FOLFIRINOX chemotherapy modulates the peripheral immune landscape in pancreatic cancer: Implications for combination therapies and early response prediction.

BACKGROUND: FOLFIRINOX chemotherapy has improved outcomes for pancreatic cancer patients, but poor long-term survival outcomes and high toxicity remain challenges. This study investigates the impact of FOLFIRINOX on plasma proteins and peripheral immune cells to guide immune-based combination therapies and, ideally, to identify a potential biomarker to predict early disease progression during FOLFIRINOX. METHODS: Blood samples were collected from 86 pancreatic cancer patients before and two weeks after the first FOLFIRINOX cycle and subjected to comprehensive immune cell and proteome profiling. Principal Component Analysis and Linear Mixed Effect Regression models were used for data analysis. FOLFIRINOX efficacy was radiologically evaluated after the fourth cycle. RESULTS: One cycle of FOLFIRINOX diminished tumour-cell-related pathways and enhanced pathways related to immune activation, illustrated by an increase in pro-inflammatory IL-18, IL-15, and TNFRSF4. Similarly, FOLFIRINOX promoted the activation of CD4 + and CD8 + T cells, the proliferation of NK(T), and the activation of antigen-presenting cells. Furthermore, high pre-treatment levels of VEGFA and PRDX3 and an elevation in FCRL3 levels after one cycle predicted early progression under FOLFIRINOX. Finally, patients with progressive disease exhibited high levels of inhibitory markers on B cells and CD8 + T cells, while responding patients exhibited high levels of activation markers on CD4 + and CD8 + T cell subsets. CONCLUSION: FOLFIRINOX has immunomodulatory effects, providing a foundation for clinical trials exploring immune-based combination therapies that harness the immune system to treat pancreatic cancer. In addition, several plasma proteins hold potential as circulating predictive biomarkers for early prediction of FOLFIRINOX response in patients with pancreatic cancer.

B cell immune profiles in dysbiotic vermiform appendixes of pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid tumors and is resistant to immunotherapy. B cells play an essential role in PDAC progression and immune responses, both locally and systemically. Moreover, increasing evidence suggests that microbial compositions inside the tumor, as well as in the oral cavity and the gut, are important factors in shaping the PDAC immune landscape. However, the gut-associated lymphoid tissue (GALT) has not previously been explored in PDAC patients. In this study, we analyzed healthy vermiform appendix (VA) from 20 patients with PDAC and 32 patients with colon diseases by gene expression immune profiling, flow cytometry analysis, and microbiome sequencing. We show that the VA GALT of PDAC patients exhibits markers of increased inflammation and cytotoxic cell activity. In contrast, B cell function is decreased in PDAC VA GALT based on gene expression profiling; B cells express significantly fewer MHC class II surface receptors, whereas plasma cells express the immune checkpoint molecule HLA-G. Additionally, the vermiform appendix microbiome of PDAC patients is enriched with Klebsiella pneumoniae, Bifidobacterium animalis, and Adlercreutzia equolifaciens, while certain commensals are depleted. Our findings may suggest impaired B cell function within the GALT of PDAC patients, which could potentially be linked to microbial dysbiosis. Additional investigations are imperative to validate our observations and explore these potential targets of future therapies.

Serum cytokine levels are associated with tumor progression during FOLFIRINOX chemotherapy and overall survival in pancreatic cancer patients.

