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Early indicators of metastatic cancer response to therapy are important for evaluating new drugs and stopping ineffective treatment. The RECIST guidelines based on repeat cancer imaging are widely adopted in clinical trials, are used to identify active regimens that may change practice, and contribute to regulatory approvals. However, these criteria do not provide insight before 6 to 12 weeks of treatment and typically require that patients have measurable disease. Recent data suggest that measuring on-treatment changes in the amount or proportion of ctDNA in peripheral blood plasma may accurately identify responding and nonresponding cancers at earlier time points. Over the past year, the RECIST working group has evaluated current evidence for plasma ctDNA kinetics as a treatment response biomarker in metastatic cancers and early endpoint in clinical trials to identify areas of focus for future research and validation. Here, we outline the requirement for large standardized trial datasets, greater scrutiny of optimal ctDNA collection time points and assay thresholds, and consideration of regulatory body guidelines and patient opinions. In particular, clinically meaningful changes in plasma ctDNA abundance are likely to differ by cancer type and therapy class and must be assessed before ctDNA can be considered a potential pan-cancer response evaluation biomarker. Despite the need for additional data, minimally invasive on-treatment ctDNA measurements hold promise to build upon existing response assessments such as RECIST and offer opportunities for developing novel early endpoints for modern clinical trials., (©2024 American Association for Cancer Research.)

Background: To better predict response to immune checkpoint therapy and toxicity in healthy tissues, insight in the in vivo behavior of immune checkpoint targeting monoclonal antibodies is essential. Therefore, we aimed to study in vivo pharmacokinetics and whole-body distribution of zirconium-89 ( 89 Zr) labeled programmed cell death protein-1 (PD-1) targeting pembrolizumab with positron-emission tomography (PET) in humanized mice., Methods: Humanized (huNOG) and non-humanized NOG mice were xenografted with human A375M melanoma cells. PET imaging was performed on day 7 post 89 Zr-pembrolizumab (10 µg, 2.5 MBq) administration, followed by ex vivo biodistribution studies. Other huNOG mice bearing A375M tumors received a co-injection of excess (90 µg) unlabeled pembrolizumab or 89 Zr-IgG 4 control (10 µg, 2.5 MBq). Tumor and spleen tissue were studied with autoradiography and immunohistochemically including PD-1., Results: PET imaging and biodistribution studies showed high 89 Zr-pembrolizumab uptake in tissues containing human immune cells, including spleen, lymph nodes and bone marrow. Tumor uptake of 89 Zr-pembrolizumab was lower than uptake in lymphoid tissues, but higher than uptake in other organs. High uptake in lymphoid tissues could be reduced by excess unlabeled pembrolizumab. Tracer activity in blood pool was increased by addition of unlabeled pembrolizumab, but tumor uptake was not affected. Autoradiography supported PET findings and immunohistochemical staining on spleen and lymph node tissue showed PD-1 positive cells, whereas tumor tissue was PD-1 negative., Conclusion: 89 Zr-pembrolizumab whole-body biodistribution showed high PD-1-mediated uptake in lymphoid tissues, such as spleen, lymph nodes and bone marrow, and modest tumor uptake. Our data may enable evaluation of 89 Zr-pembrolizumab whole-body distribution in patients., Competing Interests: Competing interests: EGEdV reports grants from IMI TRISTAN (GA no.116106), during the conduct of the study; consulting and advisory role for NSABP, Daiichi Sankyo, Pfizer, Sanofi, Merck, Synthon Biopharmaceuticals; grants from Amgen, Genentech, Roche, Chugai Pharma, CytomX Therapeutics, Nordic Nanovector, G1 Therapeutics, AstraZeneca, Radius Health, Bayer, all made available to the institution, outside the submitted work., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Tumours can escape the immune system by expressing programmed death-ligand-1 (PD-L1), which allows them to bind to PD-1 on T-cells and avoid recognition by the immune system. Regulatory T-cells (Tregs), indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) also play a role in immune suppression. Knowledge about the interaction of neuroendocrine tumours (NETs) with their immune microenvironment and the role of immunotherapy in patients with NET is scarce. Here, we investigated the immune microenvironment of serotonin-producing (SP) and non-serotonin-producing NETs (NSP-NETs). Tumours of 33 patients with SP-NET and 18 patients with NSP-NET were studied. Immunohistochemical analyses were performed for PD-L1, T-cells, IDO, TDO, mismatch repair proteins (MMRp) and activated fibroblasts. PD-L1 expression was seen in < 1% of tumour and T-cells. T-cells were present in 33% of NETs, varying between 1 and 10% T-cells per high power field. IDO was expressed in tumour cells in 55% of SP-NETs and 22% of NSP-NETs (p = 0.039). TDO was expressed in stromal cells in 64% of SP-NETs and 13% of NSP-NETs (p = 0.001). No tumours had loss of MMRp. TDO-expressing stromal cells also strongly expressed α-SMA and were identified as cancer-associated fibroblasts (CAFs). Factors that are associated with a response to checkpoint inhibitor treatment were absent or only present to a limited extent in the tumour microenvironment of NETs. The expression of IDO and TDO in a substantial part of NETs and the presence of CAFs suggest two mechanisms that could be responsible for the cold immune microenvironment, which should be explored to enhance anti-tumour immunity and clinical responses.

