Highlights of This Issue

The standard treatment for advanced urothelial carcinoma includes platinum-based chemotherapy and programmed cell death protein 1 or programmed death ligand 1 inhibitors. However, urothelial carcinomas are often associated with both intrinsic and acquired resistance to these treatments. Paclitaxel, ifosfamide, and nedaplatin (TIN) chemotherapy has been proven to be effective as the second- or third-line treatment for platinum-resistant advanced urothelial cancer. Herein, we report two cases of patients with advanced bladder cancer resistant to platinum-based chemotherapy or pembrolizumab, who were treated with TIN chemotherapy. The first case was in a 66-year-old woman treated with gemcitabine and cisplatin (GC) chemotherapy followed by gemcitabine, paclitaxel, and cisplatin chemotherapy for multiple pulmonary metastases after radical cystectomy. Following reduction in pulmonary metastases after six courses of TIN treatment, metastasectomy and two courses of adjuvant TIN treatment were administered, with no recurrence for eight years. The other case was in a 70-year-old man treated with GC chemotherapy and pembrolizumab for invasive bladder cancer and multiple pulmonary metastases. We treated this patient with salvage pelvic exenteration. Pulmonary metastases significantly decreased after six courses of TIN chemotherapy. After a partial response for seven months; the patient died due to a novel cerebellar metastasis after six courses of TIN chemotherapy. Thus, we conclude that TIN chemotherapy can be considered as a third line treatment for advanced urothelial cancer resistant to platinum-based chemotherapy and pembrolizumab.

<div>Abstract<p>Combination drug therapy is a widely used paradigm for managing numerous human malignancies. In cancer treatment, additive and/or synergistic drug combinations can convert weakly efficacious monotherapies into regimens that produce robust antitumor activity. This can be explained in part through pathway interdependencies that are critical for cancer cell proliferation and survival. However, identification of the various interdependencies is difficult due to the complex molecular circuitry that underlies tumor development and progression. Here, we present a high-throughput platform that allows for an unbiased identification of synergistic and efficacious drug combinations. In a screen of 22,737 experiments of 583 doublet combinations in 39 diverse cancer cell lines using a 4 by 4 dosing regimen, both well-known and novel synergistic and efficacious combinations were identified. Here, we present an example of one such novel combination, a Wee1 inhibitor (AZD1775) and an mTOR inhibitor (ridaforolimus), and demonstrate that the combination potently and synergistically inhibits cancer cell growth <i>in vitro</i> and <i>in vivo</i>. This approach has identified novel combinations that would be difficult to reliably predict based purely on our current understanding of cancer cell biology. <i>Mol Cancer Ther; 15(6); 1155–62. ©2016 AACR</i>.</p></div>

This is a plain language summary of an article originally published in The New England Journal of Medicine. It is about initial results (collected in October 2019) from the JAVELIN Bladder 100 study (a clinical trial), which looked at avelumab maintenance treatment in people with advanced urothelial cancer. Urothelial cancer is the most common type of bladder cancer. People with advanced urothelial cancer often receive chemotherapy. If this is the first treatment people with advanced disease are given, it is called first-line treatment. If the cancer stops growing or shrinks with first-line chemotherapy, people can be given different treatment to try to prevent the cancer from growing again. This is called maintenance treatment. It may help people live longer. In the JAVELIN Bladder 100 study, researchers wanted to find out if maintenance treatment with avelumab would help people with advanced urothelial cancer live longer. Avelumab is a type of medicine called immunotherapy. Immunotherapy helps the body's immune system fight cancer. 700 people took part in the study. To take part, they must have already been treated with first-line chemotherapy. Also, their cancer must have shrunk or not grown with this treatment. They were then treated with either avelumab maintenance treatment plus best supportive care or best supportive care alone. Best supportive care means treatments that help improve symptoms and quality of life. These treatments do not affect the cancer directly and can include medicines to relieve pain. Researchers found that people treated with avelumab maintenance treatment plus best supportive care lived, on average, 7months longer than people who received best supportive care alone. People treated with avelumab had more side effects than those not treated with avelumab, but most were not severe. Common side effects with avelumab included persistent tiredness, itchy skin, urinary tract infection, and diarrhea. Results from the JAVELIN Bladder 100 study support the use of avelumab as maintenance treatment for people with advanced urothelial cancer whose cancer has shrunk or not grown with first-line chemotherapy. ClinicalTrials.gov NCT number: NCT02603432.

