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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.

Background: The purpose of this retrospective biomarker study of the Canadian Cancer Trials Group (CCTG) MA.31 randomized phase 3 trial (lapatinib vs trastuzumab) of HER2-positive metastatic breast cancer (MBC) was to evaluate the prognostic and predictive biomarker utility of pretreatment serum programmed death ligand 1 (PD-L1) levels., Methods: CCTG MA.31 accrued 652 HER2-positive patients; 387 had serum available (185 in the trastuzumab arm and 202 in the lapatinib arm). The Ella immunoassay platform (ProteinSimple, San Jose, California) was used to quantitate serum PD-L1 levels. Stepwise forward Cox multivariable analyses were performed for progression-free survival and overall survival (OS)., Results: In the whole trial population, continuous pretreatment serum PD-L1 levels were not associated with OS. However, within the trastuzumab arm, a higher continuous pretreatment serum PD-L1 level was significant for shorter OS (hazard ratio [HR], 3.85; P = .04), but within the lapatinib arm, pretreatment serum PD-L1 was not associated with OS (P = .37). In the whole trial, in a multivariable analysis for OS, serum PD-L1 (median cut point) remained a significant independent covariate (HR, 2.38; P = .001). There was a significant interaction between treatment arm and continuous serum PD-L1 (bootstrap method; P = .0025): at or above 214.2 pg/mL (the 89th percentile), serum PD-L1 was associated with significantly shorter OS with trastuzumab treatment versus lapatinib treatment., Conclusions: In the CCTG MA.31 trial, serum PD-L1 was a significant predictive factor: a higher pretreatment serum PD-L1 level was associated with shorter OS with trastuzumab treatment but with longer OS with lapatinib treatment. Immune evasion may decrease the effectiveness of trastuzumab therapy. Further evaluation of elevated serum PD-L1 in advanced breast cancer is warranted to identify patients with HER2-positive MBC who may benefit from novel immune-targeted therapies in addition to trastuzumab., (© 2020 American Cancer Society.)

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.

Increased extracellular matrix (ECM) formation and matrix metalloprotease (MMP)-mediated ECM degradation are parts of tumorgenesis and generates collagen fragments that are released into circulation. We evaluated the association of specific collagen fragments measured in serum with outcomes in two independent metastatic breast cancer (MBC) cohorts. ELISAs were used to measure C1M (MMP-generated type I collagen fragment), C3M (MMP-generated type III collagen fragment), C4M (MMP-generated type IV collagen fragment), and PRO-C3 (pro-peptide of type III collagen) in pretreatment serum from a phase 3 randomized clinical trial of second-line hormone therapy (HR+, n = 148), and a first-line trastuzumab-treated cohort (HER2+, n = 55). All sites of metastases were included. The collagen fragments were evaluated by Cox-regression analysis for their association with time-to-progression (TTP) and overall survival (OS). In the HR+ cohort, higher C1M and C3M levels (75th percentile cut-off) were associated with shorter TTP; all fragments were associated with shorter OS. In the HER2+ cohort, higher levels of all fragments were associated with shorter TTP; higher PRO-C3 was associated with shorter OS. In multivariate analysis of the HR+ trial for OS, higher levels of all fragments were significant for reduced OS when added separately (C1M HR = 2.1, p < 0.001; C3M HR = 1.8, p = 0.028; C4M HR = 1.8, p = 0.018; PRO-C3 HR = 1.8, p = 0.017); none other clinical covariates were significant. In conclusion, collagen fragments quantified in pretreatment serum was associated with shorter TTP and OS in two independent MBC cohorts receiving systemic therapy. If validated, quantification of ECM remodeling in serum has potential as prognostic and/or predictive biomarkers in MBC., (© 2018 UICC.)

