Standard Cytotoxic Agents Research Articles

CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GC), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-offunction experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GC-resistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GC against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

Establishment and characterization of multiple patient-derived organoids from a case of advanced endometrial cancer.

Patient-derived organoids (PDOs) retain the original tumor's characteristics to a large degree and allow direct evaluation of the drug sensitivity, thereby emerging as a valuable resource for both basic and preclinical researches. Whereas most past studies stereotypically adopted a single PDO as an avatar of the patient, it remains to be investigated whether this assumption can be justified even for the tumor with spatial diversity. To address this issue, we established and characterized multiple PDOs originating from various sites of a patient with advanced uterine carcinosarcoma (UCS). Specifically, cancer cells were separately sampled from three sites; resected UCS tumor tissue, the peritoneal lavage fluid, and an intra-uterine brushing of the tumor. The three derived PDOs were morphologically undistinguishable, displaying typical carcinoma organoids-like appearance, but two of them proliferated at a faster rate. The primary tumor harbored mutations in TP53 and STK11 along with amplifications in CCNE1, ERBB2, and KRAS. These two mutations and the CCNE1 amplification were detected in all PDOs, while either KRAS or ERBB2 amplification was selectively observed in each PDO in a mutually exclusive manner. Observed intra-tumor heterogeneity in HER2 expression was differentially reproduced in the PDOs, which mirrored each PDO's sensitivity to HER2 inhibitors. Inter-PDO heterogeneity was also evident in sensitivity to standard cytotoxic agents. Lastly, a drug screening identified four candidate reagents commonly effective to all PDOs. Collectively, we showed that multiple PDOs could help reproduce the spatial diversity of a tumor and serve as a valuable resource in UCS research in many respects.

Derivation of pancreatic acinar cell carcinoma cell line HS-1 as a patient-derived tumor organoid.

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long-term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy-derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS-1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS-1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.

Gamma secretase inhibitors as potential therapeutic targets for the notch signaling pathway in uterine leiomyosarcoma (199)

Gamma secretase inhibitors as potential therapeutic targets for the notch signaling pathway in uterine leiomyosarcoma (199)

Experimental and computational assessment of the synergistic pharmacodynamic drug–drug interactions of a triple combination therapy in refractory HER2-positive breast cancer cells

The development of innate and/or acquired resistance to human epidermal growth factor receptor type-2 (HER2)-targeted therapy in HER2-positive breast cancer (HER2 + BC) is a major clinical challenge that needs to be addressed. One of the main mechanisms of resistance includes aberrant activation of the HER2 and phosphatidylinositol 3-kinase/AKT8 virus oncogene cellular homolog/mammalian target of rapamycin (PI3K/Akt/mTOR) pathways. In the present work, we propose to use a triple combination therapy to combat this resistance phenomenon. Our strategy involves evaluation of two targeted small molecule agents, everolimus and dasatinib, with complementary inhibitory circuitries in the PI3K/Akt/mTOR pathway, along with a standard cytotoxic agent, paclitaxel. Everolimus inhibits mTOR, while dasatinib inhibits Src, which is a protein upstream of Akt. An over-activation of these two proteins has been implicated in approximately 50% of HER2 + BC cases. Hence, we hypothesize that their simultaneous inhibition may lead to enhanced cell-growth inhibition. Moreover, the potent apoptotic effects of paclitaxel may help augment the overall cytotoxicity of the proposed triple combination in HER2 + BC cells. To this end, we investigated experimentally and assessed computationally the in vitro pharmacodynamic drug-drug interactions of the various dual and triple combinations to assess their subsequent combinatorial effects (synergistic/additive/antagonistic) in a HER2-therapy resistant BC cell line, JIMT-1. Our proposed triple combination therapy demonstrated synergism in JIMT-1 cells, thus corroborating our hypothesis. This effort may form the basis for further investigation of the triple combination therapy in vivo at a mechanistic level in HER2-therapy resistant BC cells.

Neoadjuvant treatment as a window to chemotherapy driven alterations to the genome and transcriptome of high-grade serous ovarian cancer