5-Fluorouracil, interferon-α-2b and cisplatin (FAP) for advanced urothelial cancer. A phase II study

Attempts were made to identify factors related to the response of patients treated with intravenous methotrexate, vinblastine, Adriamycin, and cisplatin (M-VAC). The subjects consisted of 54 patients with advanced urothelial cancer whose histological type was transitional-cell carcinoma. The effects of various factors on the response were studied using univariate analysis and a multiple logistic regression model. The following factors were included in the analyses: (1) age, (2) sex, (3) performance status (PS), (4) primary site, (5) histological grade, (6) T category, (7) N category, (8) M category, (9) tumor status, and (10) dose of drugs. In all, 9 patients achieved a complete response and 23 showed a partial response, for an overall response rate of 59% (95% confidence limits, 46%-72%). Univariate analysis revealed that the PS, M category, and dose of drugs were related to the response, and there was a significant correlation among these three factors. In the multiple logistic regression model, the absolute value of t was high for the M category. The presence of distant metastases is an important factor in predicting poor efficacy for the present regimen. The management of metastatic disease will be the subject of further study in the treatment of advanced urothelial cancer.

Carboplatin Based Induction Chemotherapy for Nonorgan Confined Bladder Cancer—A Reasonable Alternative for Cisplatin Unfit Patients?

Objective: The efficacy of second-line treatment after failure of platinum-based chemotherapy in patients with advanced urothelial cancer is limited. Based on encouraging preclinical and clinical phase I data, we evaluated the safety and efficacy of the combination of paclitaxel and everolimus in these patients. Methods: In this trial, patients having failed to respond to prior platinum-based combination treatment of urothelial cancer were treated with paclitaxel (175 mg/m² i.v., 3-weekly) and the mTOR-inhibitor everolimus (10 mg p.o., once daily). The patients were treated until tumor progression or until a maximum of 6 cycles was completed. A one-stage design was used to evaluate the objective response rate (ORR) as the primary endpoint. Results: A total of 27 patients (67% male; median age 63 years) were enrolled. The most frequent grade III/IV toxicities were anemia (28%), peripheral neuropathy (28%), and fatigue (24%). No treatment-related deaths were reported. Complete and partial remissions were observed in 0/24 and 3/24 patients eligible for efficacy analysis, respectively (ORR 13%). Progression-free survival was 2.9 months [95% confidence interval (95% CI) 1.9-4.2], and the median overall survival was 5.6 months (95% CI 4.8-10.2). Conclusion: The combination of paclitaxel and everolimus has not achieved the expected efficacy in second-line treatment of urothelial cancer and should not be further explored.

Blockade of the PD-1/PD-L1 pathway is part of the standard treatment for advanced urothelial cancer, but reliable predictive biomarkers have not been identified. Here, we analyze with Multiplexed Quantitative Immunofluorescence the pretreatment tumor microenvironment (TME) of urothelial cancer samples from patients treated with atezolizumab to identify correlations with treatment efficacy in a Real-World-Evidence (RWE) study. We assessed with Multiplexed Quantitative Immunofluorescence the expression of CD8, PD-1, TIM-3 and LAG-3 on T-cells in the different compartments of the TME (tumor, stroma and whole tissue) in pre-treatment tissue microarrays. We studied associations between the expression of the markers and clinical efficacy. One hundred-nine patients received atezolizumab, showing an overall response rate of 23.8%. Safety was comparable to previous studies with atezolizumab. Pre-treatment tumor samples were available from 45 patients. CD8+ T-cell density was significantly increased in the tumor compartment, but not in the stromal compartment, of patients experiencing complete and partial responses, as compared with patients presenting stable disease or progression. Similar results were observed for co-expression of CD8/PD-1, CD8/TIM-3 and CD8/PD-1/TIM-3/LAG-3. Our findings support the relevance of the density and spatial distribution of CD8+ T-cells and its co-receptors for the clinical efficacy of single-agent PD-L1 blockade in patients with advanced urothelial cancer. RWE studies are a valuable tool for identifying predictive biomarkers.