Background: Biomarkers predicting treatment response may be used to stratify patients with pancreatic ductal adenocarcinoma (PDAC) for available therapies. The aim of this study was to evaluate the association of circulating cytokines with FOLFIRINOX response and with overall survival (OS). Methods: Serum samples were collected before start and after the first cycle of FOLFIRINOX from patients with PDAC (n=83) of all disease stages. Overall, 34 circulating cytokines were analyzed with a multiplex immunoassay. In addition, changes in peripheral blood immune cell counts were determined by flow cytometry to correlate with differences in cytokine levels. Chemotherapy response was determined by CT scans with the RECIST 1.1 criteria, as disease control (n=64) or progressive disease (n=19) within eight cycles of FOLFIRINOX. Results: Patients with high serum IL-1RA concentrations after one cycle of chemotherapy were less likely to have tumor progression during FOLFIRINOX (OR 0.25, P=0.040). Increase of circulating IL-1RA concentrations correlated with increase of total, classical (CD14+CD16-), and non-classical monocytes (CD14-CD16+), and dendritic cells. In multivariable cox regression, including the variables chemotherapy response outcome and baseline CA19-9 level, serum concentrations of IL-7 (HR 2.14, P=0.010), IL-18 (HR 2.00, P=0.020), and MIP-1ß (HR 0.51, P=0.025) after one cycle of FOLFIRINOX showed correlations with OS. Conclusions: Circulating IL-1RA, IL-7, IL-18, and MIP-1ß concentrations are biomarkers associated with FOLFIRINOX response in PDAC patients, suggesting an important role for specific immune cells in chemotherapy response and PDAC progression. Cytokine-based treatment might improve patient outcome and should be evaluated in future studies.

Distinct Response of Circulating microRNAs to the Treatment of Pancreatic Cancer Xenografts with FGFR and ALK Kinase Inhibitors.

Pancreatic adenocarcinoma is typically detected at a late stage and thus shows only limited sensitivity to treatment, making it one of the deadliest malignancies. In this study, we evaluate changes in microRNA (miR) patterns in peripheral blood as a potential readout of treatment responses of pancreatic cancer to inhibitors that target tumor-stroma interactions. Mice with pancreatic cancer cell (COLO357PL) xenografts were treated with inhibitors of either fibroblast growth factor receptor kinase (FGFR; PD173074) or anaplastic lymphoma kinase receptor (ALK; TAE684). While both treatments inhibited tumor angiogenesis, signal transduction, and mitogenesis to a similar extent, they resulted in distinct changes in circulating miR signatures. Comparison of the miR pattern in the tumor versus that in circulation showed that the inhibitors can be distinguished by their differential impact on tumor-derived miRs as well as host-derived circulating miRs. Distinct signatures that include circulating miR-1 and miR-22 are associated with the efficacy of ALK and FGFR inhibition, respectively. We propose that monitoring changes in circulating miR profiles can provide an early signature of treatment response or resistance to pathway-targeted drugs, and thus provide a non-invasive measurement to rapidly assess the efficacy of candidate therapies.

Impaired CXCL12 signaling contributes to resistance of pancreatic cancer subpopulations to T cell-mediated cytotoxicity.

Pancreatic cancer remains largely unresponsive to immune modulatory therapy attributable in part to an immunosuppressive, desmoplastic tumor microenvironment. Here, we analyze mechanisms of cancer cell-autonomous resistance to T cells. We used a 3D co-culture model of cancer cell spheroids from the KPC (LSL-KrasG12D/+ /LSL-Trp53R172H/+ /p48-Cre) pancreatic ductal adenocarcinoma (PDAC) model, to examine interactions with tumor-educated T cells isolated from draining lymph nodes of PDAC-bearing mice. Subpopulations of cancer cells resistant to these tumor-educated T cells were isolated from the in vitro co-culture and their properties compared with sensitive cancer cells. In co-culture with resistant cancer cell subpopulations, tumor-educated T cells showed reduced effector T cell functionality, reduced infiltration into tumor cell spheroids and decreased induction of apoptosis. A combination of comparative transcriptomic analyses, cytometric and immunohistochemistry techniques allowed us to dissect the role of differential gene expression and signaling pathways between sensitive and resistant cells. A decreased expression of the chemokine CXCL12 (SDF-1) was revealed as a common feature in the resistant cell subpopulations. Adding back CXCL12 reversed the resistant phenotype and was inhibited by the CXCR4 inhibitor AMD3100 (plerixafor). We conclude that reduced CXCL12 signaling contributes to PDAC subpopulation resistance to T cell-mediated attack.

Serum miR-373-3p and miR-194-5p Are Associated with Early Tumor Progression during FOLFIRINOX Treatment in Pancreatic Cancer Patients: A Prospective Multicenter Study.

