Harnessing imaging tools to guide immunotherapy trials: summary from the National Cancer Institute Cancer Imaging Steering Committee workshop.

As the immuno-oncology field continues the rapid growth witnessed over the past decade, optimising patient outcomes requires an evolution in the current response-assessment guidelines for phase 2 and 3 immunotherapy clinical trials and clinical care. Additionally, investigational tools-including image analysis of standard-of-care scans (such as CT, magnetic resonance, and PET) with analytics, such as radiomics, functional magnetic resonance agents, and novel molecular-imaging PET agents-offer promising advancements for assessment of immunotherapy. To document current challenges and opportunities and identify next steps in immunotherapy diagnostic imaging, the National Cancer Institute Clinical Imaging Steering Committee convened a meeting with diverse representation among imaging experts and oncologists to generate a comprehensive review of the state of the field.

Assessing the role of tumour-associated macrophage subsets in breast cancer subtypes using digital image analysis.

PURPOSE: The number of M1-like and M2-like tumour-associated macrophages (TAMs) and their ratio can play a role in breast cancer development and progression. Early clinical trials using macrophage targeting compounds are currently ongoing. However, the most optimal detection method of M1-like and M2-like macrophage subsets and their clinical relevance in breast cancer is still unclear. We aimed to optimize the assessment of TAM subsets in different breast cancer subtypes, and therefore related TAM subset numbers and ratio to clinicopathological characteristics and clinical outcome. METHODS: Tissue microarrays of 347 consecutive primary Luminal-A, Luminal-B, HER2-positive and triple-negative tumours of patients with early-stage breast cancer were serially sectioned and immunohistochemically stained for the pan-macrophage marker CD68 and the M2-like macrophage markers CD163, CSF-1R and CD206. TAM numbers were quantified using a digital image analysis algorithm. M1-like macrophage numbers were calculated by subtracting M2-like TAM numbers from the total TAM number. RESULTS: M2-like markers CD163 and CSF-1R showed a moderate positive association with each other and with CD68 (r ≥ 0.47), but only weakly with CD206 (r ≤ 0.06). CD68 + , CD163 + and CSF-1R + macrophages correlated with tumour grade in Luminal-B tumours (P < 0.001). Total or subset TAM numbers did not correlate with disease outcome in any breast cancer subtype. CONCLUSION: In conclusion, macrophages and their subsets can be detected by means of a panel of TAM markers and are related to unfavourable clinicopathological characteristics in Luminal-B breast cancer. However, their impact on outcome remains unclear. Preferably, this should be determined in prospective series.

Optimisation of scan duration and image quality in oncological 89Zr immunoPET imaging using the Biograph Vision PET/CT.

PURPOSE: Monoclonal antibody (mAb)-based PET (immunoPET) imaging can characterise tumour lesions non-invasively. It may be a valuable tool to determine which patients may benefit from treatment with a specific monoclonal antibody (mAb) and evaluate treatment response. For 89Zr immunoPET imaging, higher sensitivity of state-of-the art PET/CT systems equipped with silicon photomultiplier (SiPM)-based detector elements may be beneficial as the low positron abundance of 89Zr causes a low signal-to-noise level. Moreover, the long physical half-life limits the amount of activity that can be administered to the patients leading to poor image quality even when using long scan durations. Here, we investigated the difference in semiquantitative performance between the PMT-based Biograph mCT, our clinical reference system, and the SiPM-based Biograph Vision PET/CT in 89Zr immunoPET imaging. Furthermore, the effects of scan duration reduction using the Vision on semiquantitative imaging parameters and its influence on image quality assessment were evaluated. METHODS: Data were acquired on day 4 post 37 MBq 89Zr-labelled mAb injection. Five patients underwent a double scan protocol on both systems. Ten patients were scanned only on the Vision. For PET image reconstruction, three protocols were used, i.e. one camera-dependent protocol and European Association of Nuclear Medicine Research Limited (EARL) standards 1 and 2 compliant protocols. Vision data were acquired in listmode and were reprocessed to obtain images at shorter scan durations. Semiquantitative PET image parameters were derived from tumour lesions and healthy tissues to assess differences between systems and scan durations. Differently reconstructed images obtained using the Vision were visually scored regarding image quality by two nuclear medicine physicians. RESULTS: When images were reconstructed using 100% acquisition time on both systems following EARL standard 1 compliant reconstruction protocols, results regarding semiquantification were comparable. For Vision data, reconstructed images that conform to EARL1 standards still resulted in comparable semiquantification at shorter scan durations (75% and 50%) regarding 100% acquisition time. CONCLUSION: Scan duration of 89Zr immunoPET imaging using the Vision can be decreased up to 50% compared with using the mCT while maintaining image quality using the EARL1 compliant reconstruction protocol.