Antibody-drug conjugate (ADC) and programmed death-1 (PD-1) inhibitors play crucial roles in the treatment of advanced urothelial cancer (aUC). Increasingly, combination treatment modalities are used in patients with aUC intolerant to platinum-based chemotherapy (PBC). However, clinical evidence on the efficacy and safety of disitamab vedotin plus PD-1 inhibitors for aUC is limited. This case series aims to address this knowledge gap. Patients with aUC who were refractory or intolerant to PBC were included. All patients received combined treatment with disitamab vedotin (one of the ADC drugs) and PD-1 inhibitors for at least three cycles. The clinical characteristics of examination, histopathology, outcomes, and adverse events (AEs) were retrospectively collected. Among this case series, eight patients received disitamab vedotin plus PD-1 inhibitors, of which three achieved a complete response (CR) and two had a partial response (PR). The most common AE was peripheral neuropathy (4/8); the remaining AEs were mostly of mild to moderate severity or unknown and were manageable by supportive care. Disitamab vedotin combined with PD-1 inhibitors exhibits a favorable efficacy and safety profile, but subsequent larger cohort clinical studies are required to provide evidence-based medicine for the universal application of this regimen.

Neuroblastoma is the most common tumor in infants younger than 1 year of age and one of the most common solid tumors in childhood. The disease is highly variable: while spontaneous tumor regression occurs more often than in any other cancer type, patients with more severe variant face therapy-resistant recurrence. Therefore we have set out to develop new therapeutic approaches to the disease with a particular focus on aggressive subtypes. Using a high-throughput systems level approach we have identified a novel synergistic combination of approved drugs and compounds in clinical trials. Our initial screen assessed the potency of a panel of clinically applicable drugs by proliferation assays versus a defined panel of isogenic cell lines that are representative of the different neuroblastoma variants. We identified potential drugs combinations by three-dimensional dose response matrices and adopted Bliss values as a quantitative measure of the synergy. The striking synergy was observed in SH-SY5Y cell line using combinations of EGFR/HER2 inhibitors with the apoptosis inducing agents. We were able to validate the observation using colony formation assay. In contrast to single drug treatment, the combination completely abolished the clonogenic growth of neuroblastoma cells, showing the most tangible evidence of the synergy. In order to evaluate our results in more aggressive disease subtypes, we tested our combination in SH-SY5Y cell line with transiently overexpressed N-Myc and in N-Myc-amplified IMR5-75 cell line. Notably, even in this setting we could recapitulate our findings, suggesting that this combination of drugs is highly potent in the chemo-resistant tumor subtypes as well. The same effect was observed when we transiently overexpressed TrkA in SH-SY5Y, mimicking yet another neuroblastoma variant. Pursuing the exact mechanism of the synergy, we have tested the combination in breast cancer cells. To our surprise, the synergistic effect was still present in HER2 negative cell line, but completely absent in the HER2 positive one. We also exchanged the dual EGFR/HER2 inhibitor drug with more potent EGFR inhibitors to conclude that a combination seems to be specific to a particular set of compounds and not necessarily to their main target spectra. Our synergistic combination is highly potent in a large number of neuroblastoma conditions. Strong synergistic effect can be reached at doses much lower than required for the single drug treatment. Animal models that we are currently testing will enable quick translation of our data in clinics. Citation Format: Branka Radic, Uwe Rix, Stefan Kubicek, Giulio Superti-Furga. Probing network fragilities in neuroblastoma by synergistic drug combinations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5038. doi:10.1158/1538-7445.AM2013-5038