In this study, we explored the predictive value of serum microRNA (miRNA) expression for early tumor progression during FOLFIRINOX chemotherapy and its association with overall survival (OS) in patients with pancreatic ductal adenocarcinoma (PDAC). A total of 132 PDAC patients of all disease stages were included in this study, of whom 25% showed progressive disease during FOLFIRINOX according to the RECIST criteria. MiRNA expression was analyzed in serum collected before the start and after one cycle of chemotherapy. In the discovery cohort (n = 12), a 352-miRNA RT-qPCR panel was used. In the validation cohorts (total n = 120), miRNA expression was detected using individual RT-qPCR miRNA primers. Before the start of FOLFIRINOX, serum miR-373-3p expression was higher in patients with progressive disease compared to patients with disease control after FOLFIRINOX (Log2 fold difference (FD) 0.88, p = 0.006). MiR-194-5p expression after one cycle of FOLFIRINOX was lower in patients with progressive disease (Log2 FD -0.29, p = 0.044). Both miRNAs were predictors of early tumor progression in a multivariable model including disease stage and baseline CA19-9 level (miR-373-3p odds ratio (OR) 3.99, 95% CI 1.10-14.49; miR-194-5p OR 0.91, 95% CI 0.83-0.99). MiR-373-3p and miR-194-5p did not show an association with OS after adjustment for disease stage, baseline CA19-9, and chemotherapy response. In conclusion, high serum miR-373-3p before the start and low serum miR-194-5p after one cycle are associated with early tumor progression during FOLFIRINOX.

Circulating Immunological Biomarkers: Prognosis of Pancreatic Cancer Patients Reflected by the Immune System.

ABSTRACT: To date, little advances have been made toward new and more effective therapies for pancreatic ductal adenocarcinoma (PDAC). Discovery of prognostic and predictive biomarkers is needed to stratify patients for available treatments and to elucidate how new therapies could be developed. Recent studies have made clear that the immune system is not only affected in the microenvironment of the primary tumor and it is also systemically disrupted in PDAC patients. Under normal circumstances, the immune system is in perfect balance with both proinflammatory and anti-inflammatory components present. In this review, we focus on circulating immunological characteristics including immune cells and their subtypes, cytokines, and immune checkpoints in the peripheral blood not only to understand the poor prognosis of PDAC patients but also to find new leads for new innovative therapies.

Organoids Derived from Neoadjuvant FOLFIRINOX Patients Recapitulate Therapy Resistance in Pancreatic Ductal Adenocarcinoma.

PURPOSE: We investigated whether organoids can be generated from resected tumors of patients who received eight cycles of neoadjuvant FOLFIRINOX chemotherapy before surgery, and evaluated the sensitivity/resistance of these surviving cancer cells to cancer therapy. EXPERIMENTAL DESIGN: We generated a library of 10 pancreatic ductal adenocarcinoma (PDAC) organoid lines: five each from treatment-naïve and FOLFIRINOX-treated patients. We first assessed the histologic, genetic, and transcriptional characteristics of the organoids and their matched primary PDAC tissue. Next, the organoids' response to treatment with single agents-5-FU, irinotecan, and oxaliplatin-of the FOLFIRINOX regimen as well as combined regimen was evaluated. Finally, global mRNA-seq analyses were performed to identify FOLFIRINOX resistance pathways. RESULTS: All 10 patient-derived PDAC organoids recapitulate histologic, genetic, and transcriptional characteristics of their primary tumor tissue. Neoadjuvant FOLFIRINOX-treated organoids display resistance to FOLFIRINOX (5/5), irinotecan (5/5), and oxaliplatin (4/5) when compared with treatment-naïve organoids (FOLFIRINOX: 1/5, irinotecan: 2/5, oxaliplatin: 0/5). 5-Fluorouracil treatment responses between naïve and treated organoids were similar. Comparative global transcriptome analysis of treatment-naïve and FOLFIRINOX samples-in both organoids and corresponding matched tumor tissues-uncovered modulated pathways mainly involved in genomic instability, energy metabolism, and innate immune system. CONCLUSIONS: Resistance development in neoadjuvant FOLFIRINOX organoids, recapitulating their primary tumor resistance, suggests continuation of FOLFIRINOX therapy as an adjuvant treatment may not be advantageous for these patients. Gene-expression profiles of PDAC organoids identify targetable pathways involved in chemoresistance development upon neoadjuvant FOLFIRINOX treatment, thus opening up combination therapy possibilities.