<h3>Objectives:</h3> The growing use of neoadjuvant chemotherapy to treat advanced stage high-grade serous ovarian cancer (HGSOC) creates an opportunity to better understand chemotherapy-induced mutational and gene expression changes. A tumor's response to chemotherapy may provide prognostic information or allow prediction of future response to therapy. We performed a cohort study to assess changes in the tumor genome and transcriptome in women receiving neoadjuvant chemotherapy and explored the role of the AP-1 pathway on chemotherapy response. <h3>Methods:</h3> In our single-institution study, we included 50 patients with stage IIIC or IV HGSOC who received neoadjuvant chemotherapy and had archived pre- and post-chemotherapy tumor samples. We performed whole exome RNA-sequencing and panel sequencing of 596 cancer-related genes from FFPE specimens. Significantly differentially expressed genes (DEGs) between pre- and post-chemotherapy samples were identified using paired statistical analysis (GFOLD). An AP-1 inhibitor, SR 11302, was used to explore the in vitro role of AP-1 signaling on chemotherapy response. <h3>Results:</h3> Following tissue and sequencing quality control, the final patient cohort consisted of a total of 32 paired DNA and 20 paired RNA samples from pre- and post-neoadjuvant chemotherapy. Eighteen patients within the cohort had both DNA and RNA available. Mutations in <i>TP53</i> were identified in 31/32 patients. There was a shift in mutational class after chemotherapy from C>G to C>T (p=0.035). Gene expression analyses found that most DEGs were upregulated by chemotherapy, primarily in the chemotherapy resistant specimens. Several genes were solely upregulated in chemotherapy resistant patients, including AP-1 transcription factor family genes (FOS, FOSB, FRA-1), genes implicated in chemotherapy resistance (DUSP1, SERPINE1, MEOX2, ERRFI1), and genes previously associated with cell survival (CCL14, HAS1, NR4A1). The in vitro role of AP-1 was demonstrated by observing an increase in c-Jun expression after treatment with paclitaxel, but not carboplatin. Combining paclitaxel or carboplatin with SR 11302 decreased the IC50 of both therapies. Pairing these therapies additionally demonstrated synergistic effects with a favorable combination index of <1, at effect sizes >75% for paclitaxel and >90% for carboplatin. Additionally, favorable drug dose reduction indices were found at large effect sizes. These observations were most pronounced with the SR 11302 and paclitaxel combination. <h3>Conclusions:</h3> Genomic analysis of tumors before and after chemotherapy did not reveal any consistent mutations associated with chemotherapy response. Gene expression analysis suggested a link between upregulation of the AP-1 pathway in chemotherapy resistant patients after treatment. In vitro experiments indicated that AP-1 inhibition synergizes with standard cytotoxic agents, particularly paclitaxel, and might be a novel approach to overcome or delay paclitaxel resistance in HGSOC.

Systemic Options for Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are rare mesenchymal neoplasms that represent a profound therapeutic challenge due to their high proclivity for recurrence and metastasis and relatively poor response to systemic therapy regimens. While our understanding of the pathophysiology of MPNST is growing, including loss of the tumor suppressor gene neurofibromin and subsequent activation of the Ras pathway, targeted therapy to modify the poor prognosis seen in MPNST patients has thus far been without success. Correspondingly, MPNST patients are treated as per soft tissue sarcoma treatment algorithms with anthracycline-based therapy as the front-line therapy of choice for patients with unresectable, locally advanced, or metastatic MPNST. Beyond first-line anthracycline-based therapy, other standard cytotoxic chemotherapy agents used in advanced MPNST include the alkylating agent ifosfamide and the topoisomerase II inhibitor etoposide. Notably, soft tissue sarcoma regimens are used in MPNST despite distinct systemic therapy sensitivity and prognosis. This is particularly notable for neurofibromatosis type 1 (NF1)-associated MPNST, which is associated with poorer response to systemic therapy and prognosis than sporadic MPNST. As such, NF1-associated MPNST is a particular area in need of novel therapeutic strategies. Given the lack of benefit in the targeting of unique aspects of MPNST disease biology thus far, pre-clinical studies to identify novel rational therapies are critical to inform future clinical trials.

Effects of Maackia amurensis seed lectin (MASL) on oral squamous cell carcinoma (OSCC) gene expression and transcriptional signaling pathways.

Oral cancer causes over 120,000 deaths annually and affects the quality of life for survivors. Over 90% of oral cancers are derived from oral squamous cell carcinoma cells (OSCCs) which are generally resistant to standard cytotoxic chemotherapy agents. OSCC cells often exhibit increased TGFβ and PDPN receptor activity compared to nontransformed oral epithelial cells. Maackia amurensis seed lectin (MASL) can target the PDPN receptor and has been identified as a novel agent that can be used to treat oral cancer. However, mechanisms by which MASL inhibits OSCC progression are not yet clearly defined. Here, we performed cell migration and cytotoxicity assays to assess the effects of MASL on OSCC motility and viability at physiologically relevant concentrations. We then performed comprehensive transcriptome analysis combined with transcription factor reporter assays to investigate the how MASL affects OSCC gene expression at these concentration. Key data were then confirmed by western blotting to evaluate the effects of MASL on gene expression and kinase signaling activity at the protein level. MASL significantly affected the expression of about 27% of approximately 15,000 genes found to be expressed by HSC-2 cells used to model OSCC cells in this study. These genes affected by MASL include members of the TGFβ-SMAD, JAK-STAT, and Wnt-βCTN signaling pathways. In particular, MASL decreased expression of PDPN, SOX2, and SMAD5 at the RNA and protein levels. MASL also inhibited SMAD and MAPK activity, and exhibited potential for combination therapy with doxorubicin and 5-fluorouracil. Taken together, results from this study indicate that MASL decreases activity of JAK-STAT, TGFβ-SMAD, and Wnt-βCTN signaling pathways to inhibit OSCC growth and motility. These data suggest that further studies should be undertaken to determine how MASL may also be used alone and in combination with other agents to treat oral cancer.

