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Nanoparticle albumin-bound (nab)-paclitaxel, an albumin-stabilized paclitaxel formulation, demonstrates clinical activity when administered in combination with gemcitabine in patients with metastatic pancreatic ductal adenocarcinoma (PDA). The limited availability of patient tissue and exquisite sensitivity of xenografts to chemotherapeutics have limited our ability to address the mechanistic basis of this treatment regimen. Here, we used a mouse model of PDA to show that the coadministration of nab-paclitaxel and gemcitabine uniquely demonstrates evidence of tumor regression. Combination treatment increases intratumoral gemcitabine levels attributable to a marked decrease in the primary gemcitabine metabolizing enzyme, cytidine deaminase. Correspondingly, paclitaxel reduced the levels of cytidine deaminase protein in cultured cells through reactive oxygen species–mediated degradation, resulting in the increased stabilization of gemcitabine. Our findings support the concept that suboptimal intratumoral concentrations of gemcitabine represent a crucial mechanism of therapeutic resistance in PDA and highlight the advantages of genetically engineered mouse models in preclinical therapeutic trials.Significance: This study provides mechanistic insight into the clinical cooperation observed between gemcitabine and nab-paclitaxel in the treatment of pancreatic cancer. Cancer Discovery; 2(3); 260–9. ©2012 AACR.Read the Commentary on this article by Clark, p. 208This article is highlighted in the In This Issue feature, p. 193

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Table S1. Pharmacokinetic parameters of encorafenib and alpelisib in patients at steady state (cycle 2 day 1). Table S2. Criteria for defining dose-limited toxicities. Supplementary Figure 1. Radiological images of response for a patient treated with the dual-combination therapy of encorafenib and cetuximab. Supplementary Figure 2. Time on study by response for patients treated with the dual-combination therapy of encorafenib and cetuximab and patients treated with the triple-combination therapy of encorafenib, alpelisib, and cetuximab.

mp3 file (10 MB). In the March edition of the Cancer Discovery podcast, Executive Editor Mark Landis talks with David A. Tuveson about his paper, which provides mechanistic insight into the clinical cooperation observed between gemcitabine and nab-paclitaxel in the treatment of pancreatic cancer.

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Purpose:PD-1 inhibition results in durable antitumor responses in a proportion of patients with metastatic urothelial cancer (mUC). The majority of patients, however, do not experience clinical benefit. In this study, we aimed to identify early changes in T-cell subsets that underlie anti–PD-1 efficacy in patients with mUC.Experimental Design:Paired samples were collected from peripheral blood, plasma, and metastatic lesions of 56 patients with mUC at baseline and weeks 6 and 12 after initiating pembrolizumab treatment (200 mg intravenously, every 3 weeks). Samples were analyzed using multiplex flow cytometry, ELISA, and in situ stainings, including cellular network analysis. Treatment response was evaluated as best overall response according to RECIST v1.1, and patients were classified as responder (complete or partial response) or nonresponder (progressive disease).Results:In responders, baseline fractions of CD4+ T cells expressing cosignaling receptors were higher compared with nonresponders. The fraction of circulating PD-1+ CD4+ T cells decreased at weeks 6 and 12, whereas the fraction of 4-1BB+ CD28+ CD4+ T cells increased at week 12. In metastatic lesions of responders, the baseline density of T helper-type 1 (Th1) cells, defined as T-bet+ CD4+ T cells, was higher as compared to non-responders. Upon treatment, Th1 cells became localized in close proximity to CD8+ T cells, CD11b+ myeloid cells, and tumor cells.Conclusions:A decrease in the fraction of circulating PD-1+ CD4+ T cells, and juxtaposition of Th1, CD8+, and myeloid cells was associated with response to anti–PD-1 treatment in patients with mUC.

Purpose: This study investigated the safety, clinical activity, and target-associated biomarkers of lumretuzumab, a humanized, glycoengineered, anti-HER3 monoclonal antibody (mAb), in combination with the EGFR-blocking agents erlotinib or cetuximab in patients with advanced HER3-positive carcinomas.Experimental Design: The study included two parts: dose escalation and dose extension phases with lumretuzumab in combination with either cetuximab or erlotinib, respectively. In both parts, patients received lumretuzumab doses from 400 to 2,000 mg plus cetuximab or erlotinib according to standard posology, respectively. The effect of HRG mRNA and HER3 mRNA and protein expression were investigated in a dedicated extension cohort of squamous non–small cell lung cancer (sqNSCLC) patients treated with lumretuzumab and erlotinib.Results: Altogether, 120 patients were treated. One dose-limiting toxicity (DLT) in the cetuximab part and two DLTs in the erlotinib part were reported. The most frequent adverse events were gastrointestinal and skin toxicities, which were manageable. The objective response rate (ORR) was 6.1% in the cetuximab part and 4.2% in the erlotinib part. In the sqNSCLC extension cohort of the erlotinib part, higher tumor HRG and HER3 mRNA levels were associated with a numerically higher disease control rate but not ORR.Conclusions: The toxicity profile of lumretuzumab in combination with cetuximab and erlotinib was manageable, but only modest clinical activity was observed across tumor types. In the sqNSCLC cohort, there was no evidence of meaningful clinical benefit despite enriching for tumors with higher HRG mRNA expression levels. Clin Cancer Res; 23(18); 5406–15. ©2017 AACR.