Periostin is an extracellular matrix protein that actively contributes to tumor progression and metastasis. Here, we hypothesized that it could be a marker of bone metastasis formation. To address this question, we used two polyclonal antibodies directed against the whole molecule or its C-terminal domain to explore the expression of intact and truncated forms of periostin in the serum and tissues (lung, heart, bone) of wild-type and periostin-deficient mice. In normal bones, periostin was expressed in the periosteum and specific periostin proteolytic fragments were found in bones, but not in soft tissues. In animals bearing osteolytic lesions caused by 4T1 cells, C-terminal intact periostin (iPTN) expression disappeared at the invasive front of skeletal tumors where bone-resorbing osteoclasts were present. In vitro, we found that periostin was a substrate for osteoclast-derived cathepsin K, generating proteolytic fragments that were not recognized by anti-periostin antibodies directed against iPTN. In vivo, using an in-house sandwich immunoassay aimed at detecting iPTN only, we observed a noticeable reduction of serum periostin levels (- 26%; P < 0.002) in animals bearing osteolytic lesions caused by 4T1 cells. On the contrary, this decrease was not observed in women with breast cancer and bone metastases when periostin was measured with a human assay detecting total periostin. Collectively, these data showed that mouse periostin was degraded at the bone metastatic sites, potentially by cathepsin K, and that the specific measurement of iPTN in serum should assist in detecting bone metastasis formation in breast cancer.

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.

Background: The lapatinib-taxane combination led to shorter PFS than trastuzumab-taxane in HER2+ metastatic breast cancer. We investigated the prognostic and predictive effects of pretreatment serum HER2, CAIX, and TIMP-1., Methods: MA.31 accrued 652 patients; 537 (82%) were centrally confirmed HER2+. Biomarkers were categorized for univariate and multivariable predictive investigations with a median cut-point, ULN cut-points (15 ng/ml for HER2; 506 pg/ml for CAIX; 454 pg/ml for TIMP-1), and custom cut-points (30 and 100 ng/ml for HER2). Stratified step-wise forward Cox multivariable analysis examined continuous and categorical effects of biomarkers on PFS in the ITT and central HER2+ populations; central HER2+ biomarker results are shown., Results: Serum was banked for 472 (72%) of 652 patients. Higher serum HER2 (>median; >15; >30; or >100 ng/ml; p = 0.05-0.002); higher CAIX (>median; >506 pg/ml; p = 0.02; p = 0.001); and higher TIMP-1 (> median; > 454 pg/ml; p = 0.001; p = 0.02) had shorter univariate PFS. In multivariable analysis, higher continuous TIMP-1 was associated with significantly shorter PFS: HR = 1.001 (95% CI = 1.00-01.002; p = 0.004). Continuous serum HER2 and CAIX were not significantly associated with PFS. HER2 of 15 ng/ml or higher had shorter PFS (p = 0.02); higher categorical CAIX had shorter PFS (p = 0.01-0.08). Interaction terms of HER2, CAIX, and TIMP-1 with treatment were not significant; the predictive test power was low., Conclusions: Higher levels of serum TIMP-1, CAIX, and HER2 were significant prognostic biomarkers of shorter PFS. We found no significant interaction between serum biomarkers and response to lapatinib versus trastuzumab. Evaluation of TIMP-1 and CAIX-targeted therapy in addition to HER2-targeted therapy appears warranted in patients with elevated serum levels of these biomarkers.

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: Breast cancer patients in the MA.27 trial had similar outcomes with steroidal aromatase inhibitor (AI) exemestane and nonsteroidal anastrozole. AIs increase the risk of osteoporosis. This study examined the effects of self-reported osteoporosis and osteoporosis therapy (OPT) on outcomes., Methods: The MA.27 phase 3 adjuvant trial enrolled 7576 postmenopausal women. The primary outcome was event-free survival (EFS), and the secondary outcome was distant disease-free survival (DDFS). Patients were permitted bisphosphonates to prevent or treat osteopenia/osteoporosis. In a multivariate, stratified Cox regression, factors were significant with a 2-sided Wald test P value ≤ .05., Results: Osteoporosis was reported at the baseline by 654 of the 7576 women (8.6%) and in total by 1294 patients. Oral OPT was received at the baseline by 815 of the 7576 women (10.8%) and in total by 2711 patients (36%). With a median follow-up of 4.1 years, 693 EFS events (9.15%) and 321 DDFS events (4.2%) occurred. Osteoporosis was not associated with EFS or DDFS. Few EFS events occurred before the initiation of OPT, with no substantive evidence of a time-differing effect on outcomes (nonproportional hazards). OPT (yes vs no) was significantly associated with improved EFS (hazard ratio [HR] for yes vs no, 0.67; 95% confidence interval [CI], 0.57-0.80; P < .001) and DDFS (HR, 0.57; 95% CI, 0.44-0.73; P <. 001). Time-differing (time-dependent) OPT was not (EFS; P = .45). OPT did not alter the incidence of visceral-only metastasis (P = .31)., Conclusions: Oral OPT, administered to postmenopausal breast cancer patients receiving adjuvant AI therapy, was associated with improved EFS and DDFS; the time of OPT initiation (a time-dependent effect) did not affect the outcome. OPT did not alter the risk of visceral metastasis. Cancer 2017;123:2444-51. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

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.)