Total serum N-glycans associate with response to immune checkpoint inhibition therapy and survival in patients with advanced melanoma.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of melanoma and other cancers. However, no reliable biomarker of survival or response has entered the clinic to identify those patients with melanoma who are most likely to benefit from ICIs. Glycosylation affects proteins and lipids' structure and functions. Tumours are characterized by aberrant glycosylation which may contribute to their progression and hinder an effective antitumour immune response. METHODS: We aim at identifying novel glyco-markers of response and survival by leveraging the N-glycome of total serum proteins collected in 88 ICI-naive patients with advanced melanoma from two European countries. Samples were collected before and during ICI treatment. RESULTS: We observe that responders to ICIs present with a pre-treatment N-glycome profile significantly shifted towards higher abundancy of low-branched structures containing lower abundances of antennary fucose, and that this profile is positively associated with survival and a better predictor of response than clinical variables alone. CONCLUSION: While changes in serum protein glycosylation have been previously implicated in a pro-metastatic melanoma behaviour, we show here that they are also associated with response to ICI, opening new avenues for the stratification of patients and the design of adjunct therapies aiming at improving immune response.

Access to cancer medicines deemed essential by oncologists in 82 countries: an international, cross-sectional survey.

BACKGROUND: The WHO Essential Medicines List (EML) identifies priority medicines that are most important to public health. Over time, the EML has included an increasing number of cancer medicines. We aimed to investigate whether the cancer medicines in the EML are aligned with the priority medicines of frontline oncologists worldwide, and the extent to which these medicines are accessible in routine clinical practice. METHODS: This international, cross-sectional survey was developed by investigators from a range of clinical practice settings across low-income to high-income countries, including members of the WHO Essential Medicines Cancer Working Group. A 28-question electronic survey was developed and disseminated to a global network of oncologists in 89 countries and regions by use of a hierarchical snowball method; each primary contact distributed the survey through their national and regional oncology associations or personal networks. The survey was open from Oct 15 to Dec 7, 2020. Fully qualified physicians who prescribe systemic anticancer therapy to adults were eligible to participate in the survey. The primary question asked respondents to select the ten cancer medicines that would provide the greatest public health benefit to their country; subsequent questions explored availability and cost of cancer medicines. Descriptive statistics were used to compare access to medicines between low-income and lower-middle-income countries, upper-middle-income countries, and high-income countries. FINDINGS: 87 country-level contacts and two regional networks were invited to participate in the survey; 46 (52%) accepted the invitation and distributed the survey. 1697 respondents opened the survey link; 423 were excluded as they did not answer the primary study question and 326 were excluded because of ineligibility. 948 eligible oncologists from 82 countries completed the survey (165 [17%] in low-income and lower-middle-income countries, 165 [17%] in upper-middle-income countries, and 618 [65%] in high-income countries). The most commonly selected medicines were doxorubicin (by 499 [53%] of 948 respondents), cisplatin (by 470 [50%]), paclitaxel (by 423 [45%]), pembrolizumab (by 414 [44%]), trastuzumab (by 402 [42%]), carboplatin (by 390 [41%]), and 5-fluorouracil (by 386 [41%]). Of the 20 most frequently selected high-priority cancer medicines, 19 (95%) are currently on the WHO EML; 12 (60%) were cytotoxic agents and 13 (65%) were granted US Food and Drug Administration regulatory approval before 2000. The proportion of respondents indicating universal availability of each top 20 medication was 9-54% in low-income and lower-middle-income countries, 13-90% in upper-middle-income countries, and 68-94% in high-income countries. The risk of catastrophic expenditure (spending >40% of total consumption net of spending on food) was more common in low-income and lower-middle-income countries, with 13-68% of respondents indicating a substantial risk of catastrophic expenditures for each of the top 20 medications in lower-middle-income countries versus 2-41% of respondents in upper-middle-income countries and 0-9% in high-income countries. INTERPRETATION: These data demonstrate major barriers in access to core cancer medicines worldwide. These findings challenge the feasibility of adding additional expensive cancer medicines to the EML. There is an urgent need for global and country-level policy action to ensure patients with cancer globally have access to high priority medicines. FUNDING: None.