Purpose:KEYNOTE-158 (ClinicalTrials.gov identifier: NCT02628067) investigated the efficacy and safety of pembrolizumab across multiple cancers. We present results from patients with previously treated advanced well-differentiated neuroendocrine tumors (NET).Patients and Methods:Pembrolizumab 200 mg was administered every 3 weeks for 2 years or until progression, intolerable toxicity, or physician/patient decision. Tumor imaging was performed every 9 weeks for the first year and then every 12 weeks. Endpoints included objective response rate (ORR) per RECIST v1.1 by independent central radiologic review (primary) and duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety (secondary).Results:A total of 107 patients with NETs of the lung, appendix, small intestine, colon, rectum, or pancreas were treated. Median age was 59.0 years (range, 29–80), 44.9% had ECOG performance status 1, 40.2% had received ≥3 prior therapies for advanced disease, and 15.9% had PD-L1–positive tumors (combined positive score ≥1). Median follow-up was 24.2 months (range, 0.6–33.4). ORR was 3.7% (95% CI, 1.0–9.3), with zero complete responses and four partial responses (three pancreatic and one rectal) all in patients with PD-L1–negative tumors. Median DOR was not reached, with one of four responses ongoing after ≥21 months follow-up. Median PFS was 4.1 months (95% CI, 3.5–5.4); the 6-month PFS rate was 39.3%. Median OS was 24.2 months (95% CI, 15.8–32.5). Treatment-related adverse events (AE) occurred in 75.7% of patients, 21.5% of whom had grade 3–5 AEs.Conclusions:Pembrolizumab monotherapy showed limited antitumor activity and manageable safety in patients with previously treated advanced well-differentiated NETs.

Clusters of peripheral blood CD4+ T-cell subsets in mUC patients with and without a tumor response to anti-PD1

Figure 1 Mean ({plus minus}SD) serum lumretuzumab profiles following multiple ascending doses from 100 mg up to 2000 mg Figure 2 Percentage change in standardized uptake value maximum from baseline as assessed by FDG-PET at (a) Cycle 1 Day 14 and (b) Cycle 4 Day 14 Table 1 Summary of baseline and change in HER3 expression measured by IHC in skin and tumor biopsy samples pre and post treatment with lumretuzumab Table 2 Summary of change in expression of HER3, HER2, EGFR and cMET in fresh tumor biopsies compared to primary archival samples Table 3 Peripheral blood immunophenotyping (T, B and NK cells and NK subsets) from patients dosed with 2000 mg lumretuzumab Table 4 Ex-vivo activation potential of peripheral NK lymphocytes for all patients at ba seline and following exposure to lumretuzumab in the 2000 mg dose cohort Table 5 Summary of tumor immune effector cell infiltration Table 6 Tumor response to treatment (RECIST)

Purpose:This study examined safety, pharmacokinetics, and efficacy of veliparib, a PARP inhibitor, combined with carboplatin and etoposide in patients with extensive-stage (ED) small cell lung cancer (SCLC) and other solid tumors.Patients and Methods:The 3 + 3 design was used for dose escalation of oral veliparib in combination with carboplatin (AUC 5 on day 1) and etoposide (100 mg/m2 on days 1–3) in 21-day cycles. Veliparib dose was explored from 80 to 240 mg b.i.d. on 7-day, 14-day, or continuous schedules. Patients without disease progression continued on maintenance monotherapy (veliparib 400 mg b.i.d.) until disease progression or unacceptable toxicity.Results:Thirty-nine patients were enrolled to determine the recommended phase II dose of 240 mg veliparib for 14 days combined with carboplatin and etoposide based on long-term tolerability. Dose-limiting toxicity occurred in 1 patient (grade 2 toxic motor polyneuropathy) at veliparib 240 mg b.i.d. for 7 days. Most common adverse events related to veliparib were nausea (39%), fatigue (39%), and hematologic toxicities. Continuous dosing of veliparib 240 mg b.i.d. with carboplatin and etoposide resulted in excessive chemotherapy dose delays due to hematologic toxicity (grade 3/4 neutropenia/thrombocytopenia). Etoposide pharmacokinetics was not affected by veliparib. Confirmed responses occurred in 17 of 39 (44%) and 16 of 25 (64%) of all enrolled and ED SCLC patients, respectively. At the RP2D, confirmed responses occurred in 6 of 13 (46%) and 5 of 6 (83%) of all enrolled and ED SCLC patients, respectively.Conclusions:Veliparib (240 mg b.i.d. 14 days) plus carboplatin/etoposide can be safely combined. Phase II of this study is ongoing in first-line patients with ED SCLC.