Cancer treatment-induced bone loss treatment has an important role to prevent bone loss-related events like fracture, significant morbidity, mortality, disfigurement and loss of self-esteem, and health-care expenditure. Numerous factors, including treatment regimens and bone metastasis, increase the risk of osteoporosis or local bone destruction in most breast and prostate cancer patients. Cytotoxic chemotherapies, radiation, and hormonal therapies can lead to premature menopause and decrease bone mineral density. Over 60 % of breast cancer patients within 1 year of beginning postoperative adjuvant chemotherapy experience ovarian failure. Also, ovarian ablation and aromatase inhibitors used to treat breast cancer and orchiectomy and androgen deprivation therapy (ADT; to treat prostate cancer) cause substantial bone loss. In this article, we will focus mainly on antiresorptive therapy in the management of cancer treatment-induced bone loss (CTIBL). An understanding of CTIBL is critical for determining how to assess the risk and identify which patients may benefit from preventive therapy.

Purpose: Cancer-associated inflammation plays a driver role in pancreatic tumor development and progression. Moreover, recent studies have implicated the inflammatory tumor microenvironment in modulating therapy response and inducing resistance. The aim of this study is to investigate the prognostic and predictive value of the inflammatory biomarkers serum ferritin and C-reactive protein (CRP) in advanced pancreatic cancer patients., Methods: We measured pretreatment serum ferritin and CRP levels in 159 patients with inoperable pancreatic cancer participating in a phase III trial., Results: Serum ferritin and CRP levels were examined for correlations with overall survival using Kaplan-Meier analysis. When analyzed on a categorical basis, patients with higher ferritin (>median) or CRP (>25th percentile) had shorter overall survival. Moreover, the two biomarkers were not correlated suggesting independent mechanisms of production and release. However, when patients were evaluated by their ferritin and CRP levels, only patients with elevation in both inflammatory biomarkers showed a significant decrease in overall survival., Conclusions: Serum ferritin and CRP are independent prognostic factors for shorter survival in patients with inoperable pancreatic tumors. Moreover, the evaluation of patients based on both biomarkers suggested that their prognostic value, although independent, reflected the broader state of cancer-associated inflammation. Thus, serum ferritin and CRP should be further explored as clinical biomarkers.

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.

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: Changes in serum human epidermal growth factor receptor 2 (HER2) levels associated with clinical outcomes, including objective response rate, progression-free survival (PFS), and overall survival have been reported in patients with metastatic breast cancer (MBC) receiving trastuzumab and chemotherapy. This study investigated whether baseline or changes in serum HER2 correlated with overall response rate (ORR) and/or PFS in patients with MBC receiving first-line lapatinib monotherapy., Methods: The EGF20009 study investigated lapatinib monotherapy in 138 HER2-positive patients with MBC previously untreated for their metastatic disease. Serum was collected and assessed at baseline and every 4 weeks for 16 weeks after treatment initiation. Disease assessment was performed at weeks 8 and 12 and every 12 weeks thereafter. A ≥ 20% decrease or increase in serum HER2 was defined as a significant change., Results: Seventy-nine percent of patients had elevated baseline serum HER2. Baseline serum HER2 was associated with ORR (P = .043) but not PFS. Patients with a ≥ 20% decrease from baseline of serum HER2 at weeks 4, 8, 12, and 16 had a significantly increased ORR and prolonged PFS. Conversely, those with a ≥ 20% increase from baseline had a significantly lower ORR and shorter PFS., Conclusion: Significant decreases in serum HER2 levels during the first 16 weeks of lapatinib monotherapy were associated with better clinical outcome (longer PFS and increased ORR) in HER2-positive MBC patients., (Copyright © 2011 American Cancer Society.)

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.)