mRNA-1273 COVID-19 vaccination in patients receiving chemotherapy, immunotherapy, or chemoimmunotherapy for solid tumours: a prospective, multicentre, non-inferiority trial.

BACKGROUND: Patients with cancer have an increased risk of complications from SARS-CoV-2 infection. Vaccination to prevent COVID-19 is recommended, but data on the immunogenicity and safety of COVID-19 vaccines for patients with solid tumours receiving systemic cancer treatment are scarce. Therefore, we aimed to assess the impact of immunotherapy, chemotherapy, and chemoimmunotherapy on the immunogenicity and safety of the mRNA-1273 (Moderna Biotech, Madrid, Spain) COVID-19 vaccine as part of the Vaccination Against COVID in Cancer (VOICE) trial. METHODS: This prospective, multicentre, non-inferiority trial was done across three centres in the Netherlands. Individuals aged 18 years or older with a life expectancy of more than 12 months were enrolled into four cohorts: individuals without cancer (cohort A [control cohort]), and patients with solid tumours, regardless of stage and histology, treated with immunotherapy (cohort B), chemotherapy (cohort C), or chemoimmunotherapy (cohort D). Participants received two mRNA-1273 vaccinations of 100 µg in 0·5 mL intramuscularly, 28 days apart. The primary endpoint, analysed per protocol (excluding patients with a positive baseline sample [>10 binding antibody units (BAU)/mL], indicating previous SARS-CoV-2 infection), was defined as the SARS-CoV-2 spike S1-specific IgG serum antibody response (ie, SARS-CoV-2-binding antibody concentration of >10 BAU/mL) 28 days after the second vaccination. For the primary endpoint analysis, a non-inferiority design with a margin of 10% was used. We also assessed adverse events in all patients who received at least one vaccination, and recorded solicited adverse events in participants who received at least one vaccination but excluding those who already had seroconversion (>10 BAU/mL) at baseline. This study is ongoing and is registered with ClinicalTrials.gov, NCT04715438. FINDINGS: Between Feb 17 and March 12, 2021, 791 participants were enrolled and followed up for a median of 122 days (IQR 118 to 128). A SARS-CoV-2-binding antibody response was found in 240 (100%; 95% CI 98 to 100) of 240 evaluable participants in cohort A, 130 (99%; 96 to >99) of 131 evaluable patients in cohort B, 223 (97%; 94 to 99) of 229 evaluable patients in cohort C, and 143 (100%; 97 to 100) of 143 evaluable patients in cohort D. The SARS-CoV-2-binding antibody response in each patient cohort was non-inferior compared with cohort A. No new safety signals were observed. Grade 3 or worse serious adverse events occurred in no participants in cohort A, three (2%) of 137 patients in cohort B, six (2%) of 244 patients in cohort C, and one (1%) of 163 patients in cohort D, with four events (two of fever, and one each of diarrhoea and febrile neutropenia) potentially related to the vaccination. There were no vaccine-related deaths. INTERPRETATION: Most patients with cancer develop, while receiving chemotherapy, immunotherapy, or both for a solid tumour, an adequate antibody response to vaccination with the mRNA-1273 COVID-19 vaccine. The vaccine is also safe in these patients. The minority of patients with an inadequate response after two vaccinations might benefit from a third vaccination. FUNDING: ZonMw, The Netherlands Organisation for Health Research and Development.