Identification of effective drug combinations inhibiting multiple target genes that are essential for cancer cell survival is seen as an effective strategy to overcome drug resistance. Cellular pathways are often redundant, and network rewiring in the face of an assault promotes the emergence of resistant cells that contribute to clinical relapse. However, the number of possible drug-target combinations to investigate by conducting experiments is exponentially large and impractical, therefore warranting the need for systematic approaches to prioritize the most effective drug combinations. Here, we aimed to integrate large-scale drug screening, drug-target affinity knowledge and public RNAi screening datasets on a huge compendium of cancer cell lines to predict synergistic drug combinations. In a case study in an individual breast cancer cell line, MDA-MB-231, we identified drugs selectively active in this cell line compared to the background distribution. We find that target genes of active drugs and other genes involved in related cellular processes were also differentially essential in RNAi screening data. Further, we observe that the drug combinations of these selectively active drugs were synergistic. We propose a systematic approach for integrating chemical screening, drug target and cellular processes knowledge, and functional genetic screens to optimize drug combination testing efforts. Citation Format: Alok Jaiswal, Prson Gautam, Jing Tang, Krister Wennerberg, Tero Aittokallio. Prediction of synergistic anticancer drug combinations by integrating chemical and genetic screens [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A26.

Identifying synergistic drug combinations (SDCs) is a great challenge due to the combinatorial complexity and the fact that SDC is cell line specific. The existing computational methods either did not consider the cell line specificity of SDC, or did not perform well by building model for each cell line independently. In this paper, we present a novel encoder-decoder network named SDCNet for predicting cell line-specific SDCs. SDCNet learns common patterns across different cell lines as well as cell line-specific features in one model for drug combinations. This is realized by considering the SDC graphs of different cell lines as a relational graph, and constructing a relational graph convolutional network (R-GCN) as the encoder to learn and fuse the deep representations of drugs for different cell lines. An attention mechanism is devised to integrate the drug features from different layers of the R-GCN according to their relative importance so that representation learning is further enhanced. The common patterns are exploited through partial parameter sharing in cell line-specific decoders, which not only reconstruct the known SDCs but also predict new ones for each cell line. Experiments on various datasets demonstrate that SDCNet is superior to state-of-the-art methods and is also robust when generalized to new cell lines that are different from the training ones. Finally, the case study again confirms the effectiveness of our method in predicting novel reliable cell line-specific SDCs.

Acquired resistance to vemurafenib develops through reactivation of RAF/MEK/ERK signaling or bypass mechanisms. Recent combination therapies such as a MEK inhibitor combined with vemurafenib show improvement in major clinical end points, but the percentage of patients with adverse toxic events is higher than with vemurafenib monotherapy and most patients ultimately relapse. Therefore, there is an urgent need to develop new antimelanoma drugs and/or adjuvant agents for vemurafenib therapy. In this study, we created a novel semiorganically modified derivative, DETD-35, from deoxyelephantopin (DET), a plant sesquiterpene lactone demonstrated as an anti-inflammatory and anti-mammary tumor agent. Our results show that DETD-35 inhibited proliferation of a panel of melanoma cell lines, including acquired vemurafenib resistance A375 cells (A375-R) established in this study, with superior activities to DET and no cytotoxicity to normal melanocytes. DETD-35 suppressed tumor growth and reduced tumor mass as effectively as vemurafenib in A375 xenograft study. Furthermore, DETD-35 also reduced tumor growth in both acquired (A375-R) and intrinsic (A2058) vemurafenib resistance xenograft models, where vemurafenib showed no antitumor activity. Notably, the combination of DETD-35 and vemurafenib exhibited the most significant effects in both in vitro and in vivo xenograft studies due to synergism of the compound and the drug. Mechanistic studies suggested that DETD-35 overcame acquired vemurafenib resistance at least in part through deregulating MEK-ERK, Akt, and STAT3 signaling pathways and promoting apoptosis of cancer cells. Overall, our results suggest that DETD-35 may be useful as a therapeutic or adjuvant agent against BRAF(V600E) mutant and acquired vemurafenib resistance melanoma. Mol Cancer Ther; 15(6); 1163-76. ©2016 AACR.