Background: Tissue inhibitor of metalloproteinase-1 (TIMP-1) has paradoxical multifunctional roles in tumorigenesis: inhibition of the catalytic activity of matrix metalloproteinases and apoptosis as well as promotion of angiogenesis and tumor growth. Elevated TIMP-1 levels have been associated with a poorer prognosis in multiple cancers., Methods: Ethylenediaminetetraacetic acid plasma TIMP-1 was determined in 362 castration-resistant prostate cancer (PC) patients using a TIMP-1 enzyme-linked immunosorbent assay. All patients with castration-resistant PC and available plasma were identified from an institutional database. Overall survival was analyzed using the Kaplan-Meier method and Cox modeling on plasma TIMP-1 tertiles., Results: Patients were evaluated in pilot (n = 60) and primary (n = 302) sets. Median follow-up from diagnosis was 5.8 and 6.6 years, respectively. Median plasma TIMP-1 levels were 335 and 183 ng/mL in the pilot and primary sets, respectively. Overall survival was significantly shorter with each higher tertile of TIMP-1 in both datasets (P<.001). For the primary cohort, hazard ratio of (HR) death and median survival by plasma TIMP-1 tertile levels were: low, HR 1.0, 43 months; middle, HR 1.7, 27 months; high, HR 2.4, 19 months. In the primary set, significant covariates in the adjusted Cox regression model were: TIMP-1 level (mid or high vs low tertile), prostate-specific antigen (>20 vs ≤20 ng/mL), alkaline phosphatase (>102 vs ≤102 U/L), Eastern Cooperative Oncology Group performance status (1 + vs 0), and Gleason score (7 or 8 vs ≤6)., Conclusions: Elevated plasma TIMP-1 levels predicted decreased survival in metastatic castration-resistant PC patients, independent of known prognostic markers., (Copyright © 2010 American Cancer Society.)

Background: Only a portion of breast cancer patients currently selected for trastuzumab therapy respond., Methods: Using a novel assay (HERmark) to quantify total human epidermal growth factor receptor 2 (HER2) expression, the authors examined outcomes in 102 trastuzumab-treated metastatic breast cancer patients previously assessed as immunohistochemistry (IHC) 3+ by local but not central IHC, or fluorescence in situ hybridization (FISH) positive, and then retested by central FISH., Results: Of 102 MBC patients previously scored as IHC 3+ or 2+/FISH-positive and treated with trastuzumab-containing regimens, 98 had both central FISH and HER2 total expression values. Sixty-six of 76 central FISH-positive patients (87%) had high HER2 total expression levels (concordant positive), and 19 of 22 central FISH-negative patients (86%) were HER2 total expression low (concordant negative). Fourteen percent (3 of 22) of central FISH-negative patients were HER2 total expression high (discordant HER2 total expression high), and 13% (10 of 76) of central FISH-positive patients were HER2 total expression low (discordant HER2 total expression low). The concordant positive group had a significantly longer time to progression (TTP, median = 11.3 months) compared with the concordant negative group (median TTP, 4.5 months; hazard ratio [HR] = 0.42, P < .001), and also compared with the discordant HER2 total expression low group (median TTP, 3.7 months; HR = 0.43, P = .01). The discordant HER2 total expression low group behaved similarly compared with concordant negatives (HR = 1, P = .99). In analyses restricted to central FISH-positive patients only (n = 77), Cox proportional hazards multivariate regression identified HER2 total expression as an independent predictor of TTP (HR = 0.29, P = .0015) and overall survival (HR = 0.19, P < .001)., Conclusions: A subset of patients with HER2 gene amplification by FISH express low levels of HER2 protein and have reduced response to trastuzumab-containing therapy, similar to FISH-negative patients. This cohort represents a training dataset, and the observed relationships and derived cutoffs require validation in an independent cohort of trastuzumab-treated metastatic breast cancer patients., (Copyright © 2010 American Cancer Society.)