Circulating TP53 mutations are associated with early tumor progression and poor survival in pancreatic cancer patients treated with FOLFIRINOX.

BACKGROUND: Biomarkers predicting treatment response may be used to stratify pancreatic ductal adenocarcinoma (PDAC) patients for therapy. The aim of this study was to identify circulating tumor DNA (ctDNA) mutations that associate with tumor progression during FOLFIRINOX chemotherapy, and overall survival (OS). METHODS: Circulating cell-free DNA was analyzed with a 57 gene next-generation sequencing panel using plasma samples of 48 PDAC patients of all disease stages. Patients received FOLFIRINOX as initial treatment. Chemotherapy response was determined on CT scans as disease control (n = 30) or progressive disease (n = 18) within eight cycles of FOLFIRINOX, based on RECIST 1.1 criteria. RESULTS: Detection of a TP53 ctDNA mutation before start of FOLFIRINOX [odds ratio (OR) 10.51, 95% confidence interval (CI) 1.40-79.14] and the presence of a homozygous TP53 Pro72Arg germline variant (OR 6.98, 95% CI 1.31-37.30) were predictors of early tumor progression during FOLFIRINOX in multivariable analysis. Five patients presented with the combination of a TP53 ctDNA mutation before start of FOLFIRINOX and the homozygous Pro72Arg variant. All five patients showed progression during FOLFIRINOX. The combination of the TP53 mutation and TP53 germline variant was associated with shorter survival (median OS 4.4 months, 95% CI 2.6-6.2 months) compared with patients without any TP53 alterations (median OS 13.0 months, 95% CI 8.6-17.4 months). CONCLUSION: The combination of a TP53 ctDNA mutation before start of FOLFIRINOX and a homozygous TP53 Pro72Arg variant is a promising biomarker, associated with early tumor progression during FOLFIRINOX and poor OS. The results of this exploratory study need to be validated in an independent cohort.

Cardiomyocyte-specific circulating cell-free methylated DNA in esophageal cancer patients treated with chemoradiation.

Thoracic high dose radiation therapy (RT) for cancer has been associated with early and late cardiac toxicity. To assess altered rates of cardiomyocyte cell death due to RT we monitored changes in cardiomyocyte-specific, cell-free methylated DNA (cfDNA) shed into the circulation. Eleven patients with distal esophageal cancer treated with neoadjuvant chemoradiation to 50.4 Gy (RT) and concurrent carboplatin and paclitaxel were enrolled. Subjects underwent fasting blood draws prior to the initiation and after completion of RT as well as 4-6 months following RT. An island of six unmethylated CpGs in the FAM101A locus was used to identify cardiomyocyte-specific cfDNA in serum. After bisulfite treatment this specific cfDNA was quantified by amplicon sequencing at a depth of >35,000 reads/molecule. Cardiomyocyte-specific cfDNA was detectable before RT in the majority of patient samples and showed some distinct changes during the course of treatment and recovery. We propose that patient-specific cardiac damages in response to the treatment are indicated by these changes although co-morbidities may obscure treatment-specific events.