Background: Breast cancer is rare in men, but management is focused on tumor characteristics commonly found in female breast cancer. The tumor microenvironment of male breast cancer is less well understood, and insight may improve male breast cancer management. The hepatocyte growth factor (HGF)/c-MET axis and the stromal cell-derived factor-1 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis are prognostic in women with breast cancer. We aimed to investigate these factors in male breast cancer and correlate them with patient survival., Methods: From 841 Dutch males with breast cancer who were enrolled in the EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program (NCT01101425) and diagnosed between 1990 and 2010, archival primary tumor samples were collected. Tissue microarrays were constructed with 3 cores per sample and used for immunohistochemical analysis of HGF, c-MET, CXCL12, and CXCR4. Overall survival (OS) of the patients without metastases (M0) was analyzed using the Kaplan-Meier method. The value of the markers regarding OS was determined using univariable and multivariable Cox regression analyses, providing hazard ratios (HRs) and 95% confidence intervals (95% CIs)., Results: Of 720 out of 841 patients, sufficient tissue was available for analysis; 487 out of 720 patients had M0 disease. Patients with high HGF expression and high CXCL12 expression had a superior OS (low vs high expression of both markers, 7.5 vs 13.0 years, hazard ratio [HR] 0.64, 95% CI 0.49-0.84, P = 0.001 [HGF]; 9.1 vs 15.3 years, HR 0.63, 95% CI 0.45-0.87, P = 0.005 [CXCL12]). Multivariate analysis identified HGF as an independent predictor for OS (HR 0.64, 95% CI 0.47-0.88, P = 0.001)., Conclusions: HGF and CXCL12 tumor expression appear to identify male breast cancer patients with a relatively good prognosis. Possibly, this could support male breast cancer-specific management strategies in the future.

Background: Currently, several indole markers are measured separately to support diagnosis and follow-up of patients with neuroendocrine tumors (NETs). We have developed a sensitive mass spectrometry method that simultaneously quantifies all relevant tryptophan-related indoles (tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid) in platelet-rich plasma. Direct-matrix derivatization was used to make the chemical properties of the indoles uniform and to improve the analytical sensitivity and specificity of the assay., Methods: In situ derivatization was performed directly in platelet-rich plasma with propionic anhydride at an ambient temperature. The derivatized indoles were extracted by online solid-phase extraction and eluted to the analytical column for separation followed by mass spectrometric detection. The method was validated according to international guidelines. Platelet-rich plasma samples from 68 healthy individuals and 40 NET patients were analyzed for tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid., Results: The method reproducibly quantified relevant indoles in 8.5 min, including online sample cleanup. Intra- and interassay imprecision, evaluated at 3 different concentrations, ranged from 2.0% to 12% and 1.9% to 13%, respectively. The limit of quantification was sufficient to measure endogenous concentrations of all 4 indoles. Healthy individuals and NET patients had different concentrations of 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid, but tryptophan concentrations were the same., Conclusions: Direct-matrix derivatization in combination with LC-MS/MS is a powerful tool for the simultaneous quantification of all tryptophan-related indoles in platelet-rich plasma. Simultaneous profiling of relevant indoles improves the biochemical characterization and follow-up of NETs., (© 2019 American Association for Clinical Chemistry.)