Assessing population diversity in phase III trials of cancer drugs supporting Food and Drug Administration approval in solid tumors.

Our study aimed to assess inequities in the clinical trial participation for the selected patient groups. We searched the Food and Drug Administration (FDA) database and extracted phase-III clinical trial data from MEDLINE for each approved drug by the FDA between January 1, 2006, and June 30, 2020. We analyzed the inclusion/exclusion criteria, participation according to gender, ethnic group, performance score, the positivity of HBV and HCV, and HIV, having comorbidities and brain metastasis. We compared the findings with that of the general population by retrieving data from the Surveillance, Epidemiology and End Results (SEER) database. We identified 142 phase III pivotal oncology trials that enrolled 105 397 patients. The proportion of female patients in trials was lower than their relative prevalence in the general population from SEER region (36% vs 49.6%, P < .001). The rates of black patients included were lower than their relative prevalence from SEER region (2.1% vs 9.8%, P < .001). 1.3% and 0.8% of patients had HBV and HCV infections, respectively. The patients' numbers with organ dysfunction were not established due to insufficient data from clinical trials. 1.6% of all patients had controlled brain metastasis. Black patients, women and patients with brain metastasis or with HBV and HCV were underrepresented. Our study underscores the importance of expanding the inclusion/exclusion criteria of pivotal oncology trials to be more representative of patients seen in clinical practice.

Interleukin-2 PET imaging in patients with metastatic melanoma before and during immune checkpoint inhibitor therapy.

PURPOSE: Immune checkpoint inhibitors can induce a T cell-mediated anti-tumor immune response in patients with melanoma. Visualizing T cell activity using positron emission tomography (PET) might allow early insight into treatment efficacy. Activated tumor-infiltrating T cells express the high-affinity interleukin-2 receptor (IL-2R). Therefore, we performed a pilot study, using fluorine-18-labeled IL-2 ([18F]FB-IL2 PET), to evaluate whether a treatment-induced immune response can be detected. METHODS: Patients with metastatic melanoma received ~ 200 MBq [18F]FB-IL2 intravenously, followed by a PET/CT scan before and during immune checkpoint inhibitor therapy. [18F]FB-IL2 uptake was measured as standardized uptake value in healthy tissues (SUVmean) and tumor lesions (SUVmax). Response to therapy was assessed using RECIST v1.1. Archival tumor tissues were used for immunohistochemical analyses of T cell infiltration. RESULTS: Baseline [18F]FB-IL2 PET scans were performed in 13 patients. SUVmean at baseline was highest in the kidneys (14.2, IQR: 11.6-18.0) and liver (10.6, IQR: 8.6-13.4). In lymphoid tissues, uptake was highest in spleen (10.9, IQR: 8.8-12.4) and bone marrow (2.5, IQR: 2.1-3.0). SUVmax in tumor lesions (n = 41) at baseline was 1.9 (IQR: 1.7-2.3). In 11 patients, serial imaging was performed, three at week 6, seven at week 2, and one at week 4. Median [18F]FB-IL2 tumor uptake decreased from 1.8 (IQR: 1.7-2.1) at baseline to 1.7 (IQR: 1.4-2.1) during treatment (p = 0.043). Changes in [18F]FB-IL2 tumor uptake did not correlate with response. IL-2R expression in four archival tumor tissues was low and did not correlate with baseline [18F]FB-IL2 uptake. No [18F]FB-IL2-related side effects occurred. CONCLUSION: PET imaging of the IL-2R, using [18F]FB-IL2, is safe and feasible. In this small patient group, serial [18F]FB-IL2-PET imaging did not detect a treatment-related immune response. TRIAL REGISTRATION: Clinicaltrials.gov : NCT02922283; EudraCT: 2014-003387.20.

