized into cultured primary human OCLs and their precursors within 60 min. Interestingly, PTHrP(12-48) treatment dose-dependently suppressed osteoclastogenesis, via the induction of apoptosis in both OCL precursors as well as in mature OCLs, as measured by the activation of cleaved caspase 3. Collectively, these data suggest that PTHrP(12-48) is a bioactive breast cancer-derived peptide that locally regulates the differentiation of hematopoietic cells and the activity of osteoclasts within the tumor-bone marrow microenvironment, perhaps to facilitate tumor control of bone. © 2017 American Society for Bone and Mineral Research., (© 2017 American Society for Bone and Mineral Research.)

Background: Markers of bone metabolism, such as N-telopeptide of type I collagen (NTX), have been demonstrated to be prognostic in previous trials of breast cancer (BC) patients with bone metastases (BMs). In the present study, we tested the survival effect of the NTX response to zoledronic acid (ZA) at 3 and 12 months in a contemporaneous cohort of BC patients with BMs and evaluated the influence of extraskeletal metastatic disease on NTX variation., Patients and Methods: The present study was a prospective cohort study of consecutive BC patients diagnosed and treated at a single center. Patients presenting with de novo radiological evidence of BMs who started monthly intravenous ZA were included. Urinary NTX was measured at baseline and 1, 3, 6, 9, and 12 months after ZA introduction., Results: Overall, 71 patients were enrolled, 32 with BMs and 39 with BMs plus extraskeletal metastases. The proportion of patients with elevated NTX at baseline and 3 and 12 months was 49.3%, 26.6%, and 34.2%, respectively. The variables associated with survival included age at diagnosis, tumor estrogen receptor status, and NTX at 3 and 12 months. Multivariate analysis showed that, in addition to age at diagnosis, only the 3-month NTX level was significantly associated with survival. Patients with BMs plus extraskeletal metastases had an erratic NTX variation pattern, unrelated to survival., Conclusion: In the present contemporaneous cohort of BC patients with BMs, the NTX response at 3 months was strongly associated with survival. Furthermore, an early response to ZA was strongly associated with long-term NTX control. Finally, patients with BMs plus extraskeletal metastases had an erratic NTX variation., Implications for Practice: The present study showed that when accommodating recent therapy innovations and longer patient survival, the N-telopeptide (NTX) variation at 3 months is strongly associated with survival. In this setting, in addition to a few other clinicopathological features, NTX is a powerful prognostic marker. Moreover, early NTX correction associates with persistently normal NTX. This might identify a subgroup of patients with a good prognosis who are eligible for premature zoledronic acid (ZA) de-escalation. Finally, patients with bone plus extraskeletal metastases showed an erratic variation of NTX, raising concerns that a single ZA regimen might not fit all patients. Future trials should test its effect according to the presence of extraskeletal involvement., Competing Interests: of potential conflicts of interest may be found at the end of this article., (©AlphaMed Press.)

Background: Breast cancer bone metastasis is a complication that significantly compromises patient survival due, in part, to the lack of disease-specific biomarkers that allow early and accurate diagnosis., Methods: Using mass spectrometry protein profiling, plasma samples were screened from three independent breast cancer patient cohorts with and without clinical evidence of bone metastasis., Results: The results identified 13 biomarkers that classified all 110 patients with a sensitivity of 91% and specificity of 93% [receiver operating characteristics area under the curve (AUC = 1.00)]. The most discriminatory protein was subsequently identified as a unique 12-48aa peptide fragment of parathyroid hormone-related protein (PTHrP). PTHrP(12-48) was significantly increased in plasma of patients with bone metastasis compared with patients without bone metastasis (P < 0.0001). Logistic regression models were used to evaluate the diagnostic potential of PTHrP(12-48) as a single biomarker or in combination with the measurement of the clinical marker N-telopeptide of type I collagen (NTx). The PTHrP(12-48) and NTx logistic regression models were not significantly different and classified the patient groups with high accuracy (AUC = 0.85 and 0.95), respectively. Interestingly, in combination with serum NTx, the plasma concentration of PTHrP(12-48) increased diagnostic specificity and accuracy (AUC = 0.99)., Conclusions: These data show that PTHrP(12-48) circulates in plasma of patient with breast cancer and is a novel and predictive biomarker of breast cancer bone metastasis. Importantly, the clinical measurement of PTHrP(12-48) in combination with NTx improves the detection of breast cancer bone metastasis., Impact: In summary, we present the first validated, plasma biomarker signature for diagnosis of breast cancer bone metastasis that may improve the early diagnosis of high-risk individuals.

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.