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.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) was evaluated in the pre-treatment serum of 55 newly diagnosed patients with symptomatic myeloma. TIMP-1 was elevated in 47% of patients and correlated with lytic bone disease and increased bone resorption. Importantly, TIMP-1 correlated with ISS stage (p=0.005) and was an independent prognostic covariate for survival [HR: 1.003 (1-1.006), p=0.004] in these patients who were all treated with novel agents (bortezomib and/or IMiDs) during their disease course. Our study provides evidence that pre-treatment serum TIMP-1 is associated with advanced myeloma and suggests the further evaluation of this molecule to better determine its prognostic potential in MM., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Background Endoglin (CD105) is a co-receptor for TGF-beta, is expressed by human vascular endothelial cells, and plays a major role in angiogenesis. Materials and methods Pretreatment EDTA plasma from 224 metastatic breast cancer patients enrolled in a phase III 2nd-line hormone therapy trial and 50 control subjects were assayed for endoglin using an ELISA. Results The female control group (n = 50) plasma endoglin upper limit of normal was defined as the mean + 2 SD (8.7 ng/ml). The breast cancer patient plasma endoglin was 6.40 +/- 2.23 ng/ml (range 3.00-19.79 ng/ml). Elevated plasma endoglin levels were detected in 26 of 224 patients (11.6%). Patients with elevated plasma endoglin had a reduced clinical benefit rate (CR + PR + Stable) (15 vs. 42%) (P = 0.01) to hormone therapy. TTP was shorter for patients with elevated plasma endoglin, but did not reach statistical significance (P = 0.2). Patients with elevated plasma endoglin had decreased overall survival (median 645 vs. 947 days) (P = 0.005). Conclusion Elevated pretreatment plasma endoglin levels predicted for decreased clinical benefit and a shorter overall survival in metastatic breast cancer patients treated with 2nd-line hormone therapy.

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.

Purpose: To determine the effect of elevated serum TIMP-1 on the response of patients with metastatic breast cancer to an aromatase inhibitor versus tamoxifen., Patients and Methods: Five hundred twenty-two patients estrogen receptor-positive metastatic breast cancer were randomly assigned to receive first-line hormone therapy with letrozole or tamoxifen. Serum tissue inhibitor of metalloproteinases-1 (TIMP-1) levels were measured using an enzyme-linked immunosorbent assay., Results: Pretreatment serum TIMP-1 was elevated in 120 (23%) of 522 patients. Patients with elevated serum TIMP-1 had a significantly reduced objective response rate (19.2% v 30.6%; odds ratio, 0.54; P = .01), duration of response (median, 15.5 v 26.2 months; P = .001), time to treatment progression (TTP; median, 4.5 v 9.2 months; HR, 1.78; P = .0001), time to treatment failure (median, 3.5 v 9.0 months; HR, 1.77; P = .0001), and overall survival (median, 20.3 v 35.8 months; HR, 1.77; P = .0001) compared with patients with normal pretreatment TIMP-1 levels. Letrozole was superior to tamoxifen in both the normal serum TIMP-1 group (median TTP, 11.8 v 8.6 months; P = .003) and in the elevated serum TIMP-1 group (median, 6.1 v 3.2 months; P = .03) In multivariate analysis, elevated serum TIMP-1 remained an independent predictor of both shorter TTP (HR, 1.46; P = .002) and survival (HR, 1.44; P = .002), as did serum HER-2. Combined analysis of both serum TIMP-1 and HER-2/neu conferred additional ability to predict significantly different clinical outcomes compared to using either biomarker alone., Conclusion: Patients with elevated pretreatment serum TIMP-1 had a significantly reduced response and survival. Serum TIMP-1 was an independent predictive and prognostic factor. Blockade of TIMP-1 and HER-2/neu activity may be beneficial in a subset of patients with breast cancer.

Background: Tissue inhibitors of metalloproteinase (TIMPs) have at least 2 different functions. They inhibit the catalytic activity of matrix metalloproteinases, and they act as growth factors., Methods: Pretreatment ethylenediamine tetracetic acid plasma TIMP-1 was assayed from 251 patients who were enrolled in a Phase III, second-line, hormone therapy trial, and from a control group of 50 healthy, postmenopausal women by using the TIMP-1 enzyme-linked immunosorbent assay., Results: The plasma TIMP-1 levels from the postmenopausal control group (n = 50 women) were 201 +/- 86 ng/mL mean +/- standard deviation (range, 49-455 ng/mL). The upper limit of normal was defined as the mean +/- 2 standard deviations of the control group (373 ng/mL). Patient pretreatment plasma TIMP-1 levels ranged from 70 ng/mL to 982 ng/mL. Plasma TIMP-1 was elevated above the mean + 2 standard deviations of the control group (373 ng/mL) in 19 patients (7.6%). In univariate analysis, patients who had elevated versus normal plasma TIMP-1 levels had a reduced clinical benefit rate (CBR) (16% vs 42%; P = .03). The time to progression (TTP) (84 days vs 174 days; P < .0001) and overall survival (141 days vs 860 days; P = .0001) also were significantly shorter in patients who had elevated TIMP-1 levels. TTP and overall survival also were significantly shorter in patients who had higher TIMP-1 plasma levels when it was analyzed as a continuous variable. In multivariate analysis, elevated plasma TIMP-1 level remained a prognostic factor for reduced overall survival (P < .0001) along with elevated serum HER-2/neu (P < .0001) and the presence of visceral metastases (P = .008)., Conclusions: Elevated pretreatment plasma levels of TIMP-1 predicted a decreased response to second-line hormone therapy and reduced survival in women with metastatic breast cancer., ((c) 2007 American Cancer Society)