Cadherin 11 Promotes Immunosuppression and Extracellular Matrix Deposition to Support Growth of Pancreatic Tumors and Resistance to Gemcitabine in Mice.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule CDH11, which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. METHODS: We compared levels of CDH11 messenger RNA in human pancreatitis and pancreatic cancer tissues and cells with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11+/+ and Cdh11-/- mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11+/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. RESULTS: Levels of CDH11 messenger RNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11+/- and KPC/Cdh11-/- mice survived significantly longer than KPC/Cdh11+/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11+/+ mice and incompletely in KPC/Cdh11+/- and KPC/Cdh11-/- mice, whose lesions also contained fewer FOXP3+ cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11+/+ mice, tumors of KPC/Cdh11+/- mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival of KPC/Cdh11+/- and KPC/Cdh11-/- mice only or reduced subcutaneous tumor growth in mT3 engrafted Cdh11+/+ mice when given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. CONCLUSIONS: Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

Serum miR-338-3p and miR-199b-5p are associated with the absolute neutrophil count in patients with resectable pancreatic cancer.

BACKGROUND: Several peripheral blood cell counts and immune ratios, such as the systemic immune-inflammation index (SII = platelet x neutrophil count/lymphocyte count) have prognostic value in patients with resectable pancreatic cancer (PDAC). Circulating microRNAs (miRNAs) are involved in various aspects of cancer and inflammation. We aimed to identify measurable circulating miRNAs in PDAC patients correlating with systemic inflammation parameters. METHODS: A total of 42 PDAC patients was included in this study: twelve in the discovery (n = 6 SII low; n = 6 SII high) and 30 patients in the validation cohort (n = 19 SII low, n = 11 SII high). MiRNAs isolated from preoperative serum samples were measured with a 352 miRNA panel in the discovery cohort and individual miRNA primers in the validation cohort, using RT-qPCR (ID3EAL assays, MiRXES). RESULTS: Only in the discovery cohort miR-328-3p, miR-338-3p, miR-1258 and miR-199b-5p were upregulated in high compared to low SII patients (fold difference ≥ 2, P<0.05). In the total cohort (n = 42) correlations were found between miR-338-3p (r = 0.48, P = 0.002) and miR-199b-5p (r = 0.44, P = 0.005) and the absolute neutrophil count. CONCLUSION: Circulating miR-338-3p and miR-199b-5p are correlated to the neutrophil count in the blood of PDAC patients, suggesting a potential role of circulating miRNAs in cancer immune evasion and systemic inflammation.

Endogenous Gastrin Collaborates With Mutant KRAS in Pancreatic Carcinogenesis.

OBJECTIVE: The KRAS gene is the most frequently mutated gene in pancreatic cancer, and no successful anti-Ras therapy has been developed. Gastrin has been shown to stimulate pancreatic cancer in an autocrine fashion. We hypothesized that reactivation of the peptide gastrin collaborates with KRAS during pancreatic carcinogenesis. METHODS: LSL-Kras; P48-Cre (KC) mutant KRAS transgenic mice were crossed with gastrin-KO (GKO) mice to develop GKO/KC mice. Pancreata were examined for 8 months for stage of pancreatic intraepithelial neoplasia lesions, inflammation, fibrosis, gastrin peptide, and microRNA expression. Pancreatic intraepithelial neoplasias from mice were collected by laser capture microdissection and subjected to reverse-phase protein microarray, for gastrin and protein kinases associated with signal transduction. Gastrin mRNA was measured by RNAseq in human pancreatic cancer tissues and compared to that in normal pancreas. RESULTS: In the absence of gastrin, PanIN progression, inflammation, and fibrosis were significantly decreased and signal transduction was reversed to the canonical pathway with decreased KRAS. Gastrin re-expression in the PanINs was mediated by miR-27a. Gastrin mRNA expression was significantly increased in human pancreatic cancer samples compared to normal human pancreas controls. CONCLUSIONS: This study supports the mitogenic role of gastrin in activation of KRAS during pancreatic carcinogenesis.

Circulating Biomarkers for Prediction of Objective Response to Chemotherapy in Pancreatic Cancer Patients.

Pancreatic cancer is a lethal disease with increasing incidence. Most patients present with advanced disease, for which palliative systemic chemotherapy is the only therapeutic option. Despite improved median survival rates with FOLFIRINOX or gemcitabine chemotherapy compared to the best supportive care, many individual patients may not benefit from chemotherapy. Biomarkers are needed to predict who will benefit from chemotherapy and to monitor a patient's response to chemotherapy. This review summarizes current research and future perspectives on circulating biomarkers for systemic chemotherapy response.