Background: Patients with metastatic breast cancer (MBC) overexpressing HER2 (human epidermal growth factor receptor 2) are currently selected for treatment with trastuzumab, but not all patients respond., Patients and Methods: Using a novel assay, HER2 protein expression (H2T) was measured in formalin-fixed, paraffin-embedded primary breast tumors from 98 women treated with trastuzumab-based therapy for MBC. Using subpopulation treatment effect pattern plots, the population was divided into H2T low (H2T < 13.8), H2T high (H2T ≥ 68.5), and H2T intermediate (13.8 ≤ H2T < 68.5) subgroups. Kaplan-Meier (KM) analyses were carried out comparing the groups for time to progression (TTP) and overall survival (OS). Cox multivariate analyses were carried out to identify correlates of clinical outcome. Bootstrapping analyses were carried out to test the robustness of the results., Results: TTP improved with increasing H2T until, at the highest levels of H2T, an abrupt decrease in the TTP was observed. KM analyses demonstrated that patients with H2T low tumors [median TTP 4.2 months, hazard ratio (HR) = 3.7, P < 0.0001] or H2T high tumors (median TTP 4.6 months, HR = 2.7, P = 0.008) had significantly shorter TTP than patients whose tumors were H2T intermediate (median TTP 12 months). OS analyses yielded similar results., Conclusions: MBC patients with very high levels of H2T may represent a subgroup with de novo resistance to trastuzumab. These results are preliminary and require confirmation in larger controlled clinical cohorts.

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 nontransformed 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. 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, the 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 patients with HER2-overexpressing breast cancer. Together, our findings confirm the notion that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers may reflect their inability to activate the PI3K (phosphoinositide 3-kinase)/AKT pathway. A clinical implication of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab., (©2011 AACR.)

Purpose: p95HER2 is an NH(2)-terminally truncated form of HER2 that lacks the trastuzumab binding site and is therefore thought to confer resistance to trastuzumab treatment. In this report, we introduce a new antibody that has enabled the first direct quantitative measurement of p95HER2 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. We sought to show that quantitative p95HER2 levels would correlate with outcome in trastuzumab-treated HER2-positive metastatic breast cancer., Experimental Design: The novel p95HER2 antibody used here was characterized for sensitivity, specificity, and selectivity over full-length HER2. Quantitative p95HER2 levels were measured in 93 metastatic breast tumors using a VeraTag FFPE assay to determine the correlation of p95HER2 levels with outcomes., Results: Within a cohort of trastuzumab-treated metastatic breast cancer patients, high levels of p95HER2 were found to correlate with shorter progression-free survival [hazard ratio (HR), 1.9; P = 0.017] and overall survival (HR, 2.2; P = 0.012) in patients with tumors selected to be HER2 positive by the VeraTag HER2 assay. For those with tumors found to be fluorescence in situ hybridization positive, elevated p95HER2 correlated similarly with shorter progression-free survival (HR, 1.8; P = 0.022) and overall survival (HR, 2.2; P = 0.009)., Conclusions: We have successfully generated an antibody that can specifically detect p95HER2, and developed an assay to quantify expression in FFPE tumor specimens. Using this novel assay, we have identified a group of HER2-positive patients expressing p95HER2 that have a worse outcome while on trastuzumab. As p95HER2 retains sensitivity to kinase inhibitors, measurement of p95HER2 in breast tumor sections may be useful in guiding treatment for patients with HER2-positive breast cancer.

Background: This phase Ib trial assessed safety, tolerability, and maximum tolerated dose (MTD) of figitumumab (CP-751,871), a fully human monoclonal antibody targeting the insulin-like growth factor type 1 receptor (IGF-IR), in combination with docetaxel., Methods: Patients with advanced solid tumours were treated with escalating dose levels of figitumumab plus 75 mg m(-2) docetaxel every 21 days. Safety, efficacy, pharmacokinetics (PKs), and biomarker responses were evaluated., Results: In 46 patients, no dose-limiting toxicities were attributable to the treatment combination. Grade 3 and 4 toxicities included neutropaenia (n=28), febrile neutropaenia (n=11), fatigue (n=10), leukopaenia (n=7), diarrhoea (n=5), hyperglycaemia, lymphopaenia, cellulitis, DVT, and pain (all n=1). The MTD was not reached. Four partial responses were observed; 12 patients had disease stabilisation of > or =6 months. Pharmacokinetic and biomarker analyses showed a dose-dependent increase in plasma exposure, and complete sIGF-IR downregulation at doses of >or =3 mg kg(-1). Pharmacokinetics of docetaxel in combination was similar to when given alone. Out of 18 castration-resistant prostate cancer patients, 10 (56%) had > or =5 circulating tumour cells (CTCs) per 7.5 ml of blood at baseline: 6 out of 10 (60%) had a decline from > or =5 to <5 CTCs and 9 out of 10 (90%) had a > or =30% decline in CTCs after therapy., Conclusions: Figitumumab and docetaxel in combination are well tolerated. Further evaluation is warranted.