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: Epidermal growth factor receptor (EGFR, HER-1, and erbB1) is overexpressed in primary breast cancer and had been identified as a poor prognostic factor., Methods: Pretreatment serum EGFR levels were quantified by using an enzyme-linked immunoadsorbent assay in a Phase III first-line trial of letrozole and tamoxifen and were correlated with patient outcomes., Results: Serum EGFR levels in a control group of 117 healthy, postmenopausal women measured 64.1 +/- 13.3 ng/mL (mean +/- standard deviation). Using a cutoff EGFR level of 44.1 ng/mL from the control group (5% nonparametric method), 53 of 535 patients (10%) had decreased serum levels of EGFR. Patients with decreased serum EGFR had no significant difference in objective response rate (ORR), clinical benefit rate (CBR), time to progression (TTP), or time to treatment failure (TTF); however, they did have significantly reduced survival compared with patients who had normal serum EGFR levels (median survival, 23.3 months vs. 30.9 months; P = .007). A combined analysis of pretreatment serum EGFR and HER-2 yielded no additional predictive information for ORR, CBR, TTP, or TTF compared to serum HER-2 alone. However, in the current analysis, a subgroup of patients who had decreased serum EGFR and normal serum HER-2 was identified (n = 39 of 535 patients; 7.3%) that had significantly reduced survival compared with patients who had normal serum levels of both EGFR and HER-2 (median survival, 23.5 months vs. 37.1 months; P = .005). In multivariate analysis, a decreased serum EGFR level remained a significant independent prognostic factor for decreased survival (hazards ratio, 1.58; P = .007)., Conclusions: In patients who had metastatic breast cancer, decreased serum EGFR/normal serum HER-2 predicted shorter survival compared with patients who had normal levels of serum EGFR/HER-2. This patient subgroup deserves further study to assess their response to and selection for anti-EGFR-directed therapies.

Background: Prolonged exposure of breast carcinoma cells in vitro to tamoxifen results in tamoxifen resistance. Tamoxifen-resistant cells express increased HER-2/neu mRNA and protein. The objective of this study was to determine whether patients with metastatic or locally advanced breast carcinoma who have negative serum HER-2/neu status at the initiation of first-line hormone therapy with letrozole or tamoxifen convert to positive serum HER-2/neu status at the time of disease progression and to determine whether serum HER-2/neu conversion to positive status is associated with response to therapy and overall survival., Methods: Serum samples were obtained at baseline and at the time of disease progression from 240 patients who initially had negative serum HER-2/neu status (< 15 ng/mL). A manual microtiter, enzyme-linked immunosorbent assay that was specific for the extracellular domain of the HER-2/neu (c-erbB-2) oncoprotein product was used to quantitate serum levels., Results: Among 240 patients, 61 patients (26%) converted from serum HER-2/neu negative to positive (> 15 ng/mL) at the time of disease progression. Thirty-two of 129 patients (25%) who were treated with tamoxifen and 29 of 111 patients (26%) who were treated with letrozole became converted to positive serum HER-2/neu status at the time of disease progression. The response rate and the time to disease progression on first-line hormone therapy were not affected by serum HER-2/neu conversion. The survival of patients who converted to positive serum HER-2/neu status was significantly shorter compared with the survival of patients who remained negative for serum HER-2/neu. A multivariate analysis revealed that conversion to positive serum HER-2/neu status was an independent prognostic variable for survival., Conclusions: Conversion to positive serum HER-2/neu status occurred in approximately 25% of patients who received first-line hormone therapy. Conversion to serum HER-2/neu-positive status occurred with equal frequency in antiestrogen and aromatase-inhibitor therapy. The current results showed that serum conversion to HER-2/neu-positive status was an independent risk factor for decreased survival in patients with breast carcinoma.

