Background: Long-term anti-EGFR antibody treatment increases the risk of severe dermatologic toxicities. This single-arm, phase II trial aimed to investigate the strategy of switching from cetuximab to bevacizumab in combination with FOLFIRI based on early tumor shrinkage (ETS) in patients with RAS wild-type metastatic colorectal cancer (mCRC). Methods: Radiologic assessment was performed to evaluate ETS, defined as >= 20% reduction in the sum of the largest diameters of target lesions 8 weeks after the introduction of FOLFIRI plus cetuximab. ETS-negative patients switched to FOLFIRI plus bevacizumab, whereas ETS-positive patients continued FOLFIRI plus cetuximab for eight more weeks, with a switch to FOLFIRI plus bevacizumab thereafter. The primary endpoint was progression-free survival. Results: This trial was prematurely terminated due to poor accrual after a total enrollment of 30 patients. In 29 eligible patients, 7 were ETS-negative and 22 were ETS-positive. Two ETS-negative patients and 17 ETS-positive patients switched to FOLFIRI plus bevacizumab 8 weeks and 16 weeks after initial FOLFIRI plus cetuximab, respectively. Median progression-free and overall survival durations were 13.4 and 34.7 months, respectively. Six (20%) patients experienced grade >= 3 paronychia, which improved to grade <= 2 by 18 weeks. Grade >= 3 acneiform rash, dry skin, and pruritus were not observed in any patients. Conclusions: Our novel treatment strategy delivered acceptable survival outcomes and reduced severe dermatologic toxicities.

Supplementary Table 1 from An In vivo Platform for Translational Drug Development in Pancreatic Cancer

BACKGROUND:Few prospective studies have used liquid biopsy testing in RAS-mutant metastatic colorectal cancer (mCRC), and its clinical significance remains unknown. Therefore, this study aimed to carry out a biomarker analysis by liquid biopsy using updated data of the phase II trial of FOLFOXIRI plus bevacizumab as first-line chemotherapy for RAS-mutant mCRC.MATERIALS AND METHODS:A total of 64 patients who received modified FOLFOXIRI regimen (irinotecan 150 mg/m2, oxaliplatin 85 mg/m2, levofolinate 200 mg/m2, and fluorouracil 2400 mg/m2) plus bevacizumab biweekly were enrolled. The primary endpoint was the objective response rate (ORR). Plasma samples were collected at pre-treatment, 8 weeks after treatment, and progression in participants included in the biomarker study. The levels of circulating tumour DNA (ctDNA) and specific KRAS and NRAS variants were evaluated using real-time PCR assays.RESULTS:There were 62 patients (median age: 62.5 years, 92% performance status 0, 27% right side) who were assessable for efficacy and 51 for biomarker analysis. ORR was 75.8% (95% confidence interval 65.1% to 86.5%). The median progression-free survival was 12.1 months, and the median overall survival (OS) was 30.2 months. In 78% of patients, RAS mutations disappeared in the ctDNA at 8 weeks after treatment; these patients tended to have better outcomes than those with RAS mutations. Interestingly, RAS mutations remained undetectable during progression in 62% of patients. Survival analysis indicated that the median OS from progression was significantly longer in patients with RAS mutation clearance than in those with RAS mutation in the ctDNA at disease progression (15.1 versus 7.3 months, hazard ratio: 0.21, P = 0.0046).CONCLUSIONS:Our biomarker study demonstrated no RAS mutations in ctDNA at disease progression in 62% of patients with RAS-mutant mCRC. Both OS and post-progression survival were better in patients with clearance of RAS mutations in ctDNA after triplet-based chemotherapy.