A Phase 2a Open-Label Study to Investigate Safety and Tolerability (including the MTD), Efficacy, Pharmacokinetics, Pharmacodynamics and Immunogenicity of MT-3724 in Combination with Lenalidomide in Subjects with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common histologic subtype of Non-Hodgkin Lymphoma (NHL) accounting for approximately 30-40% of NHL cases. Approximately 40% of all newly diagnosed DLBCL patients are either refractory or relapsed following initial response to therapy and represent a population with high unmet need for new therapeutic strategies to achieve or regain disease remission. Because of the near ubiquity and persistence of CD20 expression in B-cell malignancies, there is strong rationale to develop compounds with novel mechanisms of action targeting CD20. However, CD20's non-internalizing nature has, to date, leveraged only cytotoxic mechanisms such as antibody-dependent cellular cytotoxicity to which resistance can arise, and has impeded the development of agents that internalize a cytotoxic payload. MT-3724 is a novel engineered toxin body designed to overcome this limitation by combining the specific target selectivity of a single chain variable fragment with the lethality of a genetically fused Shiga-like toxin A subunit that facilitates both internalization and cell killing by inactivating ribosomal protein synthesis. MT-3724 has been shown to specifically bind and kill CD20+ malignant human B-cells in vitro (IC50 &lt;1 nM) and in CB17 SCID and PDX mice (Rajagopalan 2016; Huang 2018). Given MT-3724's unique ability to kill CD20+ cells by ribosomal inhibition, combining MT-3724 with standard cytotoxic chemotherapy and/or immune modulatory agents may provide enhanced benefit in NHL treatment algorithms. To evaluate this possibility, MT-3724 was tested in combination with Lenalidomide (LEN) to assess additive, synergistic or antagonistic effects in CD20-positive Daudi cell lines. This combination significantly (Ki = 0.40) potentiated cytotoxicity. Based on these results and the known safety profile of MT-3724, LEN was chosen as a combination agent to maximize anti-tumor effects in patients with B-cell NHL while minimizing overlapping toxicity risks. Study Design and Methods: MT-3724 is being evaluated in this Phase 2a study (NCT03645395) in combination with LEN in adult patients with histologically confirmed, relapsed or refractory NHL. The primary objective of this study is to determine the safety and tolerability, including the maximum tolerated dose (MTD), of MT-3724 + LEN. Secondary objectives include pharmacokinetics (PK), pharmacodynamics (PD), immunogenicity and tumor response in subjects treated with MT-3724 + LEN. The study will be conducted in two parts. Part 1 will include MT-3724 dose escalation according to the modified 3+3 design to identify the MTD of MT-3724 in combination with standard doses of LEN and will include up to 24 subjects with histologically confirmed NHL. Part 2 is designed to confirm the safety and tolerability of MT-3724 + LEN in the MTD Expansion Cohort, where the dose declared as MTD of MT-3724 in Part 1 would be given in combination with LEN in up to 40 subjects with CD20+ DLBCL. In addition, the PK, PD, immunogenicity and tumor response of MT-3724 + LEN will be evaluated in Part 2. Eligible subjects will have histologically confirmed, relapsed or refractory CD20+ B-cell NHL that, in the investigator's opinion, could benefit from MT-3724 + LEN therapy. Patients must have received all approved therapies for NHL that are applicable for the patient in the opinion of the treating physician and must have measurable disease by Lugano Classification for NHL. Subjects that are refractory to treatment are eligible, as well as patients who have progressed following CAR T-cell therapy, autologous- or allogeneic stem cell transplant. Serum rituximab level must be negative (&lt;500 ng/mL) at screening. Subjects will receive MT-3724 IV infusions over 1 hour on Days 1, 3, 5, 8, 10 and 12 and will receive LEN on days 1-21 of each 28-day treatment cycle during cycles 1 and 2. Thereafter, MT-3724 will be administered weekly on Days 1, 8, 15, and 22 of each cycle with continued LEN dosing on Days 1-21 for cycles 3 to 6. The study is currently ongoing. Eligible subjects will be identified and treated through competitive enrollment at multiple study centers in North America. Disclosures Burnett: Molecular Templates, Inc.: Employment. Gordon:Abbott: Equity Ownership; Abbvie: Equity Ownership. Strack:Molecular Templates, Inc.: Employment. Lehner:Molecular Templates, Inc.: Employment. Higgins:Molecular Templates, Inc.: Employment, Equity Ownership.