Introduction: 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., Methods: 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., Results: 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., Conclusions: 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.

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.

Background: There is significant heterogeneity in survival of patients with metastatic breast cancer who have bone-only metastasis. We studied the correlation of serum N-telopeptide (NTx), a marker of bone resorption, and its correlation with clinical outcomes in patients with metastatic breast cancer with bone-only or bone plus soft tissue metastasis., Patients and Methods: Serum was taken from 250 metastatic breast cancer patients with bone-only or bone plus soft tissue metastasis who participated in two similar randomized studies of second-line hormone therapy. An enzyme-linked immunosorbent assay specific for NTx of type I bone collagen was used to detect serum levels., Results: Sixty patients (24%) had elevated serum NTx levels, using the mean + 2 standard deviations (26 nanomoles Bone Collagen Equivalents per liter) of healthy women as a cut-off. The median duration of clinical benefit was significantly shorter in the group with elevated serum NTx levels compared with the group that had normal serum NTx levels (P=0.0004). Time to progression (TTP) was also significantly shorter in the patients with elevated serum NTx at 139 days compared with 220 days (P=0.0006). Median survival was also significantly shorter in patients with elevated baseline serum NTx levels at 663 days compared with 941 days (P<0.0001)., Conclusion: In this study, breast cancer patients with bone-only or bone plus soft tissue metastasis and elevated serum NTx levels have a shorter duration of clinical benefit, TTP and overall survival.

Background: The HER-2/neu oncogene and its p185 receptor protein are indicators of a more aggressive form of breast cancer. HER-2/neu status guides Herceptin therapy, specifically directed to the extracellular domain (ECD) of the HER-2/neu oncoprotein. The HER-2/neu ECD is shed from cancer cells into the circulation and is measurable by immunoassay., Methods: We performed a systematic review of the peer-reviewed literature on circulating ECD with respect to prevalence, prognosis, prediction of response to therapy, and monitoring of breast cancer., Results: The prevalence of increased ECD in patients with primary breast cancer varied between 0% and 38% (mean, 18.5%), whereas in metastatic disease the range was from 23% to 80% (mean, 43%). Some women with HER-2/neu-negative tumors by tissue testing develop increased ECD concentrations in metastatic disease. Increased ECD has been correlated with indicators of poor prognosis, e.g., overall survival and disease-free survival. Increased ECD predicts a poor response to hormone therapy and some chemotherapy regimens but can predict improved response to combinations of Herceptin and chemotherapy. Many studies support the value of monitoring ECD during breast cancer progression because serial increases precede the appearance of metastases and longitudinal ECD changes parallel the clinical course of disease., Conclusions: The monitoring of circulating HER-2/neu ECD provides a tool for assessing prognosis, for predicting response to therapy, and for earlier detection of disease progression and timely intervention with appropriate therapy.

Background: Serum HER-2/neu antigen concentrations have been reported to correlate with increased tumor volume in patients with breast cancer. We measured serum CA 15-3, a surrogate marker of disease burden, and correlated serum CA 15-3 with serum HER-2/neu and analyzed the association of both markers with clinical outcomes., Methods: Pretreatment serum samples from 566 patients were retrospectively analyzed from 2 phase III clinical trials of estrogen receptor-positive (ER(+)), ER(-)/progesterone receptor-positive, or ER status unknown metastatic breast cancer patients randomized in two similar studies to receive second-line hormone therapy with either megestrol acetate or an aromatase inhibitor (fadrozole). The extracellular domain of the HER-2/neu (c-erbB-2) oncogene and serum CA 15-3 were measured by ELISA on the Bayer Immuno 1., Results: Serum HER-2/neu protein was increased in 168 patients (30%), and CA 15-3 was increased in 337 (60%) patients. Serum CA 15-3 and HER-2/neu were weakly correlated (r = 0.39; P <0.0001). The clinical benefit (complete responses plus partial responses plus stable disease) of endocrine therapy was significantly lower in patients with increased serum HER-2/neu. When adjusted for serum HER-2/neu, serum CA 15-3 was not predictive of response rates. The median time to progression was shorter in patients with increased serum HER-2/neu (89 days) compared with patients with normal serum HER-2/neu (176 days). Survival was significantly shorter in patients with increased serum HER-2/neu (513 vs 869 days; P <0.0001) or increased serum CA 15-3 (689 vs 939 days; P <0.0001). This observation was confirmed by multivariate analysis., Conclusions: Serum HER-2/neu is a significant independent predictive and prognostic factor in hormone receptor-positive metastatic breast cancer, even when adjusted for tumor burden as measured by CA 15-3. The combination of increased serum HER-2/neu and increased serum CA 15-3 predicts a worse prognosis than does increased CA 15-3 alone.