Supplementary Figure from Patients with Rare Cancers in the Drug Rediscovery Protocol (DRUP) Benefit from Genomics-Guided Treatment

Numbers of major T-cell subsets in peripheral blood and expression of T-cell maturation/activation markers on CD4+ T-cells

Purpose: This first-in-human phase I trial assessed the safety, tolerability, and preliminary antitumor activity of apitolisib (GDC-0980), a dual inhibitor of class I PI3K, and mTOR kinases.Experimental Design: Once-daily oral apitolisib was administered to patients with solid tumors for days 1 to 21 or 1 to 28 of 28-day cycles. Pharmacokinetic and pharmacodynamic parameters were assessed.Results: Overall, 120 patients were treated at doses between 2 and 70 mg. The commonest ≥G3 toxicities related to apitolisib at the recommended phase 2 dose (RP2D) at 40 mg once daily included hyperglycemia (18%), rash (14%), liver dysfunction (12%), diarrhea (10%), pneumonitis (8%), mucosal inflammation (6%), and fatigue (4%). Dose-limiting toxicities (1 patient each) were G4 fasting hyperglycemia at 40 mg (21/28 schedule) and G3 maculopapular rash and G3 fasting hyperglycemia at 70 mg (21/28 schedule). The pharmacokinetic profile was dose-proportional. Phosphorylated serine-473 AKT levels were suppressed by ≥90% in platelet-rich plasma within 4 hours at the MTD (50 mg). Pharmacodynamic decreases in fluorodeoxyglucose positron emission tomography uptake of >25% occurred in 66% (21/32) of patients dosed at 40 mg once daily. Evidence of single-agent activity included 10 RECIST partial responses (PR; confirmed for peritoneal mesothelioma, PIK3CA mutant head-and-neck cancer, and three pleural mesotheliomas).Conclusions: Apitolisib exhibited dose-proportional pharmacokinetics with target modulation at doses ≥16 mg. The RP2D was 40 mg once-daily 28/28 schedule; severe on-target toxicities were apparent at ≥40 mg, particularly pneumonitis. Apitolisib was reasonably tolerated at 30 mg, the selected dose for pleural mesothelioma patients given limited respiratory reserve. Modest but durable antitumor activity was demonstrated. Clin Cancer Res; 22(12); 2874–84. ©2016 AACR.

Supplementary Table 1 Downregulation of HER3 membrane protein expression in skin biopsies following treatment with lumretuzumab and cetuximab or erlotinib Supplementary Table 2 Change of tumor biomarkers in tumor biopsies following treatment with lumretuzumab and cetuximab or erlotinib

Purpose: The receptor tyrosine kinase c-Met plays an important role in tumorigenesis and is a novel target for anticancer treatment. This phase I, first-in-human trial, explored safety, pharmacokinetics, pharmacodynamics, and initial antitumor activity of JNJ-38877605, a potent and selective c-Met inhibitor.Experimental Design: We performed a phase I dose-escalation study according to the standard 3+3 design.Results: Even at subtherapeutic doses, mild though recurrent renal toxicity was observed in virtually all patients. Renal toxicity had not been observed in preclinical studies in rats and dogs. Additional preclinical studies pointed toward the rabbit as a suitable toxicology model, as the formation of the M10 metabolite of JNJ-38877605 specifically occurred in rabbits and humans. Additional toxicology studies in rabbits clearly demonstrated that JNJ-38877605 induced species-specific renal toxicity. Histopathological evaluation in rabbits revealed renal crystal formation with degenerative and inflammatory changes. Identification of the components of these renal crystals revealed M1/3 and M5/6 metabolites. Accordingly, it was found that humans and rabbits showed significantly increased systemic exposure to these metabolites relative to other species. These main culprit insoluble metabolites were generated by aldehyde oxidase activity. Alternative dosing schedules of JNJ-3877605 and concomitant probenecid administration in rabbits failed to prevent renal toxicity at dose levels that could be pharmacologically active.Conclusions: Combined clinical and correlative preclinical studies suggest that renal toxicity of JNJ-38877605 is caused by the formation of species-specific insoluble metabolites. These observations preclude further clinical development of JNJ-38877605. Clin Cancer Res; 21(10); 2297–304. ©2015 AACR.