Response rate to chemotherapy after disease progression with anti-PD-1/PD-L1 in metastatic cancer.

e14088 Background: The checkpoint inhibitor (CPI) immunotherapy class of drugs is redefining how we treat cancer. The US FDA has approved CPI drugs as 1st, 2nd or salvage line after progression on conventional chemotherapy (CTX) for multiple cancers including melanoma, lung, bladder and other cancers. However, many questions remain regarding optimal treatment post-progression. Indeed, it has been noted that the patterns of response and relapse to CPI agents are quite different from those of standard cytotoxic agents and that response to CTX AFTER CPI may be different than in the de novo setting. The purpose of this retrospective analysis is to evaluate the activity (response rate (RR), response duration (DOR) and progression free survival (PFS)) of subsequent CTX after disease progression following treatment with CPI. Methods: In this analysis, patients (pts) were enrolled under an IRB approved waiver of consent. We identified pts treated with CPI agents between Jan, 2011 and Dec, 2018 at a multi-site community cancer program who received subsequent CTX as a result of disease progression (PD). We assessed the RECIST RR to subsequent therapy, DOR from onset of response, and PFS from the onset of post CPI CTX, identifying index lesions from the most recent pre-treatment anatomic scan. Results: A total of 47 cases satisfying the above criteria were found; 31 NSCLC, 8 melanoma, 1 SCLC, 1 GEJ, 1 gastric, 2 head/neck, 1 large cell neuroendocrine tumor, 2 bladder cancer. 25 pts had PD as best response to post-CPI CTX. 9 pts (19%) achieved a partial response (PR) with a median DOR of 99 days. 22 pts achieved a PR or stable disease (SD) for a clinical benefit (CB) rate of 47%. The median duration of CB was 92 days. Of the 9 patients who achieved PR, 5/6 had achieved response to CTX prior to CPI . The median PFS for the entire cohort was 97 days. Conclusions: While an expected RR could not be calculated in this heterogenous group of pts, the number and degree of responses suggests CPI possible “priming” that may enhance response to CTX. Post-CPI CTX may be of value and in this retrospective study of a heterogenous group of pts, responses may be more frequent than expected. A larger further study is warranted.

Abstract 1622: In vivo study of dasatinib in combination with veliparib plus carboplatin in triple-negative breast cancer xenograft models

Abstract Background: Recently, poly ADP ribose polymerase (PARP) inhibitors have shown potential to increase the sensitivity of platinum-based agents in tumor cells and provide therapeutic benefit for patients with advanced malignancies in BRCA or DNA repair pathway genes altered or WT condition (Basourakos et al., 2017; Gampenrieder et al., 2017). Src, a non-receptor tyrosine kinase, is involved in the regulation of cell proliferation, survival and apoptotic ability of cancer cells (Tryfonopoulos et al., 2011; Pusztai et al., 2014). Dasatinib (D) is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which has displayed anti-proliferative and anti-metastatic effectiveness against TNBC in both preclinical and clinical studies (Finn et al., 2011). Our current study examines whether combinational inhibition of Src pathway (D) would exhibit greater antitumor activity of PARP inhibitor [veliparib (V)] plus carboplatin (C) in TNBC xenograft models with different genetic back grounds. Methodology: a) We utilized MDA-MB-231, MDA-MB-468 and SUM149PT cells for in vivo study. b) TNBC cells were implanted subcutaneously into immune-compromised mice (NCr Nu/Nu, Taconic) to generate tumor xenografts. c) We evaluated the effects on tumor growth inhibition of D (12.5 mg/kg, i.p., Qd for 4 weeks) / V (25 mg/kg, oral, Bid for 5 days) / C (40mg/kg, i.v., once) as single agent or in combinations (Chan et al., 2012; De et al., 2014). d) For IHC studies, tumor tissue sections were stained with respective antibodies for proliferation, cell survival, angiogenesis and apoptosis, including: phospho-Src, Ki-67, phospho-S6 RP, phospho-Akt, phospho-ERK, CD31, cyclin D1 and cleaved caspase3. e) Student's t test was used for data analyses. Results: We observed that 1) MDA-MB-231(KRAS/BRAF mutated) xenograft model: D alone or combined with V or C or in triple combination inhibited tumor growth, the best tumor inhibition result was induced by triple combination (p &amp;lt; 0.004); 2) MDA-MB-468 (PTEN null) xenograft model: D alone or combined with V or C or in triple combination significantly inhibited tumor growth, the best tumor inhibition result was observed by triple combination (p &amp;lt; 0.00006); 3) SUM149PT (BRCA1 mutated, PTEN null) xenograft model: D alone or combined with V or C or in triple combination inhibited tumor growth, and triple combination, as well as D plus C, showed greater (p &amp;lt; 0.0025) than other combinations / single agents; 4) Single agent of D / V / C was well tolerated with less than 10% mean body weight loss compared with control groups in all models. Conclusion: Our in vivo study suggests that D may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC xenograft tumors with different genetic backgrounds. Immunohistochemical studies of xenograft tumor samples are ongoing, the results of which will be presented at the meeting. Citation Format: Yuliang Sun, Xiaoqian Lin, Jennifer Carlson, Pradip De, Nandini Dey, Casey Williams, Brian Leyland-Jones. In vivo study of dasatinib in combination with veliparib plus carboplatin in triple-negative breast cancer xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1622.

Discovery of N2-(4-Amino-cyclohexyl)-9-cyclopentyl- N6-(4-morpholin-4-ylmethyl-phenyl)- 9H-purine-2,6-diamine as a Potent FLT3 Kinase Inhibitor for Acute Myeloid Leukemia with FLT3 Mutations.

FLT3 tyrosine kinase is a potential drug target in acute myeloid leukemia (AML) because patients with FLT3-ITD mutations respond poorly to standard cytotoxic agents and there is a clear link between the disease and the oncogenic properties of FLT3. We present novel 2,6,9-trisubstituted purine derivatives with potent FLT3 inhibitory activity. The lead compound 7d displays nanomolar activity in biochemical assays and selectively blocks proliferation of AML cell lines harboring FLT3-ITD mutations, whereas other transformed and normal human cells are several orders of magnitude less sensitive. The MV4-11 cells treated with 7d suppressed the phosphorylation of FLT3 and its downstream signaling pathways, with subsequent G1 cell cycle arrest and apoptosis. Additionally, a single dose of 7d in mice with subcutaneous MV4-11 xenografts caused sustained inhibition of FLT3 and STAT5 phosphorylation over 48 h, in contrast to the shorter effect observed after administration of the reference FLT3 inhibitor quizartinib.

Impact of microsatellite instability (MSI) on tumor clonal evolution in metastatic colorectal cancer (mCRC).

616 Background: For mCRC, the contribution of clinical and pathologic factors to concordance between formalin-fixed, paraffin-embedded (FFPE) tissue-based and ctDNA-based mutation profiling remains unclear. MSI is hypothesized to confer a higher rate of somatic alteration resulting in clonal evolution over time compared to microsatellite stable (MSS) patients, but this has not been previously confirmed. Methods: All mCRC patients were consented for a prospective genomic matching protocol (Assessment of Targeted Therapies Against Colorectal Cancer [ATTACC]) using CLIA-certified platforms. Archived tumor DNA from primary or metastatic tissue was sequenced on a 46- or 50-gene panel (Ion Torrent). Paired ctDNA samples were isolated from blood and sequenced with an ultra high-sensitivity assay (Guardant360). Mutation data were normalized by excluding genomic regions not covered on both platforms and were filtered to remove germline or synonymous variants. Results: An initial cohort of 139 patients was included in our analyses, with a median of two lines of intervening chemotherapy between FFPE and plasma DNA collection; 6 (4.3%) of the patients had MSI (MSI-H) mCRC. We detected 472 total mutations in either tissue or ctDNA, with a global concordance rate of 34.5%. Global concordance was not associated with tissue source, nor treatment with specific standard cytotoxic and/or biologic agents. Mutations detected in MSI-H CRC showed significantly greater discordance compared to MSS CRC (OR = 2.5, p = 0.025). This finding was validated using an independent cohort of 17 MSI-H mCRC patients (OR = 2.78, p = 0.00015). Among recurrently altered genes, we found that MSI-H cases were significantly more likely to have gained new TP53 mutations (OR = 8.17, p = 0.001) and to have lost PIK3CA mutations (OR = 8.04, p = 0.036) in ctDNA compared to MSS cases. Conclusions: MSI correlates with discordance between tissue DNA and ctDNA-based mutation profiling, which suggests that MSI-H CRC undergoes distinct patterns of clonal evolution including acquisition of new TP53 mutations. This may have implications for targeted and immunologic therapies in this unique population, and suggests a utility for repeated molecular testing.

The evidence framework for precision cancer medicine.

Precision cancer medicine (PCM) is a concept in which oncologists increasingly strive to tailor the use of targeted therapies in order to match the complexity of the cancer genome. This approach contradicts the historical framework used to support oncology practice that requires evidence from randomized controlled trials in order to change standards of care. This contrast demonstrates the irony of PCM: the therapies themselves are more precise than standard cytotoxic agents, although the clinical evidence supporting the benefits of these therapies is often considerably less precise. Nevertheless, the implementation of PCM should still be based on a framework of evidence-based development that supports clinical decision-making; this approach should not be simple off-label prescription of drugs following sequencing of a tumour biopsy sample. The clinical validation of increasingly complex diagnostic tests, the development of novel methods of evaluating efficacy, and the re-assessment of the standards of evidence sufficient to demonstrate the benefit of precision cancer therapies are all needed before PCM becomes the standard of care for patients with tumours harbouring genomic abnormalities of uncertain clinical significance.

Evaluation of patritumab with or without erlotinib in combination with standard cytotoxic agents against pediatric sarcoma xenograft models.

Integrating molecularly targeted agents with cytotoxic drugs used in curative treatment of pediatric cancers is complex. An evaluation was undertaken with the ERBB3/Her3-specific antibody patritumab (P) either alone or with the ERBB1/epidermal growth factor receptor inhibitor erlotinib (E) in combination with standard cytotoxic agents, cisplatin, vincristine, and cyclophosphamide, in pediatric sarcoma xenograft models that express receptors and ligands targeted by these agents. Tumor models were selected based upon ERBB3 expression and phosphorylation, and ligand (heregulin) expression. Patritumab, E, or these agents combined was evaluated without or with concomitant cytotoxic agents using procedures developed by the Pediatric Preclinical Testing Program. Full doses of cytotoxic agents were tolerated when combined with P, whereas dose reductions of 25% (vincristine, cisplatin) or 50% (cyclophosphamide) were required when combined with P + E. Patritumab, E alone, or in combination did not significantly inhibit growth of any tumor model, except for Rh18 xenografts (E alone). Patritumab had no single-agent activity and marginally enhanced the activity of vincristine and cisplatin only in Ewing sarcoma ES-4. P + E did not increase the antitumor activity of vincristine or cisplatin, whereas dose-reduced cyclophosphamide was significantly less active than cyclophosphamide administered at its maximum tolerated dose when combined with P + E. P had no single-agent activity, although it marginally potentiated the activity of vincristine and cisplatin in one of three models studied. However, the addition of E necessitated dose reduction of each cytotoxic agent, abrogating the enhancement observed with P alone.

Insulin-Like Growth Factor (IGF) Pathway Targeting in Cancer: Role of the IGF Axis and Opportunities for Future Combination Studies.

Despite a strong preclinical rationale for targeting the insulin-like growth factor (IGF) axis in cancer, clinical studies of IGF-1 receptor (IGF-1R)-targeted monotherapies have been largely disappointing, and any potential success has been limited by the lack of validated predictive biomarkers for patient enrichment. A large body of preclinical evidence suggests that the key role of the IGF axis in cancer is in driving treatment resistance, via general proliferative/survival mechanisms, interactions with other mitogenic signaling networks, and class-specific mechanisms such as DNA damage repair. Consequently, combining IGF-targeted agents with standard cytotoxic agents, other targeted agents, endocrine therapies, or immunotherapies represents an attractive therapeutic approach. Anti-IGF-1R monoclonal antibodies (mAbs) do not inhibit IGF ligand 2 (IGF-2) activation of the insulin receptor isoform-A (INSR-A), which may limit their anti-proliferative activity. In addition, due to their lack of specificity, IGF-1R tyrosine kinase inhibitors are associated with hyperglycemia as a result of interference with signaling through the classical metabolic INSR-B isoform; this may preclude their use at clinically effective doses. Conversely, IGF-1/IGF-2 ligand-neutralizing mAbs inhibit proliferative/anti-apoptotic signaling via IGF-1R and INSR-A, without compromising the metabolic function of INSR-B. Therefore, combination regimens that include these agents may be more efficacious and tolerable versus IGF-1R-targeted combinations. Herein, we review the preclinical and clinical experience with IGF-targeted therapies to-date, and discuss the rationale for future combination approaches as a means to overcome treatment resistance.

P-324 - Mitomycin-c (mtc) as salvage therapy for patients with metastatic colorectal cancer (mCRC)