Early tumor shrinkage (ETS) by anti-EGFR antibody was associated with favorable prognosis in metastatic colorectal cancer (mCRC), but continuation of anti-EGFR antibody for a long period is often difficult due to skin toxicity. We conducted a phase II study to evaluate the switch treatment strategy initiating first-line FOLFIRI+cetuximab (Cet) as induction chemotherapy followed by FOLFIRI+bevacizumab (Bev) according to ETS at 8 weeks, which was defined as > 20% decrease in the sum of the longest diameters of target lesions (RECIST ver.1.1), for patients with RAS wild-type mCRC: early switch at 8 weeks for patients without ETS and late switch at 16 weeks for patients with ETS, respectively. The primary endpoint was progression-free survival (PFS). Secondary endpoints included response rate (RR), ETS rate, depth of response (DpR), time to treatment failure (TTF), overall survival (OS), and safety. A sample size of 54 provided the power of 70% to test a hypothesis of threshold PFS of 10 months and an expected PFS of 15 months at a one-sided significance level of 0.1 using the method of Brookmeyer and Crowley. Blood samples before treatment and at 8 weeks were collected to analyze specific RAS, BRAF, and PIK3CA variants in ctDNA using real-time PCR. This trial was terminated due to slow accrual when 30 patients were enrolled with a median follow-up time of 25.4 months. In 29 evaluable patients (median age 66-y, male 72%, ECOG PS0-1 97%, left-sided tumor 72%), median PFS was 13.4 (95% CI: 9.0-15.8) months and median OS was 34.7 (95% CI: 24.16-NR) months. RR, ETS rate were 72.4% (21/29), 75.9% (22/29). Median DpR was 47.7% (range: -40-100%) with median time to DpR of 3.94 months (range: 0.43-17.02). Median PFS was 14.2 months in 22 patients with ETS and 6.1 months and in 7 patients without ETS, respectively. Ten patients with ETS and one patient without ETS underwent conversion surgery, and its indication was judged between 8 and 16 weeks in 7 patients. In 12 patients with ETS who did not undergo conversion surgery, median PFS were 14.6 months (95% CI: 6.08-25.92). Grade 2/3 paronychia was observed in 13 patients (43.3%: 12 of 22 patients with ETS and 1 of 7 patients without ETS) with median time to occurrence of 10 weeks (range: 5-22 weeks) recovering to grade 1 after median time of 7 weeks (range 4-15) from the last Cet administration. BRAF V600E mutation (n=3) and RAS G13D mutation (n=1) were detected in ctDNA at pre-treatment in 4 of 7 patients without ETS but in none of 22 patients with ETS. This ETS-based treatment switch strategy from Cet to Bev may be a clinically useful option with favorable risk benefit balance showing much better clinical outcomes but associated with higher incidence of grade 2/3 paronychia in patients with ETS than those without ETS. It is suggested that mutations in ctDNA could be a biomarker of ETS.

Rarely, scientific developments centered around the patient as a whole are published. Our multidisciplinary group, headed by gastrointestinal surgeons, applied this research philosophy considering the most important aspects of the diseases "colon- and rectal cancer" in the long-term developments. Good expert cooperation/knowledge at the Comprehensive Cancer Center Ulm (CCCU) were applied in several phase III trials for multimodal treatments of primary tumors (MMT) and metastatic diseases (involving nearly 2000 patients and 64 centers), for treatment individualization of MMT and of metastatic disease, for psycho-oncology/quality of life involving the patients' wishes, and for disease prevention. Most of the targets initially were heavily rejected/discussed in the scientific communities, but now have become standards in treatments and national guidelines or are topics in modern translational research protocols involving molecular biology for e.g., "patient centered individualized treatment". In this context we also describe the paths we had to tread in order to realize our new goals, which at the end were highly beneficial for the patients from many points of view. This description is also important for students and young researchers who, with an actual view on our recent developments, might want to know how medical progress was achieved.