A phase 1b study evaluating the effect of elacestrant treatment on estrogen receptor availability and estradiol binding to the estrogen receptor in metastatic breast cancer lesions using 18F-FES PET/CT imaging.

BACKGROUND: Elacestrant is an oral selective estrogen receptor (ER) degrader. This phase 1b open-label, non-randomized study (RAD1901-106) was initiated to determine the effect of elacestrant on the availability of ER in lesions from postmenopausal women with ER+ advanced breast cancer (ABC) using 16α-18F-fluoro-17ß-estradiol positron emission tomography with low-dose computed tomography (FES-PET/CT). METHODS: Eligible patients were postmenopausal women with ER+, HER2- ABC; tumor progression after ≥ 6 months of 1-3 lines of endocrine treatment for ABC; and measurable or evaluable disease. Two 8-patient cohorts were enrolled: one treated with 400 mg elacestrant once daily (QD) and one treated with 200 mg elacestrant QD with dose escalation to 400 mg QD after 14 days. Elacestrant was dosed continuously until progressive disease, toxicity, or withdrawal. FES-PET/CT was performed pre-dose at baseline and 4 h post-dose on day 14. The primary endpoint was the percentage difference in FES uptake in tumor lesions (maximum 20) after 14 days of treatment compared to baseline. Overall response was investigator-assessed by Response Evaluation Criteria in Solid Tumors [RECIST] version 1.1. RESULTS: Patients (n = 16; median age, 53.5 years) had ABC with a median 2.5 prior lines of endocrine therapy. Median reduction in tumor FES uptake from baseline to day 14 was 89.1% (Q1, Q3: 75.1%, 94.1%) and was similar in both cohorts (89.1% [Q1, Q3: 67.4%, 94.2%], 200/400 mg and 88.7% [Q1, Q3: 79.5%, 94.1%], 400 mg). Residual ER availability (> 25% persistence in FES uptake) on day 14 was observed in 3 patients receiving 200/400 mg (3/78, 37.5%) and 1 patient receiving 400 mg (1/8, 12.5%). The overall response rate (ORR) was 11.1% (1 partial response), and clinical benefit rate (CBR) was 30.8%. Median percentage change in FES uptake did not correlate with ORR or CBR. Adverse events occurring in > 20% of the patients were nausea (68.8%), fatigue (50.0%), dyspepsia (43.8%), vomiting (37.5%), and decreased appetite, dysphagia, and hot flush (31.3% each). Most events were grade 2 in severity. CONCLUSION: Elacestrant 200 mg and 400 mg QD greatly reduced ER availability measured by FES-PET/CT. In a heavily pretreated population, elacestrant was associated with antitumor activity. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02650817 . Registered on 08 January 2016.

High hepatocyte growth factor expression in primary tumor predicts better overall survival in male breast cancer.

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.

Neuroendocrine tumours and their microenvironment.

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.

Mass spectrometric quantification of urinary 6-sulfatoxymelatonin: age-dependent excretion and biological variation.

Objectives: Regulators of circadian rhythm, including melatonin, influence fundamental biological processes. Measuring the melatonin metabolite 6-sulfatoxymelatonin in urine can estimate melatonin production. 6-sulfatoxymelatonin is mainly analyzed by immunoassays, but these methods are hampered by cross-reactivity and poor reproducibility when used to analyze small molecules. Therefore, we validated a high-throughput liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to quantify 6-sulfatoxymelatonin in urine. We evaluated age-dependent 24-h excretion of 6-sulfatoxymelatonin into urine and the biological variation of urinary excretion in healthy individuals. Methods: The online solid phase extraction method combined with LC-MS/MS was validated according to international guidelines, and used to measure the excretion of 6-sulfatoxymelatonin into urine of 240 healthy individuals. Biological variation of 6-sulfatoxymelatonin excretion was examined in 10 healthy individuals. Results: Urinary 6-sulfatoxymelatonin results were well within the validation criteria (interassay coefficient of variation: <5.4%, quantification limit: 0.2 nmol/L). There was an age-related decrease in 6-sulfatoxymelatonin excretion into 24-h urine [F(5, 234)=13.9; p<0.001]. Within-subject variation of 6-sulfatoxymelatonin was 39.2% in day urine, 15.1% in night urine, and 12.2% in 24-h urine. Between-subject variation was 39.1% in day urine, 37.9% in night urine, and 36.8% in 24-h urine. Conclusions: This MS-based method enables straightforward, reproducible, and sensitive quantification of 6-sulfatoxymelatonin in urine. Urinary 6-sulfatoxymelatonin levels decreased with age. Biological variation of 6-sulfatoxymelatonin excretion into urine was high between subjects and lower within subjects, indicating that repeated measurements of 6-sulfatoxymelatonin in 24-h urine are needed in future studies.