Immune-Related Circulating miR-125b-5p and miR-99a-5p Reveal a High Recurrence Risk Group of Pancreatic Cancer Patients after Tumor Resection.

Clinical follow-up aided by changes in the expression of circulating microRNAs (miRs) may improve prognostication of pancreatic ductal adenocarcinoma (PDAC) patients. Changes in 179 circulating miRs due to cancer progression in the transgenic Kras G12D/+; Trp53 R172H/+; P48-Cre (KPC) animal model of PDAC were analyzed for serum miRs that are altered in metastatic disease. In addition, expression levels of 250 miRs were profiled before and after pancreaticoduodenectomy in the serum of two patients with resectable PDAC with different progression free survival (PFS) and analyzed for changes indicative of PDAC recurrence after resection. Three miRs that were upregulated ≥3-fold in progressive PDAC in both mice and patients were selected for validation in 26 additional PDAC patients before and after resection. We found that high serum miR-125b-5p and miR-99a-5p levels after resection are significantly associated with shorter PFS (HR 1.34 and HR 1.73 respectively). In situ hybridization for miR detection in the paired resected human PDAC tissues showed that miR-125b-5p and miR-99a-5p are highly expressed in inflammatory cells in the tumor stroma, located in clusters of CD79A expressing cells of the B-lymphocyte lineage. In conclusion, we found that circulating miR-125b-5p and miR-99a-5p are potential immune-cell related prognostic biomarkers in PDAC patients after surgery.

Reprint of: Circulating cell-free DNA mutation patterns in early and late stage colon and pancreatic cancer.

Cancer is a heterogeneous disease harboring diverse subclonal populations that can be discriminated by their DNA mutations. Environmental pressure selects subclones that ultimately drive disease progression and tumor relapse. Circulating cell-free DNA (ccfDNA) can be used to approximate the mutational makeup of cancer lesions and can serve as a marker for monitoring disease progression at the molecular level without the need for invasively acquired samples from primary or metastatic lesions. This potential for molecular analysis makes ccfDNA attractive for the study of clonal evolution and for uncovering emerging therapeutic resistance or sensitivity. We assessed ccfDNA from colon and pancreatic adenocarcinoma patients using next generation sequencing of 56 cancer-associated genes at the time of primary resectable disease and metastatic progression and compared this to the mutational patterns of the primary tumor. 28%-47% of non-synonymous mutations in the primary tumors were also detected in the ccfDNA while 71%-78% mutations found in ccfDNA were not detected in the primary tumors. ccfDNA collected at the time of progression harbored 3-5 new mutations not detected in ccfDNA at the earlier collection time points. We conclude that incorporation of ccfDNA analysis provides crucial insights into the changing molecular makeup of progressive colon and pancreatic cancer.

Circulating cell-free DNA mutation patterns in early and late stage colon and pancreatic cancer.

Cancer is a heterogeneous disease harboring diverse subclonal populations that can be discriminated by their DNA mutations. Environmental pressure selects subclones that ultimately drive disease progression and tumor relapse. Circulating cell-free DNA (ccfDNA) can be used to approximate the mutational makeup of cancer lesions and can serve as a marker for monitoring disease progression at the molecular level without the need for invasively acquired samples from primary or metastatic lesions. This potential for molecular analysis makes ccfDNA attractive for the study of clonal evolution and for uncovering emerging therapeutic resistance or sensitivity. We assessed ccfDNA from colon and pancreatic adenocarcinoma patients using next generation sequencing of 56 cancer-associated genes at the time of primary resectable disease and metastatic progression and compared this to the mutational patterns of the primary tumor. 28%-47% of non-synonymous mutations in the primary tumors were also detected in the ccfDNA while 71%-78% mutations found in ccfDNA were not detected in the primary tumors. ccfDNA collected at the time of progression harbored 3-5 new mutations not detected in ccfDNA at the earlier collection time points. We conclude that incorporation of ccfDNA analysis provides crucial insights into the changing molecular makeup of progressive colon and pancreatic cancer.