Aim: We report an exploratory analysis of cfRNA as a biomarker to monitor clinical responses in non-small cell lung cancer (NSCLC), breast cancer, and colorectal cancer (CRC). An analysis of cfRNA as a method for measuring PD-L1 expression with comparison to clinical responses was also performed in the NSCLC cohort. Methods: Blood samples were collected from 127 patients with metastatic disease that were undergoing therapy, 52 with NSCLC, 50 with breast cancer, and 25 with CRC. cfRNA was purified from fractionated plasma, and following reverse transcription (RT), total cfRNA and gene expression of PD-L1were analyzed by real-time polymerase chain reaction (qPCR) using beta-actin expression as a surrogate for relative amounts of cfDNA and cfRNA. For the concordance study of liquid biopsies and tissue biopsies, the isolated RNA was analyzed by RNAseq for the expressions of 13 genes. We had to close the study early due to a lack of follow-up during the Covid-19 pandemic. Results: We collected a total of 373 blood samples. Mean cfRNA PCR signals after RT were about 50-fold higher than those of cfDNA. cfRNA was detected in all patients, while cfDNA was detected in 88% of them. A high concordance was found for the expression levels of 13 genes between blood and solid tumor tissue. Changes in cfRNA levels followed over the course of treatments were associated with response to therapy, increasing in progressive disease (PD) and falling when a partial response (PR) occurred. The expression of PD-L1 over time in patients treated with immunotherapy decreased with PR but increased with PD. Pre-treatment levels of PD-L1 were predictive of response in patients treated with immunotherapy. Conclusion: Changes in cfRNA correlate with clinical response to the therapy. Total cfRNA may be useful in predicting clinical outcomes. PD-L1 gene expression may provide a biomarker to predict response to PD-L1 inhibition.

KRAS mutant NSCLC is heterogeneous with differential responses to MEK inhibition and immune checkpoint blockade that may be dependent on: KRAS amino acid substitution, associated co-mutations, smoking status, and tumor mutational burden. Preclinical models suggest sequencing of MEK inhibitor before or after PD-1 blockade differentially modulate the immune microenvironment, impacting anti-tumor activity of PD-1 blockade. This phase 1 study examined intercalated sequencing schemes of the MEK inhibitor trametinib and the PD-1 antibody pembrolizumab with planned dose expansion in KRAS mutant NSCLC.

The most analyzed nucleic acid in liquid biopsies is cfDNA due to its presumed greater stability compared with cfRNA. However, sometimes it is difficult to isolate sufficient tumor cfDNA from blood for analyses. In theory, cfRNA representing a particular gene expression should be present at much higher levels than the corresponding gene in cfDNA because a single gene is normally transcribed many times. cfRNA should contain only those mutations that are consequential for tumor development and also allows the measurement of tumor gene expressions.

Molecular profiling of prostate cancer with liquid biopsies, such as circulating tumor cells (CTCs) and cell-free nucleic acid analysis, yields informative yet distinct data sets. Additional insights may be gained by simultaneously interrogating multiple liquid biopsy components to construct a more comprehensive molecular disease profile. We conducted an initial proof-of-principle study aimed at piloting this multiparametric approach. Peripheral blood samples from men with metastatic castrate-resistant prostate cancer were analyzed simultaneously for CTC enumeration, single-cell copy number variations, CTC DNA and matched cell-free DNA mutations, and plasma cell-free RNA levels of androgen receptor (AR) and AR splice variant (ARV7). In addition, liquid biopsies were compared with matched tumor profiles when available, and a second liquid biopsy was drawn and analyzed at disease progression in a subset of patients. In this manner, multiparametric liquid biopsy profiles were successfully generated for each patient and time point, demonstrating the feasibility of this approach and highlighting shared as well as unique cancer-relevant alterations. With further refinement and validation in large cohorts, multiparametric liquid biopsies can optimally integrate disparate but clinically informative data sets and maximize their utility for molecularly directed, real-time patient management.