Trastuzumab duocarmazine in locally advanced and metastatic solid tumours and HER2-expressing breast cancer: a phase 1 dose-escalation and dose-expansion study.

BACKGROUND: Trastuzumab duocarmazine is a novel HER2-targeting antibody-drug conjugate comprised of trastuzumab covalently bound to a linker drug containing duocarmycin. Preclinical studies showed promising antitumour activity in various models. In this first-in-human study, we assessed the safety and activity of trastuzumab duocarmazine in patients with advanced solid tumours. METHODS: We did a phase 1 dose-escalation and dose-expansion study. The dose-escalation cohort comprised patients aged 18 years or older enrolled from three academic hospitals in Belgium, the Netherlands, and the UK with locally advanced or metastatic solid tumours with variable HER2 status who were refractory to standard cancer treatment. A separate cohort of patients were enrolled to the dose-expansion phase from 15 hospitals in Belgium, the Netherlands, Spain, and the UK. Dose-expansion cohorts included patients aged 18 years or older with breast, gastric, urothelial, or endometrial cancer with at least HER2 immunohistochemistry 1+ expression and measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST). Trastuzumab duocarmazine was administered intravenously on day 1 of each 3-week cycle. In the dose-escalation phase, trastuzumab duocarmazine was given at doses of 0·3 mg/kg to 2·4 mg/kg (3 + 3 design) until disease progression or unacceptable toxicity. The primary endpoint of the dose-escalation phase was to assess safety and ascertain the recommended phase 2 dose, which would be the dose used in the dose-expansion phase. The primary endpoint of the dose-expansion phase was the proportion of patients achieving an objective response (complete response or partial response), as assessed by the investigator using RECIST version 1.1. This ongoing study is registered with ClinicalTrials.gov, number NCT02277717, and is fully recruited. FINDINGS: Between Oct 30, 2014, and April 2, 2018, 39 patients were enrolled and treated in the dose-escalation phase and 146 patients were enrolled and treated in the dose-expansion phase. One dose-limiting toxic effect (death from pneumonitis) occurred at the highest administered dose (2·4 mg/kg) in the dose-escalation phase. One further death occurred in the dose-escalation phase (1·5 mg/kg cohort) due to disease progression, which was attributed to general physical health decline. Grade 3-4 treatment-related adverse events reported more than once in the dose-escalation phase were keratitis (n=3) and fatigue (n=2). Based on all available data, the recommended phase 2 dose was set at 1·2 mg/kg. In the dose-expansion phase, treatment-related serious adverse events were reported in 16 (11%) of 146 patients, most commonly infusion-related reactions (two [1%]) and dyspnoea (two [1%]). The most common treatment-related adverse events (grades 1-4) were fatigue (48 [33%] of 146 patients), conjunctivitis (45 [31%]), and dry eye (45 [31%]). Most patients (104 [71%] of 146) had at least one ocular adverse event, with grade 3 events reported in ten (7%) of 146 patients. No patients died from treatment-related adverse events and four patients died due to disease progression, which were attributed to hepatic failure (n=1), upper gastrointestinal haemorrhage (n=1), neurological decompensation (n=1), and renal failure (n=1). In the breast cancer dose-expansion cohorts, 16 (33%, 95% CI 20·4-48·4) of 48 assessable patients with HER2-positive breast cancer achieved an objective response (all partial responses) according to RECIST. Nine (28%, 95% CI 13·8-46·8) of 32 patients with HER2-low, hormone receptor-positive breast cancer and six (40%, 16·3-67·6) of 15 patients with HER2-low, hormone receptor-negative breast cancer achieved an objective response (all partial responses). Partial responses were also observed in one (6%, 95% CI 0·2-30·2) of 16 patients with gastric cancer, four (25%, 7·3-52·4) of 16 patients with urothelial cancer, and five (39%, 13·9-68·4) of 13 patients with endometrial cancer. INTERPRETATION: Trastuzumab duocarmazine shows notable clinical activity in heavily pretreated patients with HER2-expressing metastatic cancer, including HER2-positive trastuzumab emtansine-resistant and HER2-low breast cancer, with a manageable safety profile. Further investigation of trastuzumab duocarmazine for HER2-positive breast cancer is ongoing and trials for HER2-low breast cancer and other HER2-expressing cancers are in preparation. FUNDING: Synthon Biopharmaceuticals.