In metastatic breast cancer (MBC), expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) guides treatment selection. In case of bone-only metastatic disease, ER, PR, and HER2 status assessment may be hampered by decalcification. We aimed to determine the optimal decalcification method, and to study discordance of receptor expression between paired primary breast tumors and optimally decalcified bone metastases. First, decalcification was simulated using acetic acid, hydrochloric/formic acid, and EDTA on 12 primary breast carcinomas. ER, PR, and HER2 immunohistochemistry (IHC) and HER2 in situ hybridization (ISH) were assessed, before and after the 3 decalcification methods. EDTA was considered the optimal method, as it did not affect IHC and as ISH failed in only 1/16 cases. Hydrochloric/formic acid altered ER and PR results, and, with acetic acid and hydrochloric/formic acid, ISH failed in, respectively, 94% and 100%. Second, ER, PR, and HER2 IHC was performed in paired primary tumors and EDTA-decalcified bone metastases obtained from patients with first presentation of MBC. Clinically relevant discordance was defined as changed receptor status with treatment implications. Paired samples of 77 patients, participating in the IMPACT-MBC trial, were evaluable. Hormonal receptor expression change was clinically relevant in 6 patients (7.9%) and HER2 expression change in 1 patient (1.3%). This study shows that EDTA decalcification minimally affects receptor expression results. The incidence of clinically relevant discordance between the primary tumor and bone metastases is low. These findings support that bone biopsies can reliably be used to assess receptor status.

Effective patient selection before or early during treatment is important to increasing the therapeutic benefits of anticancer treatments. This selection process is often predicated on biomarkers, predominantly biospecimen biomarkers derived from blood or tumour tissue; however, such biomarkers provide limited information about the true extent of disease or about the characteristics of different, potentially heterogeneous tumours present in an individual patient. Molecular imaging can also produce quantitative outputs; such imaging biomarkers can help to fill these knowledge gaps by providing complementary information on tumour characteristics, including heterogeneity and the microenvironment, as well as on pharmacokinetic parameters, drug-target engagement and responses to treatment. This integrative approach could therefore streamline biomarker and drug development, although a range of issues need to be overcome in order to enable a broader use of molecular imaging in clinical trials. In this Perspective article, we outline the multistage process of developing novel molecular imaging biomarkers. We discuss the challenges that have restricted the use of molecular imaging in clinical oncology research to date and outline future opportunities in this area.

Glypican 3 (GPC3), a membrane-bound heparan sulfate proteoglycan, is overexpressed in approximately 70% to 80% of hepatocellular carcinomas, but is not expressed commonly in healthy tissues. This raised interest in GPC3 as a drug target and several GPC3-targeting drugs are in clinical development. We therefore predicted GPC3 protein overexpression across tumors and validated these predictions. Functional genomic mRNA profiling was applied to the expression profiles of 18,055 patient-derived tumor samples to predict GPC3 overexpression at the protein level in 60 tumor types and subtypes using healthy tissues as reference. For validation, predictions were compared with immunohistochemical (IHC) staining of a breast cancer tissue microarray and literature data reporting IHC GPC3 overexpression in various solid, hematologic, and pediatric tumors. The percentage of samples with predicted GPC3 overexpression was 77% for hepatocellular carcinomas (n = 364), 45% for squamous cell lung cancers (n = 405), and 19% for head and neck squamous cell cancers (n = 344). Breast cancer tissue microarray analysis showed GPC3 expression ranged from 12% to 17% in subgroups based on estrogen receptor and human epidermal growth factor receptor 2 status. In 28 of 34 tumor types for which functional genomic mRNA data could be compared with IHC there was a relative difference of ≤10%. This study provides a data-driven prioritization of tumor types and subtypes for future research with GPC3-targeting therapies., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objectives: The pharmacodynamic effects of everolimus on gastrointestinal hormone levels have not been described in patients with pancreatic neuroendocrine tumors (pNETs). We report the effects of everolimus on gastrin and glucagon levels in patients with progressive pNET in RADIANT-1 (a single-arm phase II trial) and RADIANT-3 (a placebo-controlled, randomized, phase III trial)., Methods: Serum gastrin and glucagon levels were determined by immunoassay at baseline and at predose in subsequent treatment cycles in patients with elevated baseline hormone levels. The analyses included 158 patients from RADIANT-1 and 404 patients from RADIANT-3., Results: In RADIANT-1, everolimus induced a rapid, sustained decrease in median gastrin and glucagon levels to approximately 60% and 70% of baseline levels, respectively. In RADIANT-3, everolimus consistently reduced median gastrin and glucagon levels by greater than 50% and approximately 40%, respectively (everolimus vs placebo, P < 0.0001), whereas with placebo, both hormones at each time point were essentially the same as their baseline levels. In patients with concomitant octreotide long-acting repeatable treatment, the moderate pharmacodynamic effect on lowering gastrin was greater than that seen with everolimus alone., Conclusions: In addition to prolonging progression-free survival in patients with pNET, everolimus down-regulates excess production of 2 gastrointestinal hormones, which may help control their associated clinical syndromes.