e14567 Background: Levels of cell-free circulating RNA (cfRNA) in cancer patients (pts) indicate tumor gene expressions and thus may provide a means, not only of evaluating response, but also for monitoring and predicting outcome to therapy. Methods: Blood was drawn every 6-8 weeks from pts undergoing various treatments (tx). CfRNA was extracted from resulting plasma and generated random-primed cDNA. Total cfRNA was quantitated by qPCR of β-actin, and correlated with pts response (CR/PR/SD/PD) determined by CT scans. Changes in PD-L1 expression were used to monitor response to immunotherapy in lung cancer pts. 125 pts (50 lung, 51 breast, 24 colon) were enrolled. Among the three tumor types, a total of 84 pts completed 1-3 lines of tx with an average of 5 blood draws per pt. Results: Changes in total levels of cfRNA over time correlated with pts outcomes in all three tumor types and were independent of the specific therapies. Increasing (INC) levels of cfRNA were predictive of disease progression and decreasing (DEC) levels with benefit from therapy, with an overall concordance of 81% (68/84 pts). Changes in relative PD-L1 expression were associated with immunotherapy outcomes in lung cancer (INC associated with progression, DEC associated with benefit). Lung: There was a 79% (23/29) concordance between changes in cfRNA levels and pts response. Changes in relative PD-L1 expression were predictive of outcome to immunotherapy in 9/10 pts. Breast: There was an 83% (30/36) concordance between cfRNA levels and pts response. Colon: There was a 79% (15/19) concordance between cfRNA levels and pts response. Conclusions: A noteworthy concordance was observed between clinical response and changes in cfRNA levels in lung, breast and colon cancer pts, independent of chemotherapy regimen. Changes in relative PD-L1 gene expression correlated specifically with outcome to immunotherapy (90%). We conclude that changing cfRNA levels can indicate tx response, and PD- L1 could be used to monitor response to immunotherapy.

TPS3621 Background: A randomized phase II trial, DEEPER (JACCRO CC-13) [NCT02515734], is on-going to evaluate FOLFOXIRI plus cetuximab (cet) vs. FOLFOXIRI plus bevacizumab (bev) in terms of depth of response as primary endpoint in 360 mCRC patients (pts) with RAS wild-type tumors, PS0-1, and no previous chemotherapy. This is a head-to-head comparative trial of 2 key monoclonal antibodies in mCRC treatment; therefore, it would be of interest to perform the biomarker study for developing novel predictors of cet or bev. The clinical utility of circulating tumor DNA (ctDNA) analysis has been largely validated for monitoring during treatment and companion diagnostics after chemotherapy. However, there are few published results regarding ctRNA in cancer treatment. Use of ctRNA from liquid biopsies would enhance tumor profiling through the trending of actionable biomarkers not found in ctDNA, and allow for patient monitoring by measuring dynamic changes in levels of gene expressions. Methods: This study will enroll pts with willing to undergo biopsies of both tissue and blood among participants of the DEEPER trial. The estimated number is 250. The main purpose is to find novel predictors for efficacy of cet or bev in mCRC using liquid biopsies, which are performed to obtain ctDNA and ctRNA at 6 time points: pre-treatment, 8 weeks after treatment start, beginning of maintenance phase in FOLFOXIRI-regimen, progression, before and 8 weeks after 2nd-line treatment. The tissue samples collected before chemotherapy will be used for analyzing intra-tumoral genetic alterations. Associations between analytes in blood/tissue and the clinical outcomes of each treatment (with cet or bev) will be assessed using Fisher’s exact test, Kaplan-Meier curves, and log-rank tests in univariate analyses. We will evaluate on whether transcriptomic analysis in ctRNA could predict treatment efficacy more accurately compared to genomic analysis in ctDNA, and verify the clinical utility of ctRNA testing in mCRC treatment. Accrual will continue until the DEEPER trial is completed. Clinical trial information: UMIN000018412.