The HER2/neu oncoprotein is a major target for the development of new cancer therapies and is similar to the estrogen receptor, which guides hormone therapy. The HER2/neu status is used to guide therapy decisions in patients with HER2/neu-overexpressing breast cancer tumors. The HER2/neu oncogene, or c-erbB-2, encodes a transmembrane receptor protein that is expressed on normal epithelial cells and can be overexpressed in breast cancer cells. Studies have shown that the extracellular domain (ECD) of the HER2/neu oncoprotein is released from the cell and can be measured in the circulation of women with breast cancer. Enzyme-linked immunosorbent assay methods used to measure the circulating HER2/neu ECD have shown that the prevalence of elevated ECD levels is approximately 18.1% in women with primary breast cancer and approximately 45.6% in women with metastatic breast cancer (MBC). Many studies have monitored the circulating ECD levels after surgery and indicate that increasing ECD levels can indicate recurrence of breast cancer earlier than clinical diagnosis. Studies in women with MBC showed that serial changes in circulating HER2/neu ECD levels paralleled the clinical course of disease, regardless of the treatment regimen. Several studies identified a subgroup of patients with MBC who had HER2/neu-negative disease by tissue testing but developed elevated ECD levels with MBC. In contrast to tissue testing, which is a one-time event, monitoring the circulating levels of the HER2/neu ECD in patients with breast cancer provides a real-time assessment of the HER2/neu status and provides important information for managing the therapy of patients with MBC.

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.

Purpose: To determine the effect of elevated serum HER-2/neu on the response of metastatic breast cancer patients to an aromatase inhibitor versus an antiestrogen., Patients and Methods: Five hundred sixty-two estrogen receptor-positive metastatic breast cancer patients were randomized to first-line hormone therapy with either letrozole or tamoxifen. An automated enzyme-linked immunosorbent assay was used to detect serum HER-2/neu., Results: For patients with normal serum HER-2/neu (70.5%), objective response rate (ORR; 39% in letrozole-treated patients v 26% in tamoxifen-treated patients; P =.008), clinical benefit (CB; 57% v 45%; P =.016), time to progression (TTP; median, 12.2 v 8.5 months; P =.0019), and time to treatment failure (TTF; median, 11.6 v 6.2 months; P =.0066) were significantly better in patients treated with letrozole. In the elevated HER-2/neu group (29.5%), there was no significant difference in ORR (17% in letrozole-treated patients v 13% in tamoxifen-treated patients; P =.45) or CB (33% v 26%; P =.31), but there was a strong trend in favor of a longer TTP with letrozole (median, 6.1 v 3.3 months; P =.0596) and a significantly longer TTF with letrozole (median, 6.0 v 3.2 months; P =.0418). Multivariate analysis revealed that elevated serum HER-2/neu was a negative predictor for ORR and TTP., Conclusion: Patients with normal serum HER-2/neu receiving letrozole demonstrated a significantly greater ORR and CB and longer TTP and TTF than patients receiving tamoxifen. Although in patients with elevated serum HER-2/neu there was no significant difference between letrozole and tamoxifen in ORR or CB, there was a strong trend favoring longer TTP and significantly longer TTF with letrozole.

The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. An RT-PCR assay was used to detect EGFR mRNA in the blood of 43 patients with pancreatic, lung (NSCLC), colon, and renal carcinomas. After an initial serum tube was drawn to clear the line of detached epithelial cells, a 3 ml sample of EDTA-anticoagulated blood was collected from each cancer patient and healthy control. Total RNA was isolated from each sample and from cancer cell lines. After reverse transcription using specific priming, outer and nested primers for EGFR were employed for cDNA amplification. RNA integrity was confirmed with RT-PCR amplification using beta2-microglobulin primers. PCR products were electrophoresed on agarose gels containing ethidium bromide and visualized. The assay was validated using Southern blotting and was capable of detecting a lower limit of 100 fg of total RNA from the A431 cell line. EGFR-positive cells were detected in 3/10 (30%) non-small cell lung cancer patients, 2/11 (18%) pancreatic cancer patients, and 2/16 (12.5%) colon cancer patients, but in 0/6 (0%) patients with renal carcinoma (16% of solid tumor patients overall). All 23 healthy controls were negative. This study is the first to apply RT-PCR for the detection of EGFR mRNA in the peripheral blood of patients with pancreatic and renal carcinomas, and it lends further support for the use of EGFR mRNA as a marker of CTCs in the blood of patients with certain types of solid tumors.