<div>Abstract<p>Antibody–drug conjugates are one of the most diffused targeted therapeutic modalities for cancer treatment and consist of a tumor-targeted monoclonal antibody connected to a cytotoxic payload, which is released selectively at the tumor site. Small molecule–drug conjugates (SMDC) represent an alternative approach, in which the antibody is replaced by a tumor-homing small organic ligand. Thanks to their small molecular size, SMDCs are characterized by rapid extravasation and enhanced penetration in solid tumors compared with antibody–drug conjugates. We recently developed SMDCs targeting fibroblast activation protein (FAP), a cell surface endopeptidase abundant in the tumor microenvironment, using the highly specific FAP inhibitor OncoFAP as a targeting moiety. In this study, we compared the tumor-targeting properties and <i>in vivo</i> activity of SMDCs based on OncoFAP against products based on a stronger FAP inhibitor (i.e., trivalent OncoFAP), aiming to tune the release kinetic of the cytotoxic payload to the neoplastic site. We compared the kinetic profiles of the monovalent and trivalent derivatives of OncoFAP through <i>in vivo</i> and <i>ex vivo</i> biodistribution and therapy studies. The distinct <i>in vivo</i> monomethyl auristatin E (MMAE) release obtained for OncoFAP–GlyPro–MMAE and TriOncoFAP–GlyPro–MMAE did not lead to substantial differences in therapeutic efficacy in a preclinical FAP-positive cancer model.</p></div>

Oral cancer is one of the most common forms of cancer among developing countries but despite current efforts, the survival of oral cancer has remained constant. Current research on oral cancer is centered on clinical studies focused on single drugs or very few combinations of the most common therapeutics historically used to treat oral cancer. However, there is a major gap in the literature about combinations of drug treatments for oral cancer. The goal of this study is to explore combination treatments and assess whether any synergistic effects can be produced. To accomplish this, two oral squamous cell carcinoma (OSCC) cell lines, Cal‐27 and OECM‐1 from the tongue and the oral cavity, respectively, will be used to investigate the efficacy of single drugs as well as combinations of drugs from a wide array of chemotherapy agents currently used to treat oral cancer as well as other forms of cancer. Thus far, Cell Titer Glo assays have been performed in triplicate for both Cal‐27 and OECM‐1 to analyze their viability in the presence of four drugs used to treat other forms of cancer with different mechanisms of action: PD0, a MEK inhibitor, IBET‐762, a BET inhibitor, ABT‐199, a Bcl‐2 inhibitor, and JQ1, a BRD4 inhibitor. GI50 values were gathered from each assay. Cal‐27 presented higher GI50 values than OECM‐1 for IBET‐762 and JQ1 at 1.52 mM and 1.64 mM, respectively. The GI50 values for OECM‐1 treatment with IBET‐762 and JQ1 were lower at 1.07 mM and 1.21 mM, respectively. Both cell lines were found to have similar resistance to PD0, around 1.05 mM, and ABT‐199, around 1.02 mM. High GI50 values across the cell lines indicate that both Cal‐27 and OECM‐1 are resistant to the four drugs that were tested. In the future, Cal‐27 and OECM‐1 will be treated with a panel of 10 drugs currently used clinically to treat oral cancer to gather baseline data to understand how these cell lines react to current treatment. The drugs that will be used include: Cisplatin, 5‐FU, Taxol, Docetaxel, Hydroxyurea, Methotrexate, Bleomycin, Gemcitabine, and Vincristine, all of which interfere with DNA synthesis and cell growth. Then, Cal‐27 and OECM‐1 will be treated with new combinations of drugs currently used to treat oral cancer and drugs not currently used for oral cancer but used to treat other forms of cancer. Some of these drugs include: Gefitinib, a EGFR inhibitor, Venetoclax, a BCL‐2 inhibitor, Irinotecan, a topoisomerase inhibitor, Mitoxantrone, a topoisomerase 2 and PKC inhibitor, and several other drugs. Cellular responses will then be assessed to determine if any combinations are synergistic. The data generated can aid researchers in understanding how different OSCC cells respond to drug combinations, which may lead to more successful clinical treatment options in the future.