We have shown before that cell-free circulating tumor RNA (cfRNA) extracted from plasma of cancer patients (pts) can measure dynamic changes in gene expression that can help to evaluated disease status and predict outcome to anti-tumoral therapy in solid tumors [T. Ishiba et al. Biochem Biophys Res Commun. 2018 Jun 7; 500 (3):621-625]. We want to show here that PD-L1 and other biomarkers assessed by RNA RT-PCR can be use as predictive markers that can be used to follow Immunotherapy and chemotherapy responses in non-small cell lung cancer (NSCLC). We enrolled 54 pts with advanced NSCLC undergoing systemic therapy (STX) and we follow them for 1-year. cfRNA was extracted from resulting plasma and generated random-primed cDNA. Total cfRNA was quantitated by qPCR of β-actin, and correlated with pt clinical response (CR/PR/SD/PD) determined by CT scans. All gene expressions were measured relative to β-actin. Changes in PD-L1 expression were used to monitor response to immunotherapy in lung cancer pts. Ten milliliters of blood were collected in each of two tubes and transferred to Liquid Genomics, Inc. Blood was drawn every 6-8 weeks with an average of 5 collections were done per pt. Of the 54 enrolled pts, 30 completed 1-3 lines of STX with outcomes. The overall mutation frequency was 33% (10/30), with 27% in KRAS and 6% in EGFR. Increases or emergence of mutant allele fractions were predictive of PD status (later determined by imaging), while decreases or disappearance of mutations were predictive of SD and PR status after treatment. PD-L1 expression was detected in 87% (26/30) of pts in at least one blood draw. Immunotherapy: (Nivolumab, pembrolizumab, atezolizumab), 11/30 pts underwent immunotherapy (IO) txt. Changes in PD-L1 during IO were associated with STX outcomes. Increases in PD-L1 were associated with PD, while decreases or no changes in PD-L1 were associated with SD and PR. Of the 23 blood draws from these 11 pts, the overall concordance between changes in PD-L1 and IO outcome was 91% (21/23). Chemotherapy: 19/30 pts were given carboplatin/pemetrexed as first line therapy. Increases or decreases in PD-L1 across 28 blood draws during therapy were likewise associated with resistance or sensitivity to STX outcome (increases infer resistance; decreases infer sensitivity) in 24/28 (86%). We demonstrated a strong concordance between clinical responses and changes in plasma PD-L1 done by RT-PCR RNA levels in NSCLC pts treated with IO or chemotherapy. Monitoring cfRNA expression levels of PD-L1 is a reliable method for predicting response and resistance to IO as well as chemotherapy irrespective of KRAS and EGFR.

Cell-free circulating tumor RNA (cfRNA) extracted from plasma of cancer patients (pts) can measure dynamic changes in gene expression that can help to evaluated disease status and predict outcome to anti-tumoral therapy in solid tumors [T. Ishiba et al. Biochem Biophys Res Commun. 2018 Jun 7; 500 (3):621-625]. We want to show that PD-L1 assessed by RNA RT-PCR is a potential biomarker that can be used to follow Immunotherapy responses in non-small cell lung cancer (NSCLC). 54 pts with NSCLC undergoing systemic therapy (STX) were enrolled in a 1-year study. cfRNA was extracted from resulting plasma and generated random-primed cDNA. Total cfRNA was quantitated by qPCR of β-actin and correlated with pt response (CR/PR/SD/PD) determined by CT scans. All gene expressions were measured relative to β-actin. Changes in PD-L1 expression were used to monitor response to immunotherapy in lung cancer pts. Ten milliliters of blood were collected in each of two tubes containing a proprietary nucleic acid preservation cocktail. Blood was drawn every 6-8 weeks with an average of 5 collections were done per pt. Of the 54 enrolled pts, 30 completed 1-3 lines of STX with outcomes. The overall mutation frequency was 33% (10/30), with 27% in KRAS and 6% in EGFR. Increases or emergence of mutant allele fractions were predictive of PD status (later determined by imaging), while decreases or disappearance of mutations were predictive of SD and PR status after treatment. PD-L1 expression was detected in 87% (26/30) of pts in at least one blood draw. Immunotherapy: (Nivolumab, Pembrolizumab, Atezolizumab), 11/30 pts underwent immunotherapy (IO) txt at some point. Changes in PD-L1 during IO were associated with STX outcomes. Increases in PD-L1 were associated with PD, while decreases or no changes in PD-L1 were associated with SD and PR. Of the 23 blood draws from these 11 pts, the overall concordance between changes in PD-L1 and IO outcome was 91% (21/23). Chemotherapy: 19/30 pts were given carbo/pemetrexed at some point during their STX. Increases or decreases in PD-L1 across 28 blood draws during therapy were likewise associated with resistance or sensitivity to STX outcome (increases infer resistance; decreases infer sensitivity) in 24/28 (86%). A noteworthy concordance was observed between clinical responses and changes in plasma PD-L1 done by RT-PCR cfRNA levels in NSCLC pts treated with IO or chemotherapy. Monitoring cfRNA expression levels of PD-L1 is a reliable method for predicting response and resistance to IO as well as chemotherapy irrespective of KRAS and EGFR

Abstract Background: In addition to traditional radiology tests, cell-free circulating tumor RNA (cfRNA) extracted from plasma of cancer patients (pts) provides a means of evaluating tumor response, but based on molecular changes in the tumor. Measuring dynamic changes in gene expression and levels of total cfRNA (per ml of plasma) in metastatic patients has shown great potential for evaluating disease status and predicting outcome to anti-tumoral therapy in advance of imaging. Though checkpoint inhibitors have not been assessed widely in breast cancer, TNBC has shown mild responses to pembrozilumab and atezolizumab, with significantly better responses in pts with detectable PD-L1 expression. Methods: Blood was drawn from pts at approximately 6-week intervals under various therapies and CT scans were performed at approximately 3-month intervals. CfRNA was extracted from the resulting plasma and reverse transcribed with random hexamers to cDNA. Levels of cfRNA were quantitated by RT-qPCR and correlated with pt response (PR/SD/PD), as determined by CT scans. Levels of gene expression in cfRNA (including PD-L1 and HER2) were monitored in pts across blood draws. Results: A total of 28 breast cancer pts were enrolled in a 1-year clinical study. Of pts, 39% (11/28) were Caucasian and 36% (10/28) Hispanic. 19 pts completed the first two cycles of therapy: 2 pts had PR and showed no change (NC) or decrease (DEC) in levels of cfRNA, 11 pts achieved SD with 8 showing DEC or NC in cfRNA levels, and 6 pts had PD and all underwent increases (INC) in cfRNA levels (median increase: 788 ng/mL plasma) which correlated with progressive disease status. Of pts with SD/PR, 4 showed either an emergence or significant increase in PD-L1 expression across blood draws (3.7-98 ct); of PD pts, 1 showed a significant emergence of PD-L1 expression (12.5 ct) across blood draws. 3/5 of these PD-L1 expressing pts were being treated with an everolimus combination; the emergence or increase of PD-L1 in response to this therapy suggests use of checkpoint inhibitors as an option for these pts. In response to therapy, 3 of 5 pts had PD-L1 cfRNA levels above levels predictive of response to nivolumab in lung cancer pts. In the only pt with hyperexpressed HER2, the disappearance of HER2 cfRNA matched positive response (PR) to treatment with trastuzumab. PD-L1 decreased concomitantly for this pt. Conclusion: We found a strong correlation between clinical responses and changes in plasma levels of ctRNA in breast cancer (84%). Most of these were documented several weeks before imaging was done. Levels of PD-L1 and HER2 expression in plasma can also be used to monitor pt response to specific therapies. The emergence of PD-L1 expression in response to various therapies in breast cancer may confer sensitivity to checkpoint inhibitor therapy. Citation Format: Castrellon AB, Velez M, Raez LE, Danenberg K, Rabizadeh S, Usher J, Jaimes Y, Hunis B, Bittencourt AC, Milillo A, Blaya M, Habaue C, Danenberg PV. Use of cell-free circulating RNA and expression of PD-L1 and HER2 in plasma to monitor and predict clinical response in metastatic breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-16.

e17077 Background: Cell-free circulating RNA (cfRNA) released into the bloodstream by tumors allows non-invasive identification of molecular changes via the quantity of particular genes (i.e., gene expression). In this study, we used cfRNA in order to measure PD-L1, TIM-3, PCA3, AR, and AR-V7 expression in prostate cancer patients (pts) and compared levels of expression and total cfRNA in blood to that taken from healthy individuals. Methods: Blood samples were collected from prostate cancer pts (at various times during therapy) and from healthy volunteers (controls). After plasma was fractionated from blood samples, cfRNA was extracted, reverse transcribed into cDNA using random hexamers, and then analyzed by RT-qPCR using appropriate gene-specific primers. The cDNA of each biomarker of interest was quantitated per volume of blood in cancer pts and the controls. β-actin expression was used as a surrogate for total cfRNA (ng/rxn). Results: PCA3 was detected only in cancer pts (31/55) and not in healthy controls (p = 0.0001, Fisher’s Exact). AR was detected in 41% of pts and 86% of controls, while AR-V7, signifying resistance to ADT, was detected in 7% (4/55) of prostate cancer samples and in none of the controls (0/14). PD-L1 and TIM-3 detection frequencies were similar in cancer pts and controls (35-43% and 82-86%, respectively). All gene expressions show a significantly higher overall level in the blood of cancer patients than in healthy individuals. The median cfRNA level in cancer pts was significantly higher than in healthy controls, 23.8 ng/rxn (0.4-82.5) and 8.6 ng/rxn (2.7-17.1), respectively (p < 0.0001, Pearson chi-square). Conclusions: These data indicate that using cfRNA to quantitate gene expressions of interest from blood may have potential for detection of cancer, monitoring recurrence, and in selecting therapies. The significant difference in median total cfRNA between cancer patients and healthy individuals suggests that the total cfRNA level itself may be useful as a sensitive preliminary indicator of the presence of cancer and/or for recurrence monitoring. Clinical trial information: NCT02853097.

e15013 Background: Analysis ofcell-free circulating tumor RNA (cfRNA) extracted from plasma of cancer patients (pts) provides a means of measuring dynamic changes in gene expression as well as levels of cfRNA, allowing us the evaluation of disease status and prediction of outcomes to anti-tumoral therapy. Methods: Blood was drawn from pts under various treatments (tx) every 6-8 weeks, at the same time that CT scans were done. CfRNA was extracted from the resulting plasma and reverse transcribed with random hexamers to cDNA. Levels of cfRNA were quantitated by RT-qPCR and correlated with pt response (CR/PR/SD/PD), as determined by CT scans. Results: 96 pts (48 breast, 30 lung and 18 colon cancer) were enrolled in a 2-year study. Breast: 34 pts completed the first two cycles of tx. Of these, 8/11 pts with PD showed increasing (INC) levels of cfRNA, 16/17 pts with SD showed either no change (NC) or a decrease (DEC) in levels of cfRNA, and 4/6 pts with PR had DEC cfRNA (82% concordance between cfRNA and pt response). Of pts with PR, 3 were treated with HER2 inhibitors and in 2 of these pts, HER2 cfRNA expression levels disappeared. Lung: 23 pts completed the first two cycles of tx. Of these, 6/8 pts with PD showed INC levels of cfRNA, 8/12 pts with SD showed either NC or DEC cfRNA, and 3/3 pts with PR had DEC cfRNA, corresponding to 74% concordance between cfRNA and pt response. Among 7 pts treated with immunotherapy, 3/3 pts with PD showed INC PD-L1 expression (PDL-1), 3/3 pts with SD had NC in PD-L1, and 1 pt with PR showed DEC PD-L1, corresponding to 100% correlation between PD-L1 and response. Colon: 11/18 pts completed the first 2 cycles. Of these, 2/3 pts with PD showed INC cfRNA, and 3/4 pts with SD showed DEC cfRNA, as did 4/4 pts with PR (82% concordance between cfRNA and response). Conclusions: A significant concordance was observed between clinical response and changes in cfRNA levels in breast, lung and colon cancer pts (82%, 74% and 82%). Levels of PD-L1 correlated with response in 7/7 lung pts, and HER2 expression correlated with response in 2/3 breast pts. We conclude that cfRNA levels can indicate tx response, and PD-L1 and HER2 could be used to monitor response to immunotherapy and HER2 inhibitors.

Prostate cancer is a common malignancy impacting countless men without curative options in the advanced state. Numerous therapies have been introduced in recent years improving survival and symptom control, yet optimal methods for predicting or monitoring response have not been developed. In the era of precision medicine, characterization of individual cancers is necessary to inform treatment decisions. Liquid biopsies, through evaluation of various blood-based analytes, provide a method of patient evaluation with potential applications in virtually all disease states. In this review, we will describe current approaches with a particular focus on demonstrated clinical utility in the evaluation and management of prostate cancer.

Background: We have conducted a phase II trial of 1st-line modified (m)-FOLFOXIRI plus bev for RAS mutant mCRC, which included a biomarker study using liquid biopsies [Oncotarget 2018]. There are few reports on monitoring changes in gene mutation (mt) status in mCRC harboring RAS mt. Therefore, the pre-planned analysis was performed to investigate a number of genes in ctDNA during therapy that might be determinants of therapeutic efficacy. Methods: Sixty-two patients (pts) with unresectable/measurable tumors received protocol treatment with m-FOLFOXIRI (irinotecan 150 mg/m², oxaliplatin 85 mg/m², levofolinate [LV] 200 mg/m², and fluorouracil 2400 mg/m² repeated biweekly) plus bev. The phase II trial included objective response rate (ORR) for primary endpoint and progression-free survival (PFS), overall survival, early tumor shrinkage, depth of response (DpR), and safety for secondary endpoints. In 53 pts who enrolled in the biomarker study, plasma samples for extraction of ctDNA were collected at 3 points (pre-, 8wks, and progression) and analyzed for specific KRAS and NRAS variants with real-time PCR assays. Results: Fifty-three pts had the following clinical data: median age of 61yrs, 57% male, 91% PS0, 28% right-sided tumors, ORR of 72%, median DpR of 49%, and median PFS of 10.8 months. RAS mt was detected in pre-treatment plasma in 79% (42/53) of pts. Among pts with mt in ctDNA at pre-treatment, 76% changed to mt-negative 8wks after treatment. ORR and DpR were higher in pts of mt-negative at 8wks compared to pts of mt-positive (81% vs. 50% and 55% vs. 34%, respectively). Median PFS was 11.9 and 8.8 months in pts who were mt-negative and mt-positive, respectively (HR 0.58, 95%CI 0.25-1.33, P = 0.20). Interestingly, in 26 pts who experienced progressive disease (PD) and were evaluable for ctDNA analysis, 52% (11/21) of pts with mt at pre-treatment still had no mt in plasma at PD. Pts of mt-negative at PD had longer survival time from PD compared to pts of mt-positive (9.3 vs. 7.0 months). Conclusions: Gene mt status in ctDNA during therapy may predict clinical outcome of triplet plus bev treatment in RAS mutant mCRC. Our study suggests that pts with no mt in plasma at PD may have more favorable post-treatment. Clinical trial identification: UMIN000015152. Editorial acknowledgement: We acknowledge Sachika Koyama and Yasushi Ohtake (JACCRO) for editorial assistance. Legal entity responsible for the study: Wataru Ichikawa. Funding: Japan Clinical Cancer Research Organization (JACCRO). Disclosure: Y. Sunakawa: Honoraria for talks: Taiho Pharmaceutical, Chugai Pharma, Yakult Honsha, Takeda, Merck Serono, Bayer Yakuhin, Sanofi. H. Satake: Honoraria: Bayer, Chugai Pharma, Eli Lilly Japan, Merck Serono, Takeda, Taiho Pharmaceutical, Yakult Honsha. M. Nakamura: Honoraria: Chugai Pharma, Taiho Pharmaceutical, Yakult Honsha. M. Takeuchi: Consulting fees: Hisamitsu Pharmaceutical, Kowa, Shionogi Pharma, Abbvie, Astellas. H-J. Lenz: Consulting or advisory role, travel expenses, honoraria: Roche. W. Ichikawa: Consulting fees: Ono Pharmaceutical; Research funding: Takeda, Taiho Pharmaceutical, Eisai, Chugai Pharma, Merck Serono, Shionogi Pharma, Daiichi Sankyo; Honoraria: Merck Serono, Taiho Pharmaceutical, Chugai Pharma, Takeda, Ono Pharmaceutical, Lilly. All other authors have declared no conflicts of interest.