Purpose: Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor receptor superfamily. In vitro, OPG blocks osteoclastogenesis in a dose-dependent manner. Serum OPG levels were assayed in cancer patients and healthy control subjects using an ELISA., Results: OPG levels in healthy controls were significantly higher in sera (0.17 ng/ml) than in plasma (0.14 ng/ml). OPG levels did not differ by age in either control group. Serum was available from patients with solid tumors (n = 145), hematological malignancies (n = 111), benign hematological disorders (n = 35), and rheumatologic diseases (n = 60). When adjusted for age and sex, there was no significant OPG elevation in the sera of patients with solid tumors compared with controls (0.2 versus 0.18 ng/ml). When analyzed by site of primary malignancy within the solid tumor patient group, serum OPG elevations were observed only in patients with colorectal cancer (0.29 ng/ml; P < 0.0001) and pancreatic cancer (0.35 ng/ml; P < 0.0001). When analyzed by site of metastasis within the solid tumor patient group, significant elevations in serum OPG were observed only in patients with liver metastases (0.29 ng/ml) and soft tissue metastases (0.21 ng/ml) but not in patients with bone or lung metastases. Within the hematological malignancy group, serum levels of OPG were significantly lower in patients with multiple myeloma (0.12 ng/ml) but were elevated in patients with Hodgkin's disease (0.29 ng/ml) and Non-Hodgkin's Lymphoma (0.24 ng/ml; P = 0.048)., Conclusions: Although some patients with malignancy have significant elevations of circulating OPG, these concentrations do not approach the level that would be expected to suppress osteoclast function.

The epidermal growth factor receptor (EGFR) has been reported to be expressed in high levels in primary breast cancer by immunohistochemistry. In the present study, a reverse transcription (RT)-PCR assay using EGFR primers was developed and evaluated for the detection of circulating micrometastases in the blood of breast cancer patients. Total RNA was extracted from breast cancer cell lines and from the blood of 23 control individuals and 37 breast cancer patients. After reverse transcription, outer and nested primers for EGFR were used for cDNA amplification. RNA integrity was confirmed with parallel RT-PCR amplification using beta2-microglobulin primers. PCR products were electrophoresed on agarose gels containing ethidium bromide and visualized by UV photography. Southern blotting was used to confirm EGFR specificity. The nested EGFR RT-PCR assay was capable of detecting a lower limit of 100 fg of total RNA from the A431 cell line. EGFR RNA was identified from the blood of 4 of 18 (22%) metastatic breast cancer patients, 0 of 6 locally recurrent breast cancer patients, 0 of 13 adjuvant breast cancer patients, and 0 of 23 controls (P = 0.03, metastatic versus control). The 18 metastatic breast cancer patients all had progressive disease at the time of blood sampling. The identity of the four EGFR-positive bands was confirmed by Southern blotting. The presence of RT-PCR positivity for EGFR was not a treatment-related phenomenon, because three of the four EGFR-positive patients were not receiving treatment at the time of blood collection. RT-PCR for EGFR is a sensitive and specific method for the detection of circulating micrometastases in a proportion of patients with metastatic breast cancer.

Platelet-derived growth factor (PDGF) is produced by a variety of normal and tumor cells in vitro. We have developed an enzyme-linked immunosorbent assay for the detection of the B-chain of PDGF. This assay can reliably detect 0.1 ng/ml of homodimeric recombinant PDGF B-chain and does not cross-react with recombinant PDGF-AA, epidermal growth factor, basic fibroblast growth factor, or transforming growth factor-beta. Citrated plasma from 72 control individuals had a PDGF B-chain (PDGF-B) level of 0.32 +/- 0.14 ng/ml (mean +/- SD) with a range of 0.10-0.69 ng/ml. The plasma platelet factor 4 (PF4) level was 97 +/- 70 ng/ml, with a range of 34-363 ng/ml. Citrated plasma was obtained from 131 cancer patients, and plasma PDGF-B was elevated in 19 (15%) of the patients. Both PDGF-B and PF4 were elevated in 14 (11%) of these patients, consistent with a platelet source of PDGF-B. In 5 patients (4%), however, PDGF-B was elevated and PF4 was not elevated compared to the control group. This last group of patients may have a tumor-derived source of PDGF-B which could be important in autocrine or paracrine growth stimulation of the tumor cells.

Liver cell supernatant and platelet extract can promote the in vivo growth of herpes simplex virus type 2-transformed hamster embryo fibroblast cells. Delineation of necessary growth factors for tumor cells in vivo may open up new strategies to inhibit malignant cell growth.