Purpose:Patients with rare cancers (incidence less than 6 cases per 100,000 persons per year) commonly have less treatment opportunities and are understudied at the level of genomic targets. We hypothesized that patients with rare cancer benefit from approved anticancer drugs outside their label similar to common cancers.Experimental Design:In the Drug Rediscovery Protocol (DRUP), patients with therapy-refractory metastatic cancers harboring an actionable molecular profile are matched to FDA/European Medicines Agency–approved targeted therapy or immunotherapy. Patients are enrolled in parallel cohorts based on the histologic tumor type, molecular profile and study drug. Primary endpoint is clinical benefit (complete response, partial response, stable disease ≥ 16 weeks).Results:Of 1,145 submitted cases, 500 patients, including 164 patients with rare cancers, started one of the 25 available drugs and were evaluable for treatment outcome. The overall clinical benefit rate was 33% in both the rare cancer and nonrare cancer subgroup. Inactivating alterations of CDKN2A and activating BRAF aberrations were overrepresented in patients with rare cancer compared with nonrare cancers, resulting in more matches to CDK4/6 inhibitors (14% vs. 4%; P ≤ 0.001) or BRAF inhibitors (9% vs. 1%; P ≤ 0.001). Patients with rare cancer treated with small-molecule inhibitors targeting BRAF experienced higher rates of clinical benefit (75%) than the nonrare cancer subgroup.Conclusions:Comprehensive molecular testing in patients with rare cancers may identify treatment opportunities and clinical benefit similar to patients with common cancers. Our findings highlight the importance of access to broad molecular diagnostics to ensure equal treatment opportunities for all patients with cancer.

Table S1 lists study sites and investigators who participated in the KEYNOTE-158 NET cohort

Table S1. Tolerability of veliparib, carboplatin, and etoposide over entire combination cycles treatment period Table S2. Mean {plus minus} SD (% CV) pharmacokinetic parameters of veliparib following oral administration of veliparib in combination with carboplatin and etoposide on Cycle 1 Day 1 Figure S1. Mean (+SD) dose normalized etoposide plasma concentration-time profile with and without veliparib co-administration Table S3. Pharmacokinetic parameters of etoposide {plus minus} veliparib Table S4. Objective response rate in biomarker cohort by tumor type and veliparib dose level Figure S2. Tumor response and time on study for all dosed patients (n=39) Figure S3. Progression-free survival by disease type Figure S4. Confirmed objective response by gene type in small cell lung cancer samples (n=17)

Supplementary Table from A Phase I Study of an MPS1 Inhibitor (BAY 1217389) in Combination with Paclitaxel Using a Novel Randomized Continual Reassessment Method for Dose Escalation

Study Protocol.

Study design

Supplementary figure 1

Highlighted immune parameters in blood, plasma and tumor tissue from mUC patients with stable disease as best overall response to anti-PD1

Purpose:CPC634 is a novel nanoparticle entrapping docetaxel, developed to enhance the intratumoral chemotherapy exposure. This randomized cross-over study compared the intratumoral and plasma pharmacokinetics of CPC634 with conventional docetaxel.Patients and Methods:Adult patients with solid tumors were randomized to receive CPC634 (75 mg/m2) in cycle 1, and conventional docetaxel (75 mg/m2) in cycle 2 or vice versa. The study was powered to identify a 25% increase of intratumoral total docetaxel exposure after CPC634 infusion compared with conventional docetaxel. Four patients were allocated per tumor sampling time point, that is, 24, 48, 72, and 96 hours, 7 and 14 days after infusion during both cycles. Total docetaxel and released docetaxel from the nanoparticle were determined in tumor tissue derived from a metastatic lesion and in plasma. Pharmacokinetic data were analyzed using linear mixed modeling.Results:In total, 24 evaluable patients were included. In the tumor, CPC634 exhibited a 461% higher total docetaxel (P < 0.001) and a comparable released docetaxel concentration (P = 0.43). Plasma AUCinf was 27% higher (P = 0.001) and Cmax was 91% lower (P < 0.001) for CPC634 released docetaxel. The median observed neutrophil count nadir after conventional docetaxel treatment was lower (0.50 × 109/L) compared with CPC634 (4.30 × 109/L; P < 0.001).Conclusions:Here, we demonstrated that CPC634 enhanced the intratumoral total docetaxel exposure compared with conventional docetaxel. The lower incidence of neutropenia during CPC634 treatment is presumably related to lower plasma Cmax of released docetaxel. The unique pharmacokinetic profile of CPC634 nanoparticles has the potential to improve docetaxel treatment. A phase II efficacy trial of CPC634 is currently ongoing.

Supplementary Figure from A Phase I Study of an MPS1 Inhibitor (BAY 1217389) in Combination with Paclitaxel Using a Novel Randomized Continual Reassessment Method for Dose Escalation

Transcriptomic analysis of immune cell fractions, T-cell subsets, and chemo-attractants of baseline metastatic tumor biopsies from mUC patients

Immune parameters in blood, plasma and tumor tissue from mUC patients with stable disease as best overall response to anti-PD1

Supplementary Table from Patients with Rare Cancers in the Drug Rediscovery Protocol (DRUP) Benefit from Genomics-Guided Treatment

Supplementary Table 1. Dose reductions and discontinuations in stage 2 Supplementary Table 2. Pharmacokinetic parameters of apitolisib Supplementary Table 3. Summary of radiological responses at the recommended phase 2 doses of 40mg and 30mg 28/28 day schedule for solid tumors and malignant pleural mesothelioma (MPM) patients, respectively. Supplementary Table 4. Characteristics of pleural mesothelioma patients treated with apitolisib Supplementary Figure 1: Apitolisib study schematic Supplementary Figure 2 (A) Waterfall of FDG-PET responses to apitolisib for subjects with higher (black bars) or lower (grey bars) steady-state AUC0-24h than the median exposure for the RP2D of 40 mg QD. (B) The corresponding change in pre-scan fasting blood glucose (FBG) for each patient relative to baseline values during PET procedure. Supplementary Figure 3 CT thorax and MRI pelvis images demonstrate confirmed partial response in E545K PIK3CA mutant clear cell ovarian carcinoma with lung and right common iliac lymphadenopathy on third line apitolisib.

Supplementary Data from A Phase I Study of an MPS1 Inhibitor (BAY 1217389) in Combination with Paclitaxel Using a Novel Randomized Continual Reassessment Method for Dose Escalation

Proposed mechanism of action of pembrolizumab in mUC patients

Supplemental methods. additional description of methods

Figure S1 shows the participant enrollment and treatment disposition.

Correlations between immune parameters in blood, plasma and tumor tissue

Legends to supplementary figure 1 - 9

Supplemental tables 1-6. Supplemental table 1: Patient Demographics and Baseline Characteristics. Supplemental table 2. Plasma Pharmacokinetic parameters. Supplemental table 3. Identification of metabolites of JNJ-38877605. Supplemental table 3. Identification of metabolites of JNJ-38877605. Supplementary table 5: noteworthy post-mortem changes – 1-month recovery phase. Supplementary table 6: noteworthy microscopic changes (Probenecid study.

CD20+ B-cells are present in immune cell niches in lesions of responders upon anti-PD1 treatment

Purpose: A first-in-human phase I study was conducted to characterize safety, efficacy, and pharmacokinetic (PK) and pharmacodynamic (PD) properties of lumretuzumab, a humanized and glycoengineered anti-HER3 monoclonal antibody, in patients with advanced cancer.Experimental Design: Twenty-five patients with histologically confirmed HER3-expressing tumors received lumretuzumab (100, 200, 400, 800, 1,600, and 2,000 mg) every two weeks (q2w) in 3+3 dose-escalation phase. In addition, 22 patients were enrolled into an extension cohort at 2,000 mg q2w.Results: There were no dose-limiting toxicities. Common adverse events (any grade) included diarrhea (22 patients, 46.8%), fatigue (21 patients, 44.7%), decreased appetite (15 patients, 31.9%), infusion-related reactions (13 patients, 27.7%), and constipation (10 patients, 21.3%). The peak concentration (Cmax) and area under the concentration–time curve up to the last measurable concentration (AUClast) of lumretuzumab increased more than dose proportionally from 100 mg up to 400 mg. Linear PK was observed with doses ≥400 mg q2w indicating target-mediated drug disposition saturation. Downregulation of HER3 membranous protein was observed in on-treatment tumor biopsies from 200 mg, and was maximal at and above 400 mg. An ex vivo assay demonstrated increased activation potential of peripheral NK lymphocytes with lumretuzumab compared with a non-glycoengineered anti-HER3 antibody. Ten patients (21.3%) had stable disease and remained on study at a median of 111 days (range, 80–225 days).Conclusions: Lumretuzumab was well tolerated and showed evidence of clinical activity. Linear serum PK properties and plateauing of PD effects in serial tumor biopsies indicate optimal biologically active doses of lumretuzumab from 400 mg onwards. Clin Cancer Res; 22(4); 877–85. ©2015 AACR.

Supplementary Figure legend

Immune cells were mainly present in stroma areas of metastatic lesions of mUC patients.

Supplementary Table 1 tracked changes

Purpose:Monopolar spindle 1 (MPS1) kinase inhibitor, BAY 1217389 (BAY) synergizes with paclitaxel. This phase I study assessed the combination of BAY with paclitaxel using a novel randomized continuous reassessment method (rCRM) to improve dose determination.Patients and Methods:Patients with solid tumors were randomized to receive oral BAY (twice daily 2-days-on/5-days-off) with weekly paclitaxel (90 mg/m2) or paclitaxel monotherapy in cycle 1. Dose escalation was guided by CRM modeling. Primary objectives were to assess safety, establish the MTD of BAY, and to evaluate the pharmacokinetic profiles for both compounds. Simulations were performed to determine the contribution of the rCRM for dose determination.Results:In total, 75 patients were enrolled. The main dose-limiting toxicities were hematologic toxicities (55.6%). The MTD of BAY was established at 64 mg twice daily with paclitaxel. Inclusion of a control arm enabled the definitive attribution of grade ≥3 neutropenia to higher BAY exposure [AUC0–12 (P< 0.001)]. After determining the MTD, we included 19 patients with breast cancer at this dose for dose expansion. Other common toxicities were nausea (45.3%), fatigue (41.3%), and diarrhea (40.0%). Overall confirmed responses were seen in 31.6% of evaluable patients. Simulations showed that rCRM outperforms traditional designs in determining the true MTD.Conclusions:The combination of BAY with paclitaxel was associated with considerable toxicity without a therapeutic window. However, the use of the rCRM design enabled us to determine the exposure–toxicity relation for BAY. Therefore, we propose that the rCRM could improve dose determination in phase I trials that combine agents with overlapping toxicities.

Supplementary Table 1 clean version

Figure 1 Mean ({plus minus}SD) serum lumretuzumab profiles following multiple ascending doses from 100 mg up to 2000 mg Figure 2 Percentage change in standardized uptake value maximum from baseline as assessed by FDG-PET at (a) Cycle 1 Day 14 and (b) Cycle 4 Day 14 Table 1 Summary of baseline and change in HER3 expression measured by IHC in skin and tumor biopsy samples pre and post treatment with lumretuzumab Table 2 Summary of change in expression of HER3, HER2, EGFR and cMET in fresh tumor biopsies compared to primary archival samples Table 3 Peripheral blood immunophenotyping (T, B and NK cells and NK subsets) from patients dosed with 2000 mg lumretuzumab Table 4 Ex-vivo activation potential of peripheral NK lymphocytes for all patients at ba seline and following exposure to lumretuzumab in the 2000 mg dose cohort Table 5 Summary of tumor immune effector cell infiltration Table 6 Tumor response to treatment (RECIST)

Supplementary Figure 1 Quantile plots of baseline membrane HER3 IRS score (A) and HER3 mRNA log expression (B) in baseline FFPE tumor biopsy samples from patients with known adeno- and squamous cell carcinoma histology Supplementary Figure 2 Quantile plots of baseline membrane HER3 immuno-reactive scores in fresh FFPE tumor biopsies from NSCLC patients with non-squamous or squamous histology Supplementary Figure 3 Quantile plots of baseline HRG mRNA log expression determined by qRT-PCR in FFPE tumor biopsies of adeno- and squamous cell carcinoma histology (data obtained using the HRG research assay) Supplementary Figure 4 Baseline HRG mRNA log expression in FFPE tumor biopsies, determined by qRT-PCR, in non-sqNSCLC and sqNSCLC (data obtained using the HRG prototype diagnostic assay). Dotted line indicates prior determined median HRG expression in sqNSCLC. Supplementary Figure 5 Best percentage change from baseline in sum of longest diameters of target lesions for patients in the dose escalation and extension phase of the cetuximab part (n = 38). Red bars indicate PD patients, blue bars SD patients, light green bars PR patients and dark green bars CR patients. Eleven patients were without target lesion assessment post baseline. Tumor type and lumretuzumab dose are indicated. * indicates patients who received previous EGFR-targeting therapy. a indicates a patient who received prior trastuzumab therapy. Supplementary Figure 6 Best percentage change from baseline in sum of longest diameters of target lesions for patients in the dose escalation phase and the two NRG1 fusion gene patients of the extension phase of the erlotinib part (n = 31). Red bars indicate PD patients, blue bars SD patients and green bars PR patients. Eight patients were without target lesion assessment post baseline. Tumor type and lumretuzumab dose are indicated. * indicates patients who received previous EGFR-targeting therapy. a indicates two patients with NRG1 gene fusion.

Background: In depth sequencing of primary breast cancer (BC) has identified a heterogeneous repertoire of disease drivers, evidence of clonal evolution and underlying mutational processes. As cancer evolves over time and under treatment pressure, whole genome sequencing (WGS) of metastatic BC (MBC) tissue is crucial to gain further insight into its genetic make-up and driving forces, thereby allowing improved patient management. Methods: Metastatic tissue and matched germline DNA of patients with MBC (N=501) were prospectively recruited under the biopsy protocol of the Center of Personalized Cancer Treatment (CPCT-02; NCT01855477) and analyzed by WGS. Sequence reads were mapped to the reference genome to call somatic single nucleotide variants (SNV), small insertions and deletions (InDels) and copy number variations from which mutational signatures and tumor mutational burden (TMB; the number of SNV and InDels relative to the genome) were derived. The incidence of called aberrations in our cohort was compared to previously reported WGS data of 560 primary BC (BASIS cohort, Nik-Zainal et al. Nature 2016) (Table 1). Results: According to routine diagnostics 303 patients (60.5%) were ER+/HER2-, 70 (14%) triple negative (TNBC), 95 (19%) HER2+ and the remaining 33 (6.6%) had as of yet an unknown subtype. Top 5 recurrently affected genes were TP53 (47%), ATM (33%), MAP2K4 (32%), NCOR1 (31%), ERBB2 (30%). In the metastatic lesions, median TMB was 2.9 per million base pairs (IQR: 1.7-5.3). Interestingly, 53 (11%) patients had a high TMB (≥10). Compared to primary BC (BASIS cohort), we found (subtype-specific) enrichment of alterations in multiple genes such as ATM (0.4% to 33%), GPS2 (1.3% to 29%), MAP2K4 (6.4% to 32%), CBFB (2.7% to 25%), and, as previously reported, ESR1 (1.3% to 20%). APOBEC signature mutations appeared to be enriched in MBC while HRD signature mutations seemed less prevalent. Analyses to reveal additional genomic features is ongoing as well as the association of genomic alterations with uniquely collected information on prior treatments and response to treatment received directly after biopsy (i.e. endocrine therapy alone or combined with CDK4/6 inhibitors and chemotherapy). Also, to exclude methodological bias the raw data of the BASIS cohort will be processed through our pipeline. Table 1:Comparison of ER+/HER2- and TNBC subtypes: BASIS versus CPCT-02 MBC BASISCPCT-02 MBC ER+/HER2-(%)TNBC(%)ER+/HER2-(%)TNBC(%)Number of samples32615530370Median TMB0.962.632.732.91SNV burden/Mbp0.892.52.412.64InDel burden/Mbp0.060.120.210.3Top 5 affected genesPIK3CA (37)TP53 (83)TP53 (43)TP53 (61) TP53 (20)PTEN (37)ATM (43)MYC (37) CCND1 (20)MYC (26)MAP2K4 (37)CDKN2A (33) GATA3 (15)RB1 (24)NCOR1 (35)CDKN2B (33) MYC (14)PIK3CA (17)CDH1 (34)RB1 (33)Mutational signatures (median relative contribution) Age23.27.213.711.8APOBEC6.97.814.68.3Homologous-recombinant deficiency4.439.94.919.8DNA mismatch Repair Deficiency0.600.30 Conclusion: WGS of this unique cohort of patients with MBC shows a genetic make-up roughly similar to primary BC, but does show subtype-specific enrichment of selected driver mutations in metastatic disease. This study provides better insight into the tumor biology of MBC potentially improving management of these patients. Citation Format: Angus L, Wilting SM, van Riet J, Smid M, Steenbruggen TG, Tjan-Heijnen VC, Labots M, van Riel JM, Bloemendal HJ, Steeghs N, van de Werken HJ, Lolkema MP, Voest EE, Jager A, Cuppen E, Sleijfer S, Martens JW. The genomic landscape of 501 metastatic breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr GS1-07.

Metastatic urothelial carcinoma (mUC) is a lethal cancer with limited therapeutic options available. To identify novel targets for therapy, large-scale sequencing efforts are needed. The Cancer Genome Atlas (TCGA) initiative substantially improved our knowledge on the genomic and transcriptomic characteristics of primary UC (Robertson et al. Cell 2017), however, the molecular landscape of mUC still remains largely unexplored. We performed whole genome sequencing (WGS) on 116 biopsies of UC metastases and matched mRNA sequencing (RNAseq) for 90 samples. We applied genomic alteration analysis on the WGS data and consensus clustering on the RNAseq data. We compared the molecular alterations of mUC with the data of the TCGA cohort and found that most affected driver genes were similar between primary UC and mUC. However, we identified CNTNAP5, RARG and MGP as exclusively mutated driver genes in the metastatic setting, and TP53 alterations were more prevalent in mUC than in primary UC. APOBEC induced mutation signatures in coding and non-coding regions were identified in 91% of the mUC samples and correlated with ploidy, mutational burden and copy number alterations. Based on the etiology of observed mutational signatures, five genomic subtypes were identified in mUC and validated in the TCGA cohort. APOBEC mutagenesis was the main mutational process in genomic subtype 1, which was the most common subtype (67%, N = 78). Moreover, APOBEC deamination mutations were found to be increased over time in paired biopsies from eight patients, indicating that APOBEC enzyme activity is ongoing in the metastatic setting. Unsupervised clustering and comparing phenotypic markers between the groups revealed five clinically-relevant transcriptomic subtypes: luminal-a (20%), luminal-b (20%), stroma-rich (24%), basal/squamous (23%) and a non-specified phenotype (12%). The basal/squamous and stroma-rich subtypes showed highly similar molecular characteristics as the TCGA subtypes, but with enrichment of the stroma-rich and reduction of the basal/squamous subtype. Based on genomic alterations, potential therapeutic targets were identified in 111/116 mUC patients, of which FGFR3 alterations (18%) and fusions (6%), and CDKN2A (44%), ERBB2 (20%), and TSC1 (14%) alterations were most common. By integrating the genomic and transcriptomic data, we propose potential novel therapeutic options per transcriptomic subtype, like addition of a TGF-β inhibitor to immune checkpoint inhibition for the stroma-rich and basal/squamous subtypes that showed the highest TGF-β pathway activity. Using WGS and RNAseq analyses, we provide a comprehensive overview of the molecular landscape of mUC that may serve as a reference for more subgroup-oriented and patient-specific research on the etiology of mUC and future drug development. Citation Format: Maud Rijnders, J Alberto Nakauma-González, Job van Riet, Michiel S. van der Heijden, Jens Voortman, Edwin P. J. G Cuppen, Niven Mehra, Sandra van Wilpe, Sjoukje F. Oosting, L Lucia Rijstenberg, Ellen C. Zwarthoff, Ronald de Wit, Astrid A. van der Veldt, Harmen J. van de Werken, Martijn P. Lolkema, Joost L. Boormans. Integrative genomic and transcriptomic characterization of metastatic urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2190.

Introduction Circulating tumor cells (CTCs) can serve as a source of metastatic tumor material, however their low numbers often limit downstream applications. Diagnostic leukapheresis (DLA) has been shown to substantially increase CTC yield. In this study we isolated CTCs from metastatic prostate cancer (mPCa) patients by DLA to propagate them in vitro as organoid cultures. Furthermore, tumor-derived organoids were used as a model for drug discovery and sensitivity-screening, thereby exploring potential treatment selection. Methods We included 44 mPCa patients into the study and 18 were selected for DLA, based on the presence of ≥5 CTCs/ 7.5 mL blood. We optimized the DLA procedure by comparing low versus high density settings and their impact on CTC isolation efficacy. As the DLA product contains a median of 8.6*10^9 white blood cells (WBC), stringent enrichment methods are needed. CTC enrichment from DLA product was performed by antibody-based WBC depletion alone, or combined with subsequent EpCAM based enrichment. Enriched CTC fractions were cultured in vitro under optimized conditions, to initiate organoid expansion. Results We show that DLA is a safe and efficient method to collect large amounts of CTCs from mPCa patients. With optimized DLA settings we were able to improve CTC enrichment and observed a non-significant increase in CTC yield from DLA (median CTC recovery 15339 vs 5796, P=0.125). WBC depletion alone was found to reduce WBCs by ~2000-fold while retaining >50% of the CTCs, resulting in a WBC to CTC ratio of 545:1. We were able to culture and confirm CTC-derived organoids in 9/18 samples, including one organoid cell line, EMC-PCa-41. Whole Genome Sequencing (WGS) of EMC-PCa-41 revealed a triploid genome characterized by focal amplification of AR, a TMPRSS2-ERG fusion, a PTEN deletion and multiple inter-chromosomal rearrangements. Next we determined copy number profiles in single CTCs and matched organoids from two patients using shallow WGS. These data confirm prior data that CTCs represent the inherent intra-patient heterogeneity and organoids resemble CTCs from the original DLA product. Moreover, we performed an in vitro drug screen with the organoid cell line EMC-PCa-41, and found that it has a limited response to Enzalutamide, which correlated with the relatively short response to Enzalutamide that was observed in the patient. Conclusion Overall our study demonstrates that DLA provides a high CTC yield which enables short-term organoid cultures that preserve the genomic hallmarks of prostate cancer. Viable CTCs obtained by DLA may serve as a (personalized) drug screening system in metastatic prostate cancer. Citation Format: Lisanne Mout, Lisanne F. van Dessel, Jaco Kraan, Anouk C. de Jong, Rui P. Neves, Sigrun Erkens-Schulze, Anieta M. Siewerts, Job van Riet, Ronald de Wit, Stefan Sleijfer, Paul Hamberg, Yorick Sandberg, Peter A. te Boekhorst, Harmen J. van de Werken, John W. Martens, Nikolas H. Stoecklein, Wytske M. van Weerden, Martijn P. Lolkema. Liquid biopsy derived organoids as a potential platform for personalized cancer therapy in metastatic prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3919.