Quantitative Profiling of Platelet-Rich Plasma Indole Markers by Direct-Matrix Derivatization Combined with LC-MS/MS in Patients with Neuroendocrine Tumors.

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.

Glypican 3 Overexpression across a Broad Spectrum of Tumor Types Discovered with Functional Genomic mRNA Profiling of a Large Cancer Database.

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.

Interobserver reproducibility of tumor uptake quantification with 89Zr-immuno-PET: a multicenter analysis.

PURPOSE: In-vivo quantification of tumor uptake of 89-zirconium (89Zr)-labelled monoclonal antibodies (mAbs) with PET provides a potential tool in strategies to optimize tumor targeting and therapeutic efficacy. A specific challenge for 89Zr-immuno-PET is low tumor contrast. This is expected to result in interobserver variation in tumor delineation. Therefore, the aim of this study was to determine interobserver reproducibility of tumor uptake measures by tumor delineation on 89Zr-immuno-PET scans. METHODS: Data were obtained from previously published clinical studies performed with 89Zr-rituximab, 89Zr-cetuximab and 89Zr-trastuzumab. Tumor lesions on 89Zr-immuno-PET were identified as focal uptake exceeding local background by a nuclear medicine physician. Three observers independently manually delineated volumes of interest (VOI). Maximum, peak and mean standardized uptake values (SUVmax, SUVpeak and SUVmean) were used to quantify tumor uptake. Interobserver variability was expressed as the coefficient of variation (CoV). The performance of semi-automatic VOI delineation using 50% of background-corrected ACpeak was described. RESULTS: In total, 103 VOI were delineated (3-6 days post injection (D3-D6)). Tumor uptake (median, interquartile range) was 9.2 (5.2-12.6), 6.9 (4.0-9.6) and 5.5 (3.3-7.8) for SUVmax, SUVpeak and SUVmean. Interobserver variability was 0% (0-12), 0% (0-2) and 7% (5-14), respectively (n = 103). The success rate of the semi-automatic method was 45%. Inclusion of background was the main reason for failure of semi-automatic VOI. CONCLUSIONS: This study shows that interobserver reproducibility of tumor uptake quantification on 89Zr-immuno-PET was excellent for SUVmax and SUVpeak using a standardized manual procedure for tumor segmentation. Semi-automatic delineation was not robust due to limited tumor contrast.

Lesion detection by [89Zr]Zr-DFO-girentuximab and [18F]FDG-PET/CT in patients with newly diagnosed metastatic renal cell carcinoma.

PURPOSE: The main objective of this preliminary analysis of the IMaging PAtients for Cancer drug selecTion (IMPACT)-renal cell cancer (RCC) study is to evaluate the lesion detection of baseline contrast-enhanced CT, [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT in detecting ccRCC lesions in patients with a good or intermediate prognosis metastatic clear cell renal cell carcinoma (mccRCC) according to the International Metastatic Database Consortium (IMDC) risk model. METHODS: Between February 2015 and March 2018, 42 newly diagnosed mccRCC patients with good or intermediate prognosis, eligible for watchful waiting, were included. Patients underwent CT, [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT at baseline. Scans were independently reviewed and lesions of ≥10 mm and lymph nodes of ≥15 mm at CT were analyzed. For lesions with [89Zr]Zr-DFO-girentuximab or [18F]FDG-uptake visually exceeding background uptake, maximum standardized uptake values (SUVmax) were measured. RESULTS: A total of 449 lesions were detected by ≥1 modality (median per patient: 7; ICR 4.25-12.75) of which 42% were in lung, 22% in lymph nodes and 10% in bone. Combined [89Zr]Zr-DFO-girentuximab-PET/CT and CT detected more lesions than CT alone: 91% (95%CI: 87-94) versus 56% (95%CI: 50-62, p = 0.001), respectively, and more than CT and [18F]FDG-PET/CT combined (84% (95%CI:79-88, p < 0.005). Both PET/CTs detected more bone and soft tissue lesions compared to CT alone. CONCLUSIONS: The addition of [89Zr]Zr-DFO-girentuximab-PET/CT and [18F]FDG-PET/CT to CT increases lesion detection compared to CT alone in newly diagnosed good and intermediate prognosis mccRCC patients eligible for watchful waiting.

Clinical trial design for systemic agents in patients with brain metastases from solid tumours: a guideline by the Response Assessment in Neuro-Oncology Brain Metastases working group.

Patients with active CNS disease are often excluded from clinical trials, and data regarding the CNS efficacy of systemic agents are usually obtained late in the drug development process or not at all. In this guideline from the Response Assessment in Neuro-Oncology Brain Metastases (RANO-BM) working group, we provide detailed recommendations on when patients with brain metastases from solid tumours should be included or excluded in clinical trials of systemic agents. We also discuss the limitations of retrospective studies in determining the CNS efficacy of systemic drugs. Inclusion of patients with brain metastases early on in the clinical development of a drug or a regimen is needed to generate appropriate CNS efficacy or non-efficacy signals. We consider how to optimally incorporate or exclude such patients in systemic therapy trials depending on the likelihood of CNS activity of the agent by considering three scenarios: drugs that are considered very unlikely to have CNS antitumour activity or efficacy; drugs that are considered very likely to have CNS activity or efficacy; and drugs with minimal baseline information on CNS activity or efficacy. We also address trial design issues unique to patients with brain metastases, including the selection of appropriate CNS endpoints in systemic therapy trials.

Influence of protein properties and protein modification on biodistribution and tumor uptake of anticancer antibodies, antibody derivatives, and non-Ig scaffolds.

Newly developed protein drugs that target tumor-associated antigens are often modified in order to increase their therapeutic effect, tumor exposure, and safety profile. During the development of protein drugs, molecular imaging is increasingly used to provide additional information on their in vivo behavior. As a result, there are increasing numbers of studies that demonstrate the effect of protein modification on whole body distribution and tumor uptake of protein drugs. However, much still remains unclear about how to interpret obtained biodistribution data correctly. Consequently, there is a need for more insight in the correct way of interpreting preclinical and clinical imaging data. Summarizing the knowledge gained to date may facilitate this interpretation. This review therefore provides an overview of specific protein properties and modifications that can affect biodistribution and tumor uptake of anticancer antibodies, antibody fragments, and nonimmunoglobulin scaffolds. Protein properties that are discussed in this review are molecular size, target interaction, FcRn binding, and charge. Protein modifications that are discussed are radiolabeling, fluorescent labeling drug conjugation, glycosylation, humanization, albumin binding, and polyethylene glycolation.

APC/C(Cdh1) controls CtIP stability during the cell cycle and in response to DNA damage.

Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/C(C) (dh1) ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage-induced G2 arrest. However, it is currently unknown whether APC/C(C) (dh1) also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA-damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA-end resection factor, as a novel APC/C(C) (dh1) target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G1 and after DNA damage in G2. Finally, we find that abrogating the CtIP-Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA-end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/C(C) (dh1) on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle- and DNA damage-dependent manner.