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Claudin 18.2 (CLDN18.2) is contained within normal gastric mucosa epithelial tight junctions; upon malignant transformation, CLDN18.2 epitopes become exposed. Zolbetuximab, a chimeric monoclonal antibody, mediates specific killing of CLDN18.2-positive cells through immune effector mechanisms.The FAST study enrolled advanced gastric/gastro-oesophageal junction and oesophageal adenocarcinoma patients (aged ≥18 years) with moderate-to-strong CLDN18.2 expression in ≥40% tumour cells. Patients received first-line epirubicin + oxaliplatin + capecitabine (EOX, arm 1, n = 84) every 3 weeks (Q3W), or zolbetuximab + EOX (loading dose, 800 mg/mIn the overall population, both PFS [hazard ratio (HR) = 0.44; 95% confidence interval (CI), 0.29-0.67; P0.0005] and OS (HR = 0.55; 95% CI, 0.39-0.77; P0.0005) were significantly improved with zolbetuximab + EOX (arm 2) compared with EOX alone (arm 1). This significant PFS benefit was retained in patients with moderate-to-strong CLDN18.2 expression in ≥70% of tumour cells (HR = 0.38; 95% CI, 0.23-0.62; P0.0005). Significant improvement in PFS was also reported in the overall population of arm 3 versus arm 1 (HR = 0.58; 95% CI, 0.39-0.85; P = 0.0114) but not in high CLDN18.2-expressing patients; no significant improvement in OS was observed in either population. Most adverse events (AEs) related to zolbetuximab + EOX (nausea, vomiting, neutropenia, anaemia) were grade 1-2. Grade ≥3 AEs showed no substantial increases overall (zolbetuximab + EOX versus EOX alone).In advanced gastric/gastro-oesophageal junction and oesophageal adenocarcinoma patients expressing CLDN18.2, adding zolbetuximab to first-line EOX provided longer PFS and OS versus EOX alone. Zolbetuximab + EOX was generally tolerated and AEs were manageable. Zolbetuximab 800/600 mg/m

The selective MEK1/2 inhibitor pimasertib has shown anti-tumour activity in a pancreatic tumour model. This phase I/II, two-part trial was conducted in patients with metastatic pancreatic adenocarcinoma (mPaCa) (NCT01016483). In the phase I part, oral pimasertib was given once daily discontinuously (5 days on/2 days off treatment) or twice daily continuously (n = 53) combined with weekly gemcitabine (1,000 mg/m2 ) in 28-day cycles to identify the recommended phase II dose (RP2D) of pimasertib. In the phase II part, patients were randomised to pimasertib (RP2D) or placebo plus weekly gemcitabine (n = 88) to investigate progression-free survival (PFS), overall survival (OS) and safety. The RP2D was determined to be 60 mg BID. PFS and OS outcomes did not indicate any treatment benefit for pimasertib over placebo in combination with gemcitabine (median PFS 3.7 and 2.8 months, respectively, HR = 0.91, 95% CI: 0.58-1.42: median OS 7.3 vs. 7.6 months, respectively). KRAS status did not influence PFS or OS. The incidence of grade ≥3 adverse events was 91.1% and 85.7% for pimasertib/gemcitabine and placebo/gemcitabine respectively, but there was a higher incidence of ocular events with pimasertib/gemcitabine (28.9% vs. 4.8% for placebo/gemcitabine). In conclusion, no clinical benefit was observed with first-line pimasertib plus gemcitabine compared with gemcitabine alone in patients with mPaCa.

16 Background: Physiologically, the tight junction protein CLDN18.2 is present only in the gastric mucosa. Upon malignant transformation, CLDN18.2 epitopes are exposed on the cell surface and accessible to targeted therapy. Zolbetuximab (formerly IMAB362) is a chimeric mAb that mediates specific killing of CLDN18.2+ cancer cells through immune effector mechanisms; single-agent activity has been reported in G/GEJ cancer. Methods: Patients (pts) with advanced HER2-negative (HER–) G/GEJ cancer with CLDN18.2 expression of ≥ 2+ staining intensity with the anti-CLDN18 43-14A mAb in ≥ 40% tumor cells were eligible (NCT01630083). Patients were randomized 1:1 to receive first-line EOX ± zolbetuximab (loading dose 800 mg/m2, then 600 mg/m2) Q3W. The study was extended with an exploratory arm of 1000 mg/m2 zolbetuximab + EOX Q3W (data not presented). Primary endpoint was PFS; secondary endpoints included OS, ORR, and safety/tolerability. Results: A total of 161 pts (G, n = 128; GEJ, n = 27; esophagus n = 6) were randomized to receive zolbetuximab (800/600 mg) + EOX (n = 77) or EOX alone (n = 84). In all, 45% of pts had diffuse type histology per Lauren’s classification. Median PFS was longer with zolbetuximab + EOX (7.5 mo) versus EOX alone (5.3 mo; P < 0.0005; HR 0.44; 95% CI 0.29, 0.67). Median OS (13 vs 8.4 mo; P = 0.0008; HR 0.56; 95% CI 0.40, 0.79) and ORR (39 vs 25%; P = 0.022) were also significantly higher with zolbetuximab + EOX versus EOX alone. Increased efficacy was more pronounced in pts with high CLDN18.2 expression (≥ 2+ staining intensity in ≥ 70% tumor cells). Zolbetuximab + EOX was generally well tolerated. Most AEs considered related to zolbetuximab and EOX (ie, nausea, vomiting, neutropenia, anemia) were of grade ≤ 2 severity; there was no significant increase in grade ≥ 3 events with the addition of zolbetuximab to EOX. Conclusions: Addition of zolbetuximab to first-line chemotherapy resulted in significantly longer PFS and OS and higher ORR with manageable toxicity. A phase III trial is evaluating the combination of zolbetuximab plus mFOLFOX6 as first-line treatment of CLDN18.2+/HER2-negative, advanced/metastatic G or GEJ adenocarcinoma (NCT03504397). Clinical trial information: NCT01630083.

Due to the gain in the average life expectancy in developed countries there is increased the incidence rate of malignant neoplasms. According to the forecast of the USA National Cancer Institute in the next 20 years every 2nd American man and every third American woman will get sick with cancer, and therefore oncology should be considered to be the main medical specialty. Molecular characterization of tumors will become routine, and it will provide an opportunity via screening to detect precancerous abnormalities and early forms of cancer. Surgery will become mostly organsaving modality, radical mutilating surgery will go down in history, traditional chemotherapy with low selectivity of action will send to the glue factory. In the arsenal there will stay well-aimed «targeted» drugs hitting right on target mechanisms of the uncontrolled growth of tumor cells and their dissemination.

Background Pancreatic ductal adenocarcinoma (PDAC) has a high mortality rate with limited treatment options. Gemcitabine provides a marginal survival benefit for patients with advanced PDAC. Dasatinib is a competitive inhibitor of Src kinase, which is overexpressed in PDAC tumors. Dasatinib and gemcitabine were combined in a phase 1 clinical trial where stable disease was achieved in two of eight patients with gemcitabine-refractory PDAC. Patients and methods This placebo-controlled, randomized, double-blind, phase II study compared the combination of gemcitabine plus dasatinib to gemcitabine plus placebo in patients with locally advanced, non-metastatic PDAC. Patients received gemcitabine 1000 mg/m2 (30-min IV infusion) on days 1, 8, 15 of a 28-day cycle combined with either 100 mg oral dasatinib or placebo tablets daily. The primary objective was overall survival (OS), with safety and progression-free survival (PFS) as secondary objectives. Exploratory endpoints included overall response rate, freedom from distant metastasis, pain and fatigue progression and response rate, and CA19-9 response rate. Results There was no statistically significant difference in OS between the two treatment groups (HR = 1.16; 95% confidence interval [CI]: 0.81–1.65; P = 0.5656). Secondary and exploratory endpoint analyses also showed no statistically significant differences. The burden of toxicity was higher in the dasatinib arm. Conclusions Dasatinib failed to show increased OS or PFS in patients with locally advanced PDAC. Alternative combinations or trial designs may show a role for src inhibition in PDAC treatment.

4038 Background: Claudin 18.2 (CLDN18.2), a gastric mucosa tight junction protein, is aberrantly expressed in various cancers. In the FAST Phase 2 trial (NCT01630083), IMAB362, an anti-CLDN18.2 monoclonal antibody, administered in combination with EOX chemotherapy, prolonged survival compared to EOX alone in patients with advanced/recurrent gastric, gastroesophageal junction (GEJ), and esophageal cancers ineligible for trastuzumab. The aim of the present analysis was to assess tumor CLDN18.2 expression and co-expression with HER2 in the FAST population. Methods: Tumor tissue samples from patients screened for inclusion into the FAST trial were analyzed for CLDN18.2 expression using a CE-marked, validated immunohistochemistry (IHC) assay. CLDN18.2 expression was centrally scored based on staining intensity and percentage of stained tumor cells. In a subset of tissue samples with known HER2 status, overall HER2 expression and co-expression with CLDN18.2 were determined. Results: Tissue samples from 730 patients with gastric, GEJ, and esophageal cancer were screened for the FAST study. Of these 730 samples, 685 (94%) were assessed by central IHC; 49% (n=333/685) met the FAST CLDN18.2 expression criterion (≥2+ intensity in ≥40% of tumor cells) for inclusion. Across the 154 tissue samples with known HER2 status, the majority (84%; n=129/154) were HER2–. Furthermore, 94 of these 154 samples (61%) met the FAST CLDN18.2 expression criterion, of which 14% (n=13/94) co-expressed HER2 (Table). Conclusions: In tissue samples from patients screened for the FAST trial, nearly half met CLDN18.2 expression inclusion criterion. In samples with known HER2 status, co-expression of CLDN18.2 and HER2 occurred in 14% of the samples that met study eligibility. These data suggest CLDN18.2 may serve as a non-HER2 overlapping targetable alteration in a distinct subpopulation of patients with gastric, GEJ, or esophageal cancers. Clinical trial information: NCT01630083. [Table: see text]

Background This retrospective study was carried out to evaluate the prognostic significance of clinical factors in patients treated for metastatic gastric cancer with second-line chemotherapy. Patients and methods We evaluated the prognostic significance of various clinical factors in 126 patients, who were treated with second-line chemotherapy. Results Median progression-free and overall survival (OS) for second-line chemotherapy were 3.3 and 5.3 months, respectively, with an overall response rate of 11.1%. Multivariate analysis identified three independent prognostic factors: performance status: Eastern Cooperative Oncology Group zero to one [hazard ratio (HR) 2.3, 95% confidence interval (CI) 1.7–5.4], hemoglobin (Hb) level: ≥10 g/dl (HR 2.2, 95% CI 2.1–2.4) and time-to-progression (TTP) under first-line therapy: ≥5 months (HR 0.5, 95% CI 0.3–0.8). From the obtained data, a prognostic index was constructed, dividing the patients into three risk groups: good (n = 40), intermediate (n = 36) and poor risk group (n = 56). The median survival for good, intermediate and poor risk groups were 13.5, 6.0 and 2.9 months, respectively, whereas the 1-year OS rates were 50.2%, 14.2% and 2.6%, respectively (P = 0.00001). Conclusions With inadequate data from randomized controlled trials at the moment, our report indicates that second-line chemotherapy is effective and beneficial in patients with good performance status, higher Hb level along with higher TTP under first-line therapy.

Background This randomized, double-blind, placebo-controlled, phase II study evaluated the efficacy and safety of mapatumumab (a human agonistic monoclonal antibody against tumor necrosis factor-related apoptosis-inducing ligand receptor 1) in combination with sorafenib in patients with advanced hepatocellular carcinoma (HCC). Patients and Methods Patients with advanced HCC (stratified by Barcelona Clinic Liver Cancer stage and Eastern Cooperative Oncology Group performance status) were randomized 1:1 to receive sorafenib (400 mg, twice daily per 21-day cycle) and either placebo (placebo–sorafenib arm) or mapatumumab (30 mg/kg on day 1 per 21-day cycle; mapatumumab–sorafenib arm). The primary end point was time to (radiologic) progression (TTP), assessed by blinded independent central review. Key secondary end points included progression-free survival, overall survival, and objective response. Results In total, 101 patients were randomized (placebo–sorafenib arm: N = 51; mapatumumab–sorafenib arm: N = 50). There was no significant difference in median TTP between both arms [5.6 versus 4.1 months, respectively; adjusted hazard ratio (one-sided 90% confidence interval) 1.192 (0–1.737)]. No mapatumumab-related benefit was identified when TTP was evaluated in the stratified subgroups. The addition of mapatumumab to sorafenib did not demonstrate improvement in the secondary efficacy end points. The reported frequency of adverse events (AEs) and serious AEs was comparable in both treatment arms. Conclusions The addition of mapatumumab to sorafenib did not improve TTP or other efficacy end points, nor did it substantially change the toxicity profile of sorafenib in patients with advanced HCC. Based on these results, further development of the combination of mapatumumab and sorafenib in HCC is not planned.

e15733 Background: Second line ChT is shown to improve outcome in selected patients with PC; however there are no approved models predicting its benefit. This retrospective study was aimed to evaluate prognostic factors in patients with PC who had disease progression following 1st line ChT and their value in prediction of 2nd line ChT benefit. Methods: Records of PC patients treated in N.N. Blokhin Russian Cancer Research Center since 2000 to 2015 were analyzed. Inclusion criteria for this retrospective analysis were: morphologically confirmed PC, disease progression after 1st line ChT or adjuvant / induction ChT with ChT-free interval 5 at the time of disease progression after 1st line ChT (Table). Administration of 2nd line ChT improved outcome of patients with favorable prognosis (score ≤1): median OS increased from 1.7 to 5.5 months in groups without (n=23) and with (n=90) ChT, respectively (p=0.02). In patients with poor prognosis (score>1) there were no benefit by administration of 2nd line ChT: medians OS were 2.3 and 1.7 months in groups with (n=20) and without (n=39) ChT, respectively (p=0.23). Conclusions: This novel prognostic model can potentially predict 2nd line ChT benefit in patients with PC, however it needs to be validated in further trials. [Table: see text]

LBA4001 Background: Claudin18.2 (CLDN18.2) is a tight junction protein expressed by several cancers including gastric and GEJ adenocarcinoma. IMAB362 is a chimeric monoclonal antibody that mediates specific killing of CLDN18.2-positive cancer cells by activation of immune effector mechanisms. IMAB362 has demonstrated single-agent activity and was safe and tolerable in patients (pts) with pretreated gastric cancer. Methods: Pts with advanced/recurrent gastric and GEJ cancer were centrally evaluated for CLDN18.2 expression by IHC (validated CLAUDETECT18.2 Kit). Eligible pts had a CLDN18.2 expression of ≥ 2+ in ≥ 40% tumor cells, an ECOG PS of 0–1 and were not eligible for trastuzumab. Pts were randomized 1:1 to first-line EOX (epirubicin 50 mg/m2 and oxaliplatin 130 mg/m2 d1, and capecitabine 625 mg/m2 bid, d1–21; qd22) with or without IMAB362 (loading dose 800 mg/m2, then 600 mg/m2 d1, qd21). The study was extended by an exploratory Arm3 (N = 85) to investigate a high dose IMAB362 (1000 mg/m2) plus EOX, (not subject here). The primary study endpoint was PFS (Arm 1 v 2, 70% power, HR 0.72, 1-sided p = 0.1). Results: 730 pts were consented, of whom 352 pts (48%) were tested CLDN18.2+ per protocol criteria. Of those, 161 pts (median age, 58 yrs; male 64%; gastric, 80%; GEJ, 16%; esophageal, 4%) were randomized into Arms1 and 2. The study met its endpoints. IMAB362 plus EOX improved PFS (median 5.7 v 7.9 mon; HR 0.5; 95% CI 0.35–0.78, 1-sided p = 0.001) and OS (median 8.7 v 12.5 mon; HR 0.5, 95% CI 0.28–0.73) compared to EOX alone. In the subpopulation with very high CLDN18.2 expression ( ≥ 2+ intensity in ≥ 70% tumor cells), efficacy was more pronounced (PFS, 6.1 vs 9.1 mon; HR 0.46; OS, 9.3 v 16.6 mon; HR 0.44). Most common IMAB362-related adverse events included vomiting, neutropenia, and anemia, which were mostly of NCI-CTC grade 1/2. Grade 3/4 events were not significantly increased by IMAB362. Conclusions: IMAB362 combined with first-line chemotherapy exhibited a clinically relevant benefit in PFS and OS and a favorable risk/benefit profile. Clinical trial information: NCT01630083.

4007Background: Hypoxia in PDAC is associated with disease progression and poor prognosis. Evo is a hypoxia-activated prodrug of Br-IPM that is preferentially activated under hypoxic conditions. Th...

193 Background: Pancreatic ductal adenocarcinoma (PDAC) is invariably diagnosed at an advanced stage and has poor clinical outcome. Hypoxia is a significant prognostic factor in PDAC progression and is associated with poor prognosis. Evofosfamide (Evo, previously known as TH-302) is a hypoxia-activated prodrug of bromo-isophosphoramide mustard (Br-IPM) that is preferentially activated under hypoxic conditions. The addition of Evo to gemcitabine (Gem) significantly improved progression-free survival (PFS) in a randomized phase II trial in advanced PDAC (NCT01144455). Methods: MAESTRO is an international, randomized, double-blind, placebo-controlled phase III trial of Evo/Gem vs Placebo/Gem in patients (pts) with measurable, locally advanced unresectable or metastatic PDAC (NCT01746979). Evo and Gem were administered intravenously at a dose of 340 mg/m2 and 1,000 mg/m2, respectively, on days 1, 8, and 15 of a 28-day cycle. Treatment continued until disease progression. Key eligibility criteria included ECOG PS 0/1 and no neoadjuvant or adjuvant chemotherapy 5% of pts in treated with Evo/Gem were: TBC. Conclusions: The data from the MAESTRO trial will make an important contribution to our understanding of PDAC treatment. Clinical trial information: NCT01746979.

Since the late 1990s, docetaxel (Dtx), an antitubular drug, has been studied as a tool for the treatment of GC. Maximum effectiveness of docetaxel as monotherapy amounted to 24%, with a median survival of 7 months. Two-drug combinations were developed containing docetaxel with 5-fluorouracil (DF) and docetaxel with cisplatin (DC). They proved effective in 43 and 33% of the cases respectively and ensured a similar median survival of 9-10 months. Clinical studies of a three-component combination containing docetaxel, 5-fluorouracil and cisplatin (DCF) as first-line therapy of metastatic GC were carried out in the XXIst century and showed its efficacy in 50% of the cases with a median survival of 10-12 months. The DCF regimen may be considered as a new standard for the treatment of patients with metastatic GC and satisfactory health status (ECOG 0-1). The combination is being modified to improve its toxicity profile by substituting oxaliplatin for cisplatin and oral fluoropyrimidines for i.v. 5-fluorouracil.

344 Background: In metastatic pancreatic cancer (mPaCa), KRAS mutations lead to constitutive activation of the MAPK pathway in the vast majority of cases. Pimasertib (Pim) is a selective, non-competitive MEK 1/2 inhibitor with potent antitumor activity in preclinical models with constitutive MAPK activation. Methods: Following a phase I, dose-finding part to the trial, 88 patients (pts) with mPaCa were randomized 1:1 to receive Pim 60 mg BID (A, 44 pts) or placebo (B, 44 pts) in combination with weekly gemcitabine (Gem) 1000 mg/m2(7 of 8 wks in cycle 1, then 3 of 4 wks in subsequent cycles), in a phase II setting (NCT01016483). The primary endpoint was progression-free survival (PFS). Response rate (RR), overall survival (OS) and safety were secondary endpoints. Biomarker analysis was an exploratory endpoint. Results: Pt characteristics were balanced (median age 63.5 yrs, males 56%, stage IV at initial diagnosis 75%) except for PS 0, which was more frequent in arm A (59 vs 41%). Time on treatment was longer in arm B (10.6 vs 8.0 wks). A higher proportion of pts in arm A discontinued treatment during the first 4 wks (31 vs 19%), predominantly due to adverse events (AEs) and PD. Median PFS was 3.7 mo in arm A and 2.8 mo in arm B (HR=0.883, 95% CI: 0.549–1.42; p=0.608). No statistically significant differences were observed between arms for OS (median 7.3 mo in arm A vs 8.3 mo in arm B) and RECIST 1.0 RR (9.1% in both arms). Grade ≥3 thrombocytopenia (20.0 vs 0%), vomiting (15.6 vs 4.8%), fatigue (15.6 vs 7.1%), stomatitis (13.3 vs 0%) and diarrhea (11.1 vs 2.4%) were more common in arm A. Typical allosteric MEK inhibitor-related AEs, such as all grade retinal detachment (24.4%) and creatine phosphokinase elevation (20.0%), were observed almost exclusively in arm A. KRAS mutational status did not influence PFS or OS. Conclusions: The primary study endpoint was not met; secondary endpoints did not suggest clinically meaningful differences between arms, except for selected toxicities observed more frequently with the combination of Gem and Pim. The outcome does not support further development of this combination in the first-line setting in mPaCa. Pim is currently in development in other solid tumors. Clinical trial information: NCT01016483.

4029 Background: TRAIL is a member of the tumor necrosis factor ligand super family that induces programmed cell death primarily in tumor cells (including liver tumors) through TRAIL death receptor...

e14674 Background: Predictive value of K-ras status for the treatment with anti-EGFR monoclonal antibodies is well established. There is a lack of data about predictive significance of K-ras status for oxaliplatin-based chemotherapy (CT). We retrospectively studied prognostic and predictive significance of mutant K-ras in mCRC pts treated with FOLFOX or XELOX in 1st line CT. Methods: We analyzed data on 127 pts with mCRC, who were treated in our department during 2008-2011 with oxaliplatin-based regimens. K-ras status was determined by PCR analysis in 51/127 (40,1%) pts. Mutant K-ras was detected in 16/51 (31,3%) pts: 13/16 (81,3%) – codon 12, 3/16 (18,7%) – codon 13. mFOLFOX6 was administered in 21/51 (41,2%), XELOX – in 30/51 (58,8%) pts. No pts received anti-EGFR mAb in the 1-st line. Median follow-up was 9 months (range, 2 - 41). The mean number of courses was 9 and 5 in FOLFOX and XELOX groups, respectively. PFS was chosen as a primary end-point. Results: Median PFS was 7,5 months for all 127 pts, 8,4 months in pts with FOLFOX and 7,5 months in pts with XELOX. Median PFS in pts with wild type K-ras was 10,3 months and 6,5 months in pts with mutant K-ras (p=0,1, HR 1,7, 95%CI 0,9-3,4). Negative prognostic significance of mutant K-ras was seen in XELOX group (PFS, 10,9 vs 5,7 months, р= 0,002) but not in FOLFOX group (PFS, 7,7 vs 9,3 months, р= 0,48). In pts with wild type K-ras XELOX lead to longer PFS than FOLFOX regimen - 10,9 vs 7,5 months, respectively (р=0,09, HR 0,52, 95%CI 0,23-1,14). And vice versa, in pts with mutant K-ras FOLFOX was more active than XELOX – median PFS was 9,3 vs 5,7 months, respectively (р=0,009, HR 0,3, 95%CI 0,05-0,64). Conclusions: Our findings probably suggest that K-ras status influences the efficacy of FOLFOX and XELOX regimens in mCRC pts. These data need to be confirmed in larger series of pts.

e14623 Background: Docetaxel is an active agent for treatment of metastatic gastric carcinoma. We launched this phase II trial to evaluate the efficacy and safety of docetaxel, cisplatin and capecitabine (DCX) combination in patients (pts) with metastatic gastric carcinoma. Methods: Pts with metastatic gastric carcinoma received docetaxel 75 mg/m2 i.v. day 1, cisplatin 75 mg/m2 i.v. day 1, capecitabine 1650 mg/m2 per os days 1-14 every 3 weeks. Up to 6 cycles were provided. G-CSF was not routinely used for primary prophylaxis. Results: From 2007 to 2011, 37 pts were included in the study, all pts were available for response. Pts characteristics and results are shown in the table. Median number of cycles delivered was 5 (range, 1-5). Grade 3-4 toxicity (per cycle) included: neutropenia – 24,9%, deep venous thrombosis – 2%, diarrhea – 6.2%, stomatitis – 3.8%, infection – 11%; anemia 14% pts. Six pts had dose reductions, 2 pts stopped chemotherapy due to toxicity. No toxic deaths were observed. Median progression-free survival (PFS) and median overall survival (OS) were 7,5 months (95% CI 6,076-8,857) and 14,5 months (95% CI 10,054-18,880), respectively. Conclusions: Compare with combination of docetaxel, cisplatin and 5-FU, evaluated in V325 study, our regimen had more favorable toxic profile with similar efficacy. Further investigation of this regimen in randomized controlled trials is warranted. [Table: see text]

Background: Cell-free DNA from dying cells recently has been discovered in human blood plasma. In experiments performed on animals and humans, we examined whether this cell-free DNA can cross the kidney barrier and be used as a diagnostic tool. Methods: Mice received subcutaneous injections of either human Raji cells or purified 32P-labeled DNA. DNA was isolated from urine and analyzed by measurement of radioactivity, agarose gel electrophoresis, and PCR. In humans, the permeability of the kidney barrier to polymeric DNA was assessed by detection in urine of sequences that were different from an organism bulk nuclear DNA. Results: In the experiments on laboratory animals, we found that ∼0.06% of injected DNA was excreted into urine within 3 days in a polymeric form and that human-specific Alu sequences that passed through the kidneys could be amplified by PCR. In humans, male-specific sequences could be detected in the urine of females who had been transfused with male blood as well as in DNA isolated from urine of women pregnant with male fetuses. