Search

Purpose: Pancreatic ductal adenocarcinoma (PDAC) remains a significant health issue. For most patients, there are no options for targeted therapy, and existing treatments are limited by toxicity. The HOPE trial (Harnessing Organoids for PErsonalized Therapy) was a pilot feasibility trial aiming to prospectively generate patient-derived organoids (PDO) from patients with PDAC and test their drug sensitivity and correlation with clinical outcomes. Experimental Design: PDOs were established from a heterogeneous population of patients with PDAC including both basal and classical PDAC subtypes. Results: A method for classifying PDOs as sensitive or resistant to chemotherapy regimens was developed to predict the clinical outcome of patients. Drug sensitivity testing on PDOs correlated with clinical responses to treatment in individual patients. Conclusions: These data support the investigation of PDOs to guide treatment in prospective interventional trials in PDAC.

BackgroundTumor-specific cytotoxic T cells and T cell receptors are effective tools for cancer immunotherapy. Most efforts to identify them rely on known antigens or lymphocytes that have infiltrated into the tumor bed. Approaches to empirically identify tumor-targeting T cells and T cell receptors by exploiting all antigens expressed on tumor cell surfaces are not well developed for most carcinomas, including pancreatic cancer.MethodsAutologous tumor organoids were stimulated with T cells from the patients’ peripheral blood for 2 weeks to generate the organoid-primed T (opT) cells. opT cell phenotype was analyzed by monitoring changes in the expression levels of 28 cell surface and checkpoint proteins. Expression of ligands of the immune checkpoints was investigated by immunohistochemistry staining. T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and assayed by flow cytometry to monitor tumor-induced T cell proliferation changes. opT cell-mediated killing of three-dimensional organoids was measured using an M30 ELISA kit. T cell receptors (TCRs) were identified by deep sequencing of gDNA isolated from T cells, and the TCR specificity was confirmed by transferring TCRs to the T cell line SKW-3 or donor T cells.ResultsThe co-culture was effective in the generation of CD8 + or CD4+opT cells. The opT cells killed autologous tumors in a granzyme B or Fas-Fas ligand-dependent manner and expressed markers of tissue-resident memory phenotype. Each patient-derived opT cell culture displayed a unique complement of checkpoint proteins. Interestingly, only NKG2A blockade showed a potent increase in the interferon-γ production compared with blocking programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1) or TIM3 or TIGIT or LAG3. Importantly, TCR sequencing demonstrated a dramatic clonal expansion of T cells with a restricted subset of TCRs. Cloning and transferring the TCRs to heterologous T cells was sufficient to confer tumor cell recognition and cytotoxic properties in a patient-specific manner.ConclusionWe report a platform for expanding tumor-targeting T cells from the peripheral blood of patients with pancreatic cancer. We identify the NKG2A-HLA-E axis as a potentially important checkpoint for CD8 +T cells for pancreatic cancer. Lastly, we demonstrate empirical identification of tumor-targeting TCRs that can be used for TCR-therapeutics.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is characterized by dense desmoplastic stroma that limits the delivery of anticancer agents. VCN-01 is an oncolytic adenovirus designed to replicate in cancer cells with a dysfunctional RB1 pathway and express hyaluronidase. Here, we evaluated the mechanism of action of VCN-01 in preclinical models and in patients with pancreatic cancer.MethodsVCN-01 replication and antitumor efficacy were evaluated alone and in combination with standard chemotherapy in immunodeficient and immunocompetent preclinical models using intravenous or intratumoral administration. Hyaluronidase activity was evaluated by histochemical staining and by measuring drug delivery into tumors. In a proof-of-concept clinical trial, VCN-01 was administered intratumorally to patients with PDAC at doses up to 1×1011 viral particles in combination with chemotherapy. Hyaluronidase expression was measured in serum by an ELISA and its activity within tumors by endoscopic ultrasound elastography.ResultsVCN-01 replicated in PDAC models and exerted antitumor effects which were improved when combined with chemotherapy. Hyaluronidase expression by VCN-01 degraded tumor stroma and facilitated delivery of a variety of therapeutic agents such as chemotherapy and therapeutic antibodies. Clinically, treatment was generally well-tolerated and resulted in disease stabilization of injected lesions. VCN-01 was detected in blood as secondary peaks and in post-treatment tumor biopsies, indicating virus replication. Patients had increasing levels of hyaluronidase in sera over time and decreased tumor stiffness, suggesting stromal disruption.ConclusionsVCN-01 is an oncolytic adenovirus with direct antitumor effects and stromal disruption capabilities, representing a new therapeutic agent for cancers with dense stroma.Trial registration numberEudraCT number: 2012-005556-42 and NCT02045589.

Previous studies have shown that metastatic colorectal carcinoma (mCRC) patients treated with bevacizumab, experience variation in the plasma levels of angiogenesis growth factors and related cytokines, called angiogenic switch (AS). The aim of the present study was to analyze the relationship between AS and the clinical response during standard chemotherapy-bevacizumab treatment.Patients with Eastern Cooperative Oncology Group 0-1 mCRC were eligible. Patients received treatment with standard dose capecitabine plus either oxaliplatin or irinotecan and bevacizumab for 6 cycles. Initial treatment was followed by maintenance therapy with bevacizumab plus capecitabine until progression. Plasma levels of angiogenic-related cytokines (hepatocyte growth factor, placental growth factor, macrophage chemoattractant protein-3, MM-9, eotaxin, basic fibroblast growth factor, and interleukin 18) were prospectively analyzed at baseline and every 8 weeks. Progression-free survival (PFS) was calculated using the Kaplan-Meier method.A total of 71 patients were enrolled. AS was observed in 45 patients (63.4%), 28 of whom experienced AS at the first evaluation after treatment start. Disease control, which includes partial/complete response and stable disease, was seen in 96% of AS patients (43/45), but only in 15/26 (58%) for the remaining patients without evidence of AS (P0.001). The median PFS of AS patients was 11.4 months (95% confidence interval, 8.6-15.8) versus 8.3 months for patients without AS (95% confidence interval, 5.6-16.4; P=0.04).Chemotherapy plus Bevacizumab combination in mCRC patients results in dynamic changes in plasma cytokines, which is associated with better disease control and longer PFS. These new findings support continuing studying AS as a potential marker of angiogenesis inhibitor effectiveness.

Patient-derived organoid models are proving to be a powerful platform for both basic and translational studies. Here we conduct a methodical analysis of pancreatic ductal adenocarcinoma (PDAC) tumor organoid drug response in paired patient-derived xenograft (PDX) and PDX-derived organoid (PXO) models grown under WNT-free culture conditions. We report a specific relationship between area under the curve value of organoid drug dose response and in vivo tumor growth, irrespective of the drug treatment. In addition, we analyzed the glycome of PDX and PXO models and demonstrate that PXOs recapitulate the in vivo glycan landscape. In addition, we identify a core set of 57 N-glycans detected in all 10 models that represent 50%–94% of the relative abundance of all N-glycans detected in each of the models. Last, we developed a secreted biomarker discovery pipeline using media supernatant of organoid cultures and identified potentially new extracellular vesicle (EV) protein markers. We validated our findings using plasma samples from patients with PDAC, benign gastrointestinal diseases, and chronic pancreatitis and discovered that 4 EV proteins are potential circulating biomarkers for PDAC. Thus, we demonstrate the utility of organoid cultures to not only model in vivo drug responses but also serve as a powerful platform for discovering clinically actionable serologic biomarkers., Pancreatic ductal adenocarcinoma tumor organoids are effective models for predicting in vivo drug response and discovering new biomarkers.

Metastasis development is the leading cause of cancer-related mortality in pancreatic ductal adenocarcinoma (PDAC) and yet, few preclinical systems to recapitulate its full spreading process are available. Thus, modeling of tumor progression to metastasis is urgently needed. In this work, we describe the generation of highly metastatic PDAC patient-derived xenograft (PDX) mouse models and subsequent single-cell RNA-sequencing (RNA-seq) of circulating tumor cells (CTC), isolated by human HLA sorting, to identify altered signaling and metabolic pathways, as well as potential therapeutic targets. The mouse models developed liver and lung metastasis with a high reproducibility rate. Isolated CTCs were highly tumorigenic, had metastatic potential, and single-cell RNA-seq showed that their expression profiles clustered separately from those of their matched primary and metastatic tumors and were characterized by low expression of cell-cycle and extracellular matrix–associated genes. CTC transcriptomics identified survivin (BIRC5), a key regulator of mitosis and apoptosis, as one of the highest upregulated genes during metastatic spread. Pharmacologic inhibition of survivin with YM155 or survivin knockdown promoted cell death in organoid models as well as anoikis, suggesting that survivin facilitates cancer cell survival in circulation. Treatment of metastatic PDX models with YM155 alone and in combination with chemotherapy hindered the metastatic development resulting in improved survival. Metastatic PDX mouse model development allowed the identification of survivin as a promising therapeutic target to prevent the metastatic dissemination in PDAC.

523 Background: Robust biomarkers are urgently needed to assist in diagnosing pancreatic cancer. Earlier cancer diagnosis could increase survival rates by an estimated 5-fold and more reliable and real-time assessment of treatment effects in patients with cancer could improve quality of life and reduce healthcare costs. Isolation of circulating extracellular vesicles (cEVs) as ‘liquid biopsies’ offers an advantageous approach to diagnose and monitor disease status. Methods: We conducted a comprehensive proteomics study of cEVs from plasma samples to identify EV proteins that may be used as biomarkers for the diagnosis and prognosis of pancreatic cancer. Patients with pancreatic ductal adenocarcinoma (PDAC) of various tumor stages, chronic pancreatitis, intraductal papillary mucinous neoplasm (IPMN), and age-matched controls were enrolled. EVs were isolated directly from plasma samples using the affinity-based EVTrap method then subject to quantitation by liquid chromatography-tandem mass spectrometry. Results: A total of 124 patients (93 with PDAC, 12 with chronic pancreatitis, 8 with IPMN and 11 controls) were included in the discovery cohort. The isolation of EVs with EVtrap allowed the identification on average of 912 EV proteins per 100µL of sample. Principal component analysis of the cEV proteome showed clear separation between PDAC and benign pancreatic diseases. Individuals with IPMN were more closely related to controls, whereas chronic pancreatitis cases were more related to PDAC. At the functional level, we noted that cytokeratin, protein folding chaperons, and actin dynamics regulators were among protein clusters more highly altered in the cEV of patients with PDAC. We further identified new cEV markers associated with metastatic disease, such as PSMB4, RUVBL2, and ANKAR, as well as other EV proteins with strong correlation to prognosis, such as CRP, RALB, and CD55. Finally, we validated a 7-protein PDACEV signature in a validation cohort of 36 separate patients (24 with PDAC, 6 with chronic pancreatitis and 6 with IPMN) which yielded an 89% prediction accuracy for the diagnosis of PDAC. Conclusions: This study provides a valuable resource to the scientific community with a comprehensive catalog of novel proteins on circulating EVs that may assist in the development of novel biomarkers and improve the outcomes of patients with pancreatic cancer.

Purpose: Despite the wide use of antiangiogenic drugs in the clinical setting, predictive biomarkers of response to these drugs are still unknown. Experimental Design: We applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens, including inhibitors to the VEGF:VEGFR2 pathway. We performed extensive functional experiments, including ectopic expression of VEGFR2 mutants in different cell lines, kinase and drug sensitivity assays, and cell- and patient-derived xenografts. Results: WES-cfDNA yielded a 77% concordance rate with tumor exome sequencing and enabled the identification of the KDR/VEGFR2 L840F clonal, somatic mutation as the cause of therapy refractoriness in our patient. In addition, we found that 1% to 3% of samples from cancer sequencing projects harbor KDR somatic mutations located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK. Our in vitro and in vivo functional assays confirmed that L840F causes strong resistance to antiangiogenic drugs, whereas the KDR hot-spot mutant R1032Q confers sensitivity to strong VEGFR2 inhibitors. Moreover, we showed that the D717V, G800D, G800R, L840F, G843D, S925F, R1022Q, R1032Q, and S1100F VEGFR2 mutants promote tumor growth in mice. Conclusions: Our study supports WES-cfDNA as a powerful platform for portraying the somatic mutation landscape of cancer and discovery of new resistance mechanisms to cancer therapies. Importantly, we discovered that VEGFR2 is somatically mutated across tumor types and that VEGFR2 mutants can be oncogenic and control sensitivity/resistance to antiangiogenic drugs. Clin Cancer Res; 24(15); 3550–9. ©2018 AACR.

Purpose: The tumor microenvironment plays a key role in cancer development and progression and is involved in resistance to chemo- and immunotherapy. Cancer-associated fibroblast expressing fibroblast-activating protein α (FAPα) is one of the predominant stroma cell types and is involved in resistance to immunotherapy. Experimental Design: We generated OMTX705, a novel antibody–drug conjugate from a humanized anti-FAP antibody linked to a new cytolysin. Here, we studied its antineoplastic activity in vitro and in preclinical mouse models alone and in combination with chemotherapy as well as immunotherapy in PD-1–resistant tumors. Results: In Avatar models, OMTX705 showed a 100% tumor growth inhibition and prolonged tumor regressions as single agent and in combination with chemotherapy. Treatment rechallenge following treatment discontinuation induced additional tumor regression, suggesting lack of treatment resistance. In a mouse model with a humanized immune system resistant to PD-1 inhibition, OMTX705 increased tumor infiltration by CD8+ T cells, induced complete regressions, and delayed tumor recurrence. Conclusions: These data suggest that FAP targeting with OMTX705 represents a novel and potent strategy for cancer treatment, including tumors resistant to immunotherapy, and support its clinical development.

The aim of this study was to investigate the feasibility and efficacy of personalizing treatment of patients with advanced untreated colorectal cancer (CRC).Patients with untreated metastatic CRC, performance status 0-1, and candidates for systemic chemotherapy were eligible. Tumor tissues were analyzed for KRAS, BRAF, and PI3K mutations and expression of topoisomerase-1 (Topo-1), excision repair cross-complementing gene 1 (ERCC1), thymidylate synthase (TS), and thymidine phosphorylase (TP). Patients with Topo-1 expression received irinotecan, whereas patients with negative Topo-1 and ERCC1 expression received oxaliplatin. Otherwise, patients received physician's choice of treatment. If TS was positive, no fluoropyrimidine was administered and if negative, 5-flurorouracil if TP was negative, or capecitabine if TP was positive. KRAS-mutated patients were treated with bevacizumab, whereas KRAS-native received cetuximab. The primary endpoint of the study was progression-free survival (PFS).A total of 74 patients were enrolled and 67 received personalized treatment including irinotecan (n=27), oxaliplatin (n=16), FOLFIRI (n=12), and FOLFOX (n=12). Thirty-eight patients received cetuximab and 29 bevacizumab. With a median follow-up time of 18.3 months (95% confidence interval [CI], 4-36), the overall median PFS was 8.3 months (95% CI, 6.9-9.7), representing a 12-month PFS rate of 36.5% (95% CI, 25-48). Overall clinical benefit, including response rate and disease stabilization, was 86% (95% CI, 73%-97%). The overall median survival was 21 months (95% CI, 11-40).Real-time target-guided personalized first-line treatment of patients with advanced CRC is feasible but, with the approached used, did not result in a clear improvement in PFS to warrant phase III testing.

The HOPE trial (Harnessing Organoids for PErsonalized Therapy) was a pilot study to test the feasibility of generating patient derived organoids (PDOs) from patients with pancreatic cancer under real world conditions, test drug sensitivity against these PDOs, and correlate these findings with clinical outcomes. Biopsies were obtained primarily during routine clinical care from surgical specimens, ascites, fine needle biopsies (FNB) of primary tumors, and IR guided core biopsies of liver and lymph node metastases. PDOs were grown in WNT free media according to our previously published methods. PDO drug sensitivity testing was performed on a panel of drugs, AUC calculated, and sensitivity ranked. Patients were followed clinically and assessed for disease control. At data cutoff (January 2020), we enrolled a total of 76 subjects representing all stages of disease. Drug testing was performed successfully on PDOs generated from 12 of these subjects (16%). Factors contributing to success obtaining sufficient cells for PDO generation included modality, body part, and tumor cellularity. H&E and IHC corresponded in matched PDOs and donor tumors, as did DNA alterations. Transcriptomes of PDOs were classified as both ‘basal' and ‘classical' subtypes. When AUC values were annotated with clinical data, the Jenks break of 1.69 segregated matched PDO/AUC values into disease control and progressive disease. We estimated that a PDO AUC value 99% probability of disease control from a regimen that contains this drug, whereas if all drugs in a regimen had an AUC > 2.75 there is a > 80% probability of accurately predicting resistance. To illustrate the potential of PDO testing to tailor treatment for an individual patient, we described a case of a subject with stage IV PDAC with a KRAS mutation and ERBB2 amplification. The subject had disease control with FOLFIRINOX, which was held for toxicity. The PDO showed resistance to oxaliplatin and the patient subsequently had an extended period of disease control with regimens which did not include oxaliplatin, highlighting the potential of PDO drug sensitivity testing to exclude ineffective treatments from combination chemotherapy and limit toxicity. In conclusion, we have shown the feasibility of collecting material via real-world clinical practice sufficient to develop PDOs suitable for rapidly screening multiple drugs, and have shown a high degree of correlation between clinical outcomes in patients with PDAC and matched PDO drug sensitivity. We determined preliminary criteria based on the AUC of individual drugs in PDOs to predict drug sensitivity in subjects. These results highlight the potential of PDOs to personalize therapy and allow for the exclusion of ineffective drugs from combination regimens thereby reducing toxicity. We anticipate this approach will be used in future trials to prospectively inform treatment selection for patients with PDAC. Citation Format: Joseph Elan Grossman, Ling Huang, Lakshmi Muthuswamy, Sofia Perea, Dipikaa Akshinthala, Raul Gonzalez, Leo Tsai, Jonah Cohen, Mandeep Sawhney, Douglas Pleskow, Tyler M. Berzin, Bruno Bockorny, Andrea Bullock, Benjamin Schlechter, Mary Linton B. Peters, Catherine Conahan, Supraja Narasimhan, Christine Lim, Roger Davis, Robert Besaw, Martin Smith, Tara Kent, Mark Callery, Senthil K. Muthuswamy, Manuel Hidalgo. Organoid sensitivity in pancreatic cancer correlates with clinical response to treatment and reveals utility for reducing toxicity: Preliminary results from the HOPE trial [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 CT119.

Purpose Gemcitabine plus nanoparticle albumin-bound (NAB) paclitaxel (GA) significantly improved survival compared with gemcitabine alone in patients with metastatic pancreatic ductal adenocarcinoma (PDAC) and a Karnofsky performance status (PS) of 70% or greater. Because of the low number of patients with reduced PS, the efficacy of this regimen in fragile patients remains unclear. This study aimed to evaluate the efficacy and tolerability of different GA dosing regimens in patients with a poor PS. Patients and Methods In the phase I part of this study, patients were randomly assigned to one of the following four parallel GA treatment arms (six patients per arm): a biweekly schedule of NAB-paclitaxel (150 mg/m2 [arm A] or 125 mg/m2 [arm C]) plus gemcitabine 1,000 mg/m2 or a standard schedule of 3 weeks on and 1 week off of NAB-paclitaxel (100 mg/m2 [arm B] or 125 mg/m2 [arm D]) plus gemcitabine 1,000 mg/m2. The two regimens with the better tolerability profile on the basis of predefined criteria were evaluated in the phase II part of the study, the primary end point of which was 6-month actuarial survival. Results Arms B and D were selected for the phase II part of the study. A total of 221 patients (111 patients in arm B and 110 patients in arm D) were enrolled. Baseline characteristics including median age (71 and 68 years in arms B and D, respectively), sex (51% and 55% men in arms B and D, respectively), and metastatic disease (88% and 84% in arms B and D, respectively) were comparable between arms. The most frequent grade 3 or 4 toxicities in arms B and D were anemia (12% and 7%, respectively), neutropenia (32% and 30%, respectively), thrombocytopenia (7% and 11%, respectively), asthenia (14% and 16%, respectively), and neurotoxicity (11% and 16%, respectively). In arms B and D, there were no significant differences in response rate (24% and 28%, respectively), median progression-free survival (5.7 and 6.7 months, respectively), and 6-month overall survival (63% and 69%, respectively). Conclusion NAB-paclitaxel administered at either 100 and 125 mg/m2 in combination with gemcitabine on days 1, 8, and 15 every 28 days is well tolerated and results in acceptable safety and efficacy in patients with metastatic pancreatic ductal adenocarcinoma and a poor PS.

Background: The PI3K/AKT/mTOR signaling pathway plays a key role for the growth and survival of breast cancer cells and aberrations such as phosphatidylinositol-3-kinase (PI3KCA) mutations are common. BKM120 is an oral pan-class I PI3KCA inhibitor. The aim of this study was to evaluate neoadjuvant treatment with BKM120 in PI3KCA mutated early breast cancer in order to assess the effect in PI3K/Akt/mTOR signalling pathway. Methods: Patients (pts) with previously untreated invasive, non-metastatic histologically confirmed breast cancer, with a tumor size ≥ 1,5cm and non-urgent surgical treatment were enrolled. Only women with ER+ / HER2- and PI3KCA gene mutated were eligible. Patients received treatment with BKM120 (100mg/day; administered orally) during 4 weeks. Subsequently, surgery was performed. Inmunohistochemical analysis of pAkt and pS6 (the H-score intensity x%) as well as KRAS mutation were evaluated on tumor tissue at surgery. H score < 150 was related with inactivation of PI3K signalling pathway. Results: To date 24 patients has been included in the study (median age, 52,5 years; range 38-69 years;). 13 out of 24 (54%) had PI3KCA mutated and 8 have completed BKM120 treatment. PI3KCA mutation: Exon 20, 4 pts; exon 9, 2 pts; exon 4, 1 pts and exon 20 and 9, 1 pts. One patient discontinued treatment due to adverse event (nausea grade 2). The most relevant adverse events included transitory increase of transaminases. No serious or grade 4 adverse events were observed. Preliminary results of 7 pts showed inactivation of pAkt (H score < 150) in 2 pts with no change in the reminder 5 pts (median H score 300). Conclusion: Our preliminary data shown that four weeks of BKM120 treatment do not inactivate PI3K/Akt/mTOR signalling pathway, measured throughout pAkt in ER+/HER2-/PI3KCA mutated breast cancer pts. However, we cannot draw any conclusion because the small number of pts included so far. KRAS mutation and pS6 analysis will be presented along with updated results. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-09.

Background: AVANTHER is a phase II trial of preoperative systemic therapy combining B with T and P in a weekly regimen in HER2 positive breast cancer to assess safety and efficacy of the combination. The primary endpoint was pathological complete response (pCR), defined as the absence of invasive disease at the time of histological study. Tissue samples were collected at baseline and the relationship between the expression of PTEN, PI3KCA, TOPO 2a, VEGF, activation of insulin growth factor 1-receptor (IGF1R) and pCR was assessed. Methods: Patients (pts) with centrally-confirmed HER2–positive (IHC 3+ or FISH amplified) breast cancer (stage II or III including locally advanced) received neoadjuvant chemotherapy (NC) with weekly P (80mg/m2/week) for 12 weeks in combination with weekly T (4mg/kg loading dose and 2 mg/kg maintenance) and B (15mg/kg every 3 weeks) for 4 cycles. After surgery all pts received T (1 year) and liposomal doxorubicin plus cyclophosphamide every 3 weeks (4 cycles). Tumor samples were evaluated for the expression of PTEN and TOPO 2α by fluorescent in situ hybridization (FISH), IGF-1R, VEGF, and EGFR using standard immunohistochemistry (IHC). Status of PI3KCA was determined using Fluidigm-based real-time PCR. Results: A total of 44 pts were enrolled. Median tumor size: 3.9 cm. Nine (21%) pts had stage IIA; 19 (45%) stage IIB; 10 (24%) stage IIIA and 4 (10%) stage IIIB. Twenty-nine (69%) pts had estrogen positive receptors. PCR achieved was 42.9%. PTEN expression was normal in all pCR group and 1 patient (pt) (3.8%) had a PTEN deletion in group with no pCR. PI3KCA was mutated in 3pts (16.7%) (1pt E542k and 2 pts H1047r) in the pCR group and 4 pts (15.4%) (1pt E545k, 1pt G1049r and 2 pts H1047r) in the no pCR. Topo 2α was amplified in 8 pts in both groups. The expression of IGFR 1 was negative in 44.4% in the pCR group and 15.4% in patients with no pCR (p = 0.04). VEGF was positive in 7pts (39%) in pCR group and 10pts (38.5%) in no pCR. Conclusions: Our results are in agreement with some prior studies that show that expression of IGFR could be correlated with trastuzumab resistance. Therefore, these findings raise a hypothesis to be validated in prospective studies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-36.

// Rodrigo A. Toledo 1,4,* , Antonio Cubillo 1,2,* , Estela Vega 1,2 , Elena Garralda 1,2 , Rafael Alvarez 1,2 , Lisardo U. de la Varga 1,2 , Jesus R. Pascual 1,2 , Gema Sanchez 1,4 , Francesca Sarno 1,4 , Susana H. Prieto 3 , Sofia Perea 1,4 , Pedro P. Lopez-Casas 4 , Fernando Lopez-Rios 3 and Manuel Hidalgo 4,5 1 Centro Integral Oncologico Clara Campal (CIOCC), Madrid, Spain 2 Universidad San Pablo CEU, Madrid, Spain 3 Laboratorio de Dianas Terapeuticas, Madrid, Spain 4 Spanish National Cancer Research Centre (CNIO), Madrid, Spain 5 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA * These authors have contributed equally to this manuscript Correspondence to: Rodrigo A. Toledo, email: // Manuel Hidalgo, email: // Keywords : colorectal cancer, cfDNA, liquid biopsy, anti-EGFR, cetuximab Received : October 13, 2016 Accepted : October 24, 2016 Published : November 11, 2016 Abstract Cancer genomics and translational medicine rely on the molecular profiling of patient’s tumor obtained during surgery or biopsy. Alternatively, blood is a less invasive source of tumor DNA shed, amongst other ways, as cell-free DNA (cfDNA). Highly-sensitive assays capable to detect cancer genetic events from patient’s blood plasma became popularly known as liquid biopsy (LqB). Importantly, retrospective studies including small number of selected patients with metastatic colorectal cancer (mCRC) patients treated with anti-EGFR therapy have shown LqB capable to detect the acquired clonal mutations in RAS genes leading to therapy resistance. However, the usefulness of LqB in the real-life clinical monitoring of these patients still lack additional validation on controlled studies. In this context, we designed a prospective LqB clinical trial to monitor newly diagnosed KRAS wild-type (wt) mCRC patients who received a standard FOLFIRI-cetuximab regimen. We used BEAMing technique for evaluate cfDNA mutations in KRAS , NRAS, BRAF, and PIK3CA in twenty-five patients during a 2-y period. A total of 2,178 cfDNA mutation analyses were performed and we observed that: a) continued wt circulating status was correlated with a prolonged response; b) smoldering increases in mutant cfDNA were correlated with acquired resistance; while c) mutation upsurge/explosion anticipated a remarkable clinical deterioration. The current study provides evidences, obtained for the first time in an unbiased and prospective manner, that reinforces the utility of LqB for monitoring mCRC patients.

Background: B in combination with T has shown meaningful activity in patients (pts) with metastatic HER2-positive breast cancer. Pathological complete response (pCR) was defined as the absence of invasive disease at the time of histological study and there is a relationship between pCR and disease-free survival (DFS) and overall survival (OS). The complete pathological response rate is between 9 to 34% of patients receiving primary treatment prior to surgery with chemotherapy and when the Herceptin was administrated, a pCR of 39% was reached. AVANTHER is a Phase II trial of preoperative systemic therapy combining B with T and P in a weekly regimen in HER2 positive breast cancer to assess safety and efficacy of the combination. Methods: Pts with centrally-confirmed HER2− positive (IHC 3+ or FISH positive) breast cancer (stage II or III including locally advanced) received neoadjuvant chemotherapy (NC) with weekly P (80mg/m2/week) for 12 weeks in combination with weekly T (4mg/kg loading dose and 2 mg/kg maintenance) and B (15mg/kg every 3 weeks) for 4 cycles. After surgery all pts received T (1 year) and liposomal doxorubicin plus cyclophosphamide every 3 weeks (4 cycles); primary endpoint was rate of pathological complete response (pCR) in breast and axilla. For all patients, a tissue sample at baseline as well as at surgery was collected for biomarker analyses. Results: A total of 44 pts have been enrolled. Median tumor size: 3.9 cm. Nine (21%) pts had stage IIA; 19 (45%) stage IIB; 10 (24%) stage IIIA and 4 (10%) stage IIIB. Twenty-nine (69%) pts had estrogen positive receptors. Data from surgery of 42 patients from a total of 44 patients enrolled were presented in this abstract, but the final results will be present in the symposium. pCR was achieved in 18 (42.9%) pts. Safety and tolerability were good, with rare adverse events of grade 3 [1 (2.4%) episode of severe hypertension, 1 (2,4%) mucosal inflammation]. We presented the results of relationship between magnetic resonance imaging (MRI) and pCR that is one of the secondary objectives of the study. 100% of the patients without pathologic response had stable disease at resonance imaging. Of all patients who had pCR only 55.6% had complete radiological response. Conclusions: These data show that the combination of P with T and B without an anthracycline for 12 weeks is very effective as NC in HER2 positive breast cancer pts with a high rate of pCR and minimal side effects. And the MRI is useful for identifying the persistence of residual disease however it only predicts half of the complete pathological responses Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-14-10.

Background: HER2 amplification is frequent in DCIS and is associated with poor prognosis features. This study aimed to determine the molecular effects in Ras/Raf/MAK and PI3K/AKT signaling pathway of the HER2 inhibitor lapatinib in patients with HER2 positive DCIS as well as correlation with radiological and pathological response. Patients and Methods: Patients (pts) with HER2 positive DCIS received 1500 mg daily of lapatinib for 4 consecutive weeks prior to surgical resection. Changes in tumor volume were evaluated by MRI. The molecular effects of lapatinib on apoptotic level (TUNEL), cell proliferation (Ki67) and HER2 signaling pathway were determined in pre and post-treatment tumor biopsies. Results: A total of 20 pts were included. Lapatinib was well tolerated with only minor and transient side effects reported. The agent effectively modulated HER2 signaling by affecting cell cycle kinetics through decreasing cytoplasm pERK1 in 11 pts with no change or slight increase activation in 5 pts. Of those 11 pts: eight had parallel inactivation of pHER2, two pts had paradoxical activation of pAkt and three pts had an increase in apoptotic level. In addition, seven pts also presented with decrease in MRI signal intensity and tumor size. Conclusions: Four weeks of lapatinib for patients with HER2-positive DCIS result in significant antiproliferative changes through RAS/MAPK signaling pathway together with a decrease in MRI signal. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-18-15.

PURPOSE: To determine whether the addition of rifampin to a quinolone-based antibacterial prophylactic regimen in patients undergoing high-dose chemotherapy (HDC) with peripheral-blood stem-cell transplantation (PBSCT) decreases the incidence of neutropenia and fever, Gram-positive bacteremia, and infection-related morbidity. PATIENTS AND METHODS: Patients with solid tumors undergoing HDC with PBSCT were randomized to receive prophylactic antibiotics with either ciprofloxacin 500 mg orally every 8 hours or the same ciprofloxacin regimen with rifampin 300 mg orally every 12 hours. Prophylaxis was started 48 hours before stem-cell reinfusion. Patients were monitored to document the occurrence of neutropenia and fever, incidence and cause of bacterial infection, time to onset and duration of fever, requirement for intravenous antimicrobials, and length of hospital admission. RESULTS: Sixty-five patients were randomized to receive ciprofloxacin and 65 to receive ciprofloxacin plus rifampin, and from these groups, 62 and 61 were assessable, respectively. The proportion of patients who developed neutropenia and fever was 87% in the group treated with ciprofloxacin and 78% in the group treated with ciprofloxacin and rifampin (P = .25). Although there was a trend toward a reduction in the overall incidence of bacteremia (12 v 4 patients), and Gram-positive bacteremia (8 v 2 patients) with the addition of rifampin, none of these comparisons was statistically significant (P = .05 and P = .09, respectively). CONCLUSION: The results of this study, which demonstrate that rifampin does not improve ciprofloxacin antibacterial prophylaxis in cancer patients undergoing HDC with PBSCT support but that it does increase the occurrence of undesirable side effects, do not support the routine use of rifampin in this setting.

BACKGROUND Hospitalization and treatment with broad-spectrum intravenous antibiotics is the standard care for patients with neutropenia and fever. This randomized clinical trial evaluated the feasibility and efficacy of ambulatory care with oral ofloxacin for patients with low risk, chemotherapy-induced neutropenia and fever. METHODS Patients with solid tumors who were treated with conventional dose chemotherapy, presented with fever (axillary temperature >38°C on 2 occasions or >38.5°C on a single occasion) and neutropenia (absolute neutrophil count

e15126Background: There is a need to develop biomarkers to predict the outcome of patients with metastatic colorectal cancer (CRC) treated with chemotherapy (Ch) plus Bevacizumab (B). It has been r...

Lung cancer is one of the leading types of cancer with an associated high mortality. Currently, treatment with conventional cytotoxic agents remains suboptimal. Inhibition of the epidermal growth factor receptor (EGFR) with monoclonal antibodies or small-molecule inhibitors of the tyrosine kinase (TK) domain of the receptor have demonstrated modest, albeit significant, antitumor activities in patients with non–small-cell lung cancer (NSCLC). The goal of any targeted therapy is to direct treatment to those patients who are more likely to derive benefit from such a treatment. Two interventions may improve the predicted sensitivity to these agents: (1) the use of a real predictor before treatment, represented by pharmacodiagnostic tests, and (2) studying the relationship between an early event after treatment and outcome, represented by pharmacodynamic tests. Pharmacodiagnostic factors that have been studied include the clinical features and histologic subtype of the tumor, the presence of somatic mutations in the TK domain of the EGFR, the expression of the receptor itself, and the activation of the receptor signaling pathway. Pharmacodynamic events associated with improved outcome include drug-induced rash and regulation of the receptor expression. Currently, the best established determinant of response to small-molecule inhibitors of the EGFR is the presence of activating mutations in the receptor TK domain. However, it is not known whether these functional mutations are involved in the response of NSCLC to monoclonal antibodies. Further investigation is needed to better predict patients who are more likely to benefit from these drugs.

This study tested the hypothesis that the number of CA single sequence repeat (CA-SSR) in the intron 1 of the epidermal growth factor receptor (egfr) gene, which affects transcription efficiency of the gene, is associated with the response to EGFR inhibitors. To this end, we determined the number of CA dinucleotides in the intron 1 of the egfr gene in a panel of 12 head and neck cancer cell lines that lack egfr gene amplification and measured the expression of EGFR (mRNA and protein), as well as response to EGFR inhibition. Cells with lower number of CA dinucleotides in the CA-SSR had higher expression of the EGFR gene and protein and were more sensitive to the inhibitory effects of erlotinib, a small molecule inhibitor of the EGFR tyrosine-kinase. Phenotypic modification by silencing EGFR mRNA expression in a susceptible cell line induced resistance to the drug. The number of CA dinucleotide was equivalent in genomic and tumor DNA obtained from 30 patients with head and neck cancer. In a clinical study in colorectal cancer, subjects with lower number of CA dinucleotide frequently developed skin toxicity, a feature that is related to the antitumor activity of this class of drugs. These results suggest that polymorphic variations in the intron 1 of the egfr gene is associated with response to EGFR inhibitors and may provide an explanation as to why the development of skin toxicity is associated with a favorable outcome in patients treated with these agents.

Aim: There is a need to develop biomarkers to predict the outcome of patients with metastatic colorectal cancer (CRC) treated with chemotherapy (Ch) plus Bevacizumab (B). It has been reported that during exposure to B there is a switch in the plasma levels of angiogenesis growth factors and related cytokines called angiogenic switch (AS). It is not known if changes in these circulating factors affect the response of patients with CRC to Ch + B. The aim of this study is to determine the influence of AS in the PFS in pts with metastatic CRC treated with Ch + B. Methods: Treatment naive, ECOG 0-1, metastatic CRC pts were eligible. Patients received induction treatment with XELOX-B or XELIRI-B at standard doses x 6 cycles followed by maintenance treatment with B plus Capecitabine until progression. Angiogenic related cytokines (HGF, PIGF, MCP-3, MM-9, Eotaxin, bFGF and IL-18) were prospectively analyzed at baseline and at the time of each CT evaluation (every eight weeks). AS + was defined by A) doubling of PIGF compared to baseline or B) PIGF elevation with a simultaneous elevation of any two of bFGF, HGF, MCP-3, IL-18 or MMP-9 compared to baseline. Results: Of 62 patients enrolled, 24 (36.7%) have progressed. Median PFS is 15.6 (95% CI: 8.3 to 16.4) months for the entire population. Twenty-five pts (40.3%) were AS + , out of which 23 were detected at first on-study evaluation (week 8). 18 were classified as AS+ based on both criteria, 4 based on criteria A) and 3 based on criteria B). One year (95% CI) PFS for AS+ pts was 82.6% (53.0% to 94.4%) versus 32.9% (13.9% to 53.5%) for AS- pts (p Conclusions: There is a statistically significant improved PFS amongst metastatic CRC AS+ pts treated with first line Ch + B combination compared with AS–pts. Disclosure: All authors have declared no conflicts of interest.

2611 Background: The PI3K/AKT/mTOR signaling pathway plays a key role for the growth and survival of breast cancer cells and aberrations such as phosphatidylinositol-3-kinase (PIK3CA) mutations are...

e11531 Background: The international guidelines include the use of Oncotype DX as a predictor of chemotherapy (CT) benefit in hormonal sensitive breast cancer patients (pts) with node negative and node positive (1-3 positive nodes) disease.The aim of this study was to assess the distribution of the RS in breast cancer pts regardless lymph nodes status, and the association with treatment recommendations. Methods: Retrospectivedata from 131 pts with invasive breast cancer for which the OncotypeDX Assay had been ordered, and pathology data were available. Estrogen (ER) and progesterone (PR) receptor was assessed by IHC (cut-off 10% nuclear staining). Ki67 by IHC [high (≥14%) and low (< 14%)]. Positive-lymph nodes pts was classified as isolated tumoral cells (ITC), micrometastasis (MIC) and macrometasis (MAC). Results: Median age: 51 (range: 35-78); premenopausal status: 74 pts (56%). Median tumor size: 1.5 cm (0.3- 6); Median Ki 67 index: 15 (3-63); Median ER: 93 (35-100) and PR: 85(0-100). 42 pts (32%) had positive-lymph nodes: 6 ITC (14%), 14 MIC (33%) and 22 MAC (52%). RS was low in 82 (63%) cases, intermediate 39 (30%), and high 10 (7%). RS according to nodal status was: positive nodes, 31 pts (74%) low RS, 10 pts (24%) intermediate and 1 pts (2%) high; negative nodes: 50 pts (57%) low RS, 26 (29%) intermediate and 12 pts (14%) high RS. ER and Ki67 was similar between both lymph-nodes groups whereas a higher PR expression (median 90) was seen in positive-lymph nodes vs 76 in negative nodes. First recommendation in positive-lymph nodes: hormonotherapy (HT) 33%, CT 55% and 12% no defined (ND); after RS, HT 83% and CT 17% (p=0.021). Negative nodes first recommendation: HT 68%, CT 23% and ND 14%; after RS, HT 68% and CT 32%. Conclusions: Although based on a small case series, the results show that a substantial number (73%) pts with positive-lymph nodes have low RS, indicating minimal if any benefit from adjuvant CT. The proportion of patients with low scores is higher than in the validation studies and selection bias can’t be excluded. The wide range of RS in both negative and positive-lymph nodes breast cancer confirm the important role of Oncotype DX in treatment decision- making.

e11006 Background: The 2011 St Gallen guidelines recommend the use of Ki67 as a surrogate marker for luminal A and Luminal B breast cancer subtypes. Pts with luminal B subtypes should be considered for adjuvant chemotherapy. The guidelines also recognize the Oncotype DX RS as a predictor of chemotherapy benefit. The aim of this study is to assess the distribution of the RS in luminal A and luminal B breast cancer subtypes as defined by Ki67. Methods: Data from 91 pts with invasive breast cancer for which Oncotype DX results and pathology data were available. Pathological assessment was evaluated by the same pathologist. Estrogen (ER) and progesterone (PR) receptor was assessed by immunostaining (cut-off 10% nuclear staining). Ki67 was assessed by IHC [high (≥14%) and low (< 14%)]. Grading was performed by using Nottingham grading system. Results: Median age: 50 years (range: 35-78); premenopausal status: 49 pts (53.8%). Median tumor size: 1.5 cm (0, 3-6); Median of Ki 67 index: 15. 5 (range: 3-63); Median ER: 93 (35-100) and PR: 85(0-100). Sixty nine pts (75.8%) had negative-lymph nodes. RS was low in 56 (61. 5%) cases, intermediate in 24 (26. 4%), and high in 11 (12. 1%). The median RS was 16 (range 3-55). Forty six (51%) tumors were classified as luminal B (according to high Ki 67) and 38 (41.7%) as Luminal A. In the Luminal B group RS was low in 28 (61%) pts, intermediate in 10 (22%) and high in 8 (17%) while in the Luminal A group 23 (61%) had low RS; 13 (34%) intermediate and 2 (5%) high RS. RS changed the first treatment recommendation in 23 (50%) cases of Luminal B subtype vs. 10 (26%) cases in Luminal A subtype (p=0.013). Conclusions: Although based in a small case series, the results show that a substantial number (61%) pts with Luminal B breast cancer subtype had a low RS, therefore preserving them from adjuvant chemotherapy treatment. In addition, 5% of patients with Luminal A breast cancer had a high RS and thereby likely to benefit from chemotherapy. The wide range of RS in both Luminal B and Luminal A breast cancer subtypes confirm the important role of Oncotype DX in treatment decision- making.

602 Background: B in combination with T has showed meaningful activity in patients (pts) with metastatic HER2-positive breast cancer. AVANTHER is a Phase II trial of preoperative systemic therapy combining B with T and P in a weekly regimen in HER2 positive breast cancer to assess safety and efficacy of the combination. Methods: Pts with centrally-confirmed HER2-positive (IHC 3+ or FISH positive) breast cancer (stage II or III including locally advanced) received neoadjuvant chemotherapy (NC) with weekly P (80mg/m2/week) for 12 weeks in combination with weekly T (4mg/kg loading dose and 2 mg/kg maintenance) and B (15mg/kg every 3 weeks) for 4 cycles. After surgery all pts received T (1 year) and liposomal doxorubicin plus cyclophosphamide every 3 weeks (4 cycles); primary endpoint was rate of pathological complete response (pCR) in breast and axilla. For all patients, a tissue sample at baseline as well as at surgery was collected for biomarker analyses. Results: A total of 44 pts have been enrolled. Median tumor size: 3.9 cm. Seven (19.4%) pts had stage IIA; 17 (47.2%) stage IIB; 8 (22.2%) stage IIIA and 4 (11.1%) stage IIIB. Twenty-one (58.3%) pts had both positive-hormonal receptors and 10 (27.8%) were hormone receptor negative. Eight (22.2%) pts had sentinel biopsy before NC, being negative in 6 (16.7%) cases. Data from surgery (only from 36 pts): pCR was achieved in 16 (44.4%) pts. Safety and tolerability were good, with rare adverse events of grade ≥3 [1 (2.8%) episode of severe hypertension]. Conclusions: These data show that the combination of P with T and B without an anthracycline for 12 weeks is very effective as NC in HER2 positive breast cancer pts with a high rate of pCR and minimal side effects.

3005 Background: Factors that predict the activity of EGFR inhibitors are not known. The EGFR gene has a highly polymorphic region in an intron 1 that consist of a variable number of CA repeats tha...

Introduction Human epidermal growth factor receptor 2 (HER2) amplification is frequent in ductal carcinoma in situ (DCIS) of the breast and is associated with poorly differentiated tumors and adverse prognosis features. This study aimed to determine the molecular effects of the HER2 inhibitor lapatinib in patients with HER2 positive DCIS. Methods Patients with HER2 positive DCIS received 1,500 mg daily of lapatinib for four consecutive weeks prior to surgical resection. Magnetic resonance imaging (MRI) was used to determine changes in tumor volume. The molecular effects of lapatinib on HER2 signaling (PI3K/AKT and RAS/MAPK pathways), cell proliferation (Ki67 and p27) and apoptosis (TUNEL) were determined in pre and post-lapatinib treatment samples. Results A total of 20 patients were included. Lapatinib was well tolerated with only minor and transient side effects. The agent effectively modulated HER2 signaling decreasing significantly pHER2 and pERK1 expression, together with a decrease in tumor size evaluated by MRI. There was no evidence of changes in Ki67. Conclusions Four weeks of neoadjuvant lapatinib in patients with HER2-positive DCIS resulted in inhibition of HER2 and RAS/MAPK signaling pathway. Trial registration 2008-004492-21 (Registered June 25th 2008). Electronic supplementary material The online version of this article (doi:10.1186/bcr3695) contains supplementary material, which is available to authorized users.