Purpose Everolimus improved median progression-free survival by 6.4 months in patients with advanced pancreatic neuroendocrine tumors (NET) compared with placebo in the RADIANT-3 study. Here, we present the final overall survival (OS) data and data on the impact of biomarkers on OS from the RADIANT-3 study. Methods Patients with advanced, progressive, low- or intermediate-grade pancreatic NET were randomly assigned to everolimus 10 mg/day (n = 207) or placebo (n = 203). Crossover from placebo to open-label everolimus was allowed on disease progression. Ongoing patients were unblinded after final progression-free survival analysis and could transition to open-label everolimus at the investigator's discretion (extension phase). OS analysis was performed using a stratified log-rank test in the intent-to-treat population. The baseline levels of chromogranin A, neuron-specific enolase, and multiple soluble angiogenic biomarkers were determined and their impact on OS was explored. Results Of 410 patients who were enrolled between July 2007 and March 2014, 225 received open-label everolimus, including 172 patients (85%) randomly assigned initially to the placebo arm. Median OS was 44.0 months (95% CI, 35.6 to 51.8 months) for those randomly assigned to everolimus and 37.7 months (95% CI, 29.1 to 45.8 months) for those randomly assigned to placebo (hazard ratio, 0.94; 95% CI, 0.73 to 1.20; P = .30). Elevated baseline chromogranin A, neuron-specific enolase, placental growth factor, and soluble vascular endothelial growth factor receptor 1 levels were poor prognostic factors for OS. The most common adverse events included stomatitis, rash, and diarrhea. Conclusion Everolimus was associated with a median OS of 44 months in patients with advanced, progressive pancreatic NET, the longest OS reported in a phase III study for this population. Everolimus was associated with a survival benefit of 6.3 months, although this finding was not statistically significant. Crossover of patients likely confounded the OS results.

The aim of our study was to detect micrometastatic breast cancer by epithelial glycoprotein-2 (EGP-2) and cytokeratin 19 (CK19), using immunostaining and real time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Fifty-eight breast cancer patients, 52 primary tumors, 75 sentinel nodes (SN) and 149 peripheral blood (PB) samples (from before, during and 4 days after operation) were examined. Immunostaining was performed with antibodies directed against EGP-2 and CK19. Detection limits were one Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line cell/2.10(6) leukocytes (immunostaining) and one MCF-7 cell/10(6) leukocytes qRT-PCR. Control noncancer lymph nodes (n = 10) showed nonspecific CK19 staining, but were qRT-PCR negative; control healthy volunteer PB (n = 11) was always negative. Primary tumor samples, all positive with immunostaining, showed a wide variation of EGP-2 (>10(4) fold) and CK19 mRNA expression (>10(3) fold). SN (n = 19) from 16 patients were tumor-positive with routine haematoxylin-eosin (H&E) and/or immunostaining. SN tumor presence was positively correlated to qRT-PCR expression, but 3 tumor-positive SN were false negative with qRT-PCR. Three SN were qRT-PCR positive, while tumor negative with H&E and/or immunostaining. No immunostaining positive PB was observed, but 19 patients (33%) had one or more qRT-PCR positive PB samples. We concluded that primary tumors have varying expressions of EGP-2 and CK19 mRNA. Both markers can be used in qRT-PCR to obtain adequate sensitivity for single tumor cell detection. In SN, immunostaining appears more sensitive/specific than H&E or qRT-PCR for tumor detection. No immunostaining positivity was found in PB, while 33% of patients had qRT-PCR positive PB. The clinical value of these findings will have to be clarified., (Copyright 2003 Wiley-Liss, Inc.)