Circulating microRNAs in patients with hormone receptor-positive, metastatic breast cancer treated with dovitinib.

BACKGROUND: Serial analysis of biomarkers in the circulation of patients undergoing treatment ("liquid biopsies") can provide new insights into drug effects. In particular the analysis of cell-free, circulating nucleic acids such as microRNAs (miRs) can reveal altered expression patterns indicative of mechanism of drug action, cancer growth, and tumor-stroma interactions. RESULTS: Here we analyzed plasma miRs in patients with hormone receptor positive, metastatic breast cancer with prior disease progression during aromatase inhibitor therapy (n = 8) in a phase I/II trial with the multiple tyrosine kinase inhibitor dovitinib (TKI258). Plasma miR levels were measured by quantitative RT-qPCR before and after treatment with dovitinib. A candidate miR signature of drug response was established from a 379 miR screen for detectable plasma miRs as well as from the published literature. Changes in miR expression patterns and tumor sizes were compared. In this analysis we identified miR-21-5p, miR-100-5p, miR-125b-5p, miR-126-3p, miR-375 and miR-424-5p as potential indicators of a response to dovitinib. The altered expression patterns observed for the six circulating miRs separated patients with resistant disease from those with drug responsive disease. There was no relationship between adverse effects of dovitinib treatment and identifiable changes in miR patterns. CONCLUSION: We conclude that changes in the expression patterns of circulating miRs can be indicators of drug responses that merit prospective studies for validation.

Circulating biomarkers to monitor cancer progression and treatment.

Tumor heterogeneity is a major challenge and the root cause of resistance to treatment. Still, the standard diagnostic approach relies on the analysis of a single tumor sample from a local or metastatic site that is obtained at a given time point. Due to intratumoral heterogeneity and selection of subpopulations in diverse lesions this will provide only a limited characterization of the makeup of the disease. On the other hand, recent developments of nucleic acid sequence analysis allows to use minimally invasive serial blood samples to assess the mutational status and altered gene expression patterns for real time monitoring in individual patients. Here, we focus on cell-free circulating tumor-specific mutant DNA and RNA (including mRNA and non-coding RNA), as well as current limitations and challenges associated with circulating nucleic acids biomarkers.

Circulating DNA and Micro-RNA in Patients with Pancreatic Cancer.

Collecting repeat samples of blood ("liquid biopsies") is a broadly used clinical approach for serial monitoring of disease or response to treatments. In patients with cancer the most distinct molecular feature are somatic mutations acquired by cancer cells present in the diseased tissue. Indeed, mutant DNA derived from dying or lysed cancer cells can be isolated from patient serum samples, subjected to DNA sequencing and to analysis of abundance as a measure of tumor burden. Also, changes in the DNA mutation patterns in serum samples collected over time can indicate altered pathways or clonal evolution of the disease and altered abundance of mutant DNA suggests an altered disease burden. In addition, during the course of treatment, changes in circulating DNA mutation patterns can indicate the emergence of resistant clones and prompt changes in treatment. In contrast to mutant DNA, microRNAs (miR) are transcribed, processed, packaged and released from cells in normal and in diseased tissues as part of the extracellular crosstalk between cells. Interestingly, released miR can function in cell-to-cell communication and as hormone-like signals that operate at a distance through their release into the circulation and subsequent uptake into cells in distant tissues. Circulating miR expression patterns can be established from serial serum samples and monitored for alterations over time. Circulating miR provide a readout of the organism's steady state and serial analyses will indicate changes in the response to therapy or an altered physiologic or disease state. Furthermore, changes in circulating miR patterns can indicate treatment efficacy or resistance as well as adverse effects associated with the respective intervention. Thus, the combined serial analysis of mutant DNA and miR in the circulation has the potential to provide a molecular footprint of pancreatic cancer and can be used to monitor treatment responses or resistance to treatment in real time with a minimally invasive procedure.