Introduction: For patients with mCRC and disease progression after the available standard cytotoxic and biologic agents, treatment options outside the context of a clinical trial are limited. Mitomycin-C is an old cytotoxic drug that has been used for a variety of solid tumor types in the past. It is still used in some instances for treatment of patients with mCRC in good performance status as salvage therapy. Methods: At the IORS, we retrospectively reviewed patients with mCRC who were treated with mitomycin-C as salvage therapy in a five-year period. All patients had previously received at least two chemotherapy regimens with or without biological agents. Data concerning demographic characteristics, previous treatment, number of cycles of mitomycin-C, toxicity and treatment outcome were analysed. Results: In the period between January 2011 and December 2016, 46 patients were treated with mitomycin-C as salvage therapy, 26 male (56.5%) and 20 female (43.5%), with median age at diagnosis 57 years, range 34 to 73. All patients had previously received oxaliplatin and irinotecan based chemotherapy, with 8 patients (17.4%) also having received bevacizumab in the first line, while 12 patients (21.4%) had treatment with cetuximab in the third line as per reimbursement availability in Serbia (K-ras wild type tumours). Mitomycin-C was administered at a dose of 8 mg/m2 every three weeks as monotherapy (10 pts, 21.7%) or in combination with fluoropyrimidines: capecitabine 1000 mg twice daily (2000 mg per day) days 1 to 14 every three weeks (11 pts, 23.9%), or infusional 5FU per DeGramont regimen (25 pts, 54.4%). ECOG performance status (PS) of our patients at the start of mitomycin-C therapy was: PS0 13 pts (28%), PS1 32 pts (70%) and PS2 1 patient (2%). Dominant metastatic sites were: liver in 35 patients, lung in 9, peritoneum in 9, ovary in 6, while 14 patients had more than one metastatic site. Median number of cycles of mitomycin-C was 4, range 1 to 12. Best response to mitomycin-C therapy was partial response in 1 patient (2%), stable disease in 15 patients (33%), and progression in 30 patients (65%). Two patients were still on mitomycin-C treatment at the cut-off time. Median progression-free survival (mPFS) during mitomycin-C treatment was 2.6 months (2.2-3.1m, CI 95%). There were no statistically significant differences in mPFS between men and women, ECOG performance status at start of MTC therapy, patients with primary left versus right colon tumors, nor depending on localisation of metastatic site(s). Median overall survival in this group of patients was 43 months (35.9-50.1m, CI 95%). Nine patients are still alive at the time of this analysis. Ten patients experienced grade 3 haematologic toxicity, all of them receiving the combination MTC-DeGramont therapy, but were able to complete treatment. There were no grade 4 toxicities. Conclusion: In this study mitomycin-C+/-fluoropyrimidines showed a modest efficacy and a favourable toxicity profile as salvage regimen in mCRC, in a population of patients with prolonged survival. It may be considered for patients with mCRC in good PS who received all available therapies and are not eligible for clinical studies.

Abstract P6-08-04: Preclinical efficacy of dasatinib in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer xenograft model

Abstract Background: Dasatinib is an orally-active ATP-competitive small molecule kinase inhibitor that potently inhibits Abl kinase, Src family kinases and other kinases (Lombardo et al., 2004). Src, one of the key targets of dasatinib is involved in the regulation of cell proliferation, survival and apoptotic ability of cancer cells (Tryfonopoulos et al., 2011; Pusztai et al., 2014). Dasatinib has shown its anti-proliferative and anti-metastatic effectiveness against triple-negative breast cancer (TNBC) in both preclinical and clinical studies (Finn et al., 2011). Several molecular targets including poly ADP ribose polymerase (PARP) are under clinical investigation for the treatment of TNBC. Recently, PARP inhibitors in combination with chemotherapy have shown promising results in this disease in clinical and preclinical studies (Tutt et al., 2010; Kim et al., 2013; De et al., 2014). Here, we hypothesize that dasatinib in combination with PARP inhibitor (ABT888) plus standard cytotoxic agent (carboplatin) will attenuate the growth of both TNBC cell lines and xenograft tumors. Methodology:We have used BT-20 (PIK3CA mutated, H1047R), HCC70 (PTEN null), HCC1937 (BRCA1 mutated, PTEN null), MDA-MB-231 (KRAS/BRAF mutated), MDA-MB-468 (PTEN null) and SUM149PT (BRCA1 mutated, PTEN null) cells for in vitro study. Survival/proliferation, colony formation and apoptosis were examined by using 2D proliferative/growth assay, 3D-ON-TOP assays, and annexinV staining respectively. We next studied the activation status of Src and its downstream signaling. We also have evaluated the effects on tumor growth inhibition of dasatinib/ABT888/carboplatin as a single agent or in combination by using mouse xenograft model. Results: We observed that 1) Dasatinib inhibited Src activation in all tested lines, induced dephosphorylation of ERK1/2 and S6 RP; 2) level of Cyclin D1 was decreased by dasatinib treatment; 3) high anti-proliferative activities were observed following the treatment of dasatinib along with ABT888 plus carboplatin in both 2D proliferation assay and 3D-ON TOP colony formation assay; 4) dasatinib in combination with ABT888 plus carboplatin inducing early stage apoptosis was seen by Annexin V staining in all tested cell lines; 5) dasatinib alone or combined with ABT888 or carboplatin or in triple combination inhibited tumor growth in TNBC xenograft models, the best tumor inhibition result was induced by triple combination (comparing to no treatment control, the mean tumor volume was decreased ~ 87% ). Conclusion: Our in vitro and in vivo studies suggest that dasatinib may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC. Mechanistic studies of xenograft tumor samples are ongoing, the results of which will be presented in the meeting. Citation Format: Sun Y, Lin X, Carlson JH, De P, Dey N, Jepperson T, R &amp; D NCI, Williams C, Leyland-Jones B. Preclinical efficacy of dasatinib in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer xenograft model [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-04.

The marine triterpene glycoside frondoside A induces p53-independent apoptosis and inhibits autophagy in urothelial carcinoma cells

BackgroundAdvanced urothelial carcinomas represent a considerable clinical challenge as they are difficult to treat. Platinum-based combination regimens obtain response rates ranging from 40 to 70% in first-line therapy of advanced urothelial carcinoma. In the majority of cases, however, the duration of these responses is limited, and when progression occurs, the outcome is generally poor. Therefore, novel therapeutic strategies are urgently needed. The purpose of the current research is to investigate the anticancer effects and the mode of action of the marine triterpene glycoside frondoside A in p53-wild type and p53-deficient human urothelial carcinoma cells.MethodsActivity of frondoside A was examined in the human urothelial carcinoma cell lines RT112, RT4, HT-1197, TCC-SUP, T-24, and 486p. Effects of frondoside A on cell viability, either alone or in combination with standard cytotoxic agents were investigated, and synergistic effects were analyzed. Pro-apoptotic activity was assessed by Western blotting and FACS, alone and in combination with a caspases-inhibitor. The impact of functional p53 was investigated by siRNA gene silencing and the p53 inhibitor pifithrin-α. Effects on autophagy were studied using LC3B-I/II and SQSTM/p62 as markers. The unpaired Student’s t-test was used for comparison of the data sets.ResultsFrondoside A shows high cytotoxicity in urothelial carcinoma cells with IC50s ranging from 0.55 to 2.33 μM while higher concentrations of cisplatin are required for comparable effects (IC50 = 2.03 ~ 5.88 μM). Induction of apoptosis by frondoside A was associated with the regulation of several pro-apoptotic factors, like caspase-3, -8, and -9, PARP, Bax, p21, DNA fragmentation, and externalization of phosphatidylserine. Remarkably, inhibition of p53 by gene silencing or pifithrin-α pretreatment, as well as caspase inhibition, did not suppress apoptotic activity of frondoside A, while cisplatin activity, in contrast, was significantly decreased. Frondoside A inhibited pro-survival autophagy, a known mechanism of drug resistance in urothelial carcinoma and showed synergistic activity with cisplatin and gemcitabine.ConclusionsA unique combination of properties makes marine compound frondoside A a promising candidate for the treatment of human urothelial carcinomas.

ActRII blockade protects mice from cancer cachexia and prolongs survival in the presence of anti-cancer treatments.

BackgroundCachexia affects the majority of patients with advanced cancer and is associated with reduced treatment tolerance, response to therapy, quality of life, and life expectancy. Cachectic patients with advanced cancer often receive anti-cancer therapies against their specific cancer type as a standard of care, and whether specific ActRII inhibition is efficacious when combined with anti-cancer agents has not been elucidated yet.MethodsIn this study, we evaluated interactions between ActRII blockade and anti-cancer agents in CT-26 mouse colon cancer-induced cachexia model. CDD866 (murinized version of bimagrumab) is a neutralizing antibody against the activin receptor type II (ActRII) preventing binding of ligands such as myostatin and activin A, which are involved in cancer cachexia. CDD866 was evaluated in association with cisplatin as a standard cytotoxic agent or with everolimus, a molecular-targeted agent against mammalian target of rapamycin (mTOR). In the early studies, the treatment effect on cachexia was investigated, and in the additional studies, the treatment effect on progression of cancer and the associated cachexia was evaluated using body weight loss or tumor volume as interruption criteria.ResultsCisplatin accelerated body weight loss and tended to exacerbate skeletal muscle loss in cachectic animals, likely due to some toxicity of this anti-cancer agent. Administration of CDD866 alone or in combination with cisplatin protected from skeletal muscle weight loss compared to animals receiving only cisplatin, corroborating that ActRII inhibition remains fully efficacious under cisplatin treatment. In contrast, everolimus treatment alone significantly protected the tumor-bearing mice against skeletal muscle weight loss caused by CT-26 tumor. CDD866 not only remains efficacious in the presence of everolimus but also showed a non-significant trend for an additive effect on reversing skeletal muscle weight loss. Importantly, both combination therapies slowed down time-to-progression.ConclusionsAnti-ActRII blockade is an effective intervention against cancer cachexia providing benefit even in the presence of anti-cancer therapies. Co-treatment comprising chemotherapies and ActRII inhibitors might constitute a promising new approach to alleviate chemotherapy- and cancer-related wasting conditions and extend survival rates in cachectic cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0098-2) contains supplementary material, which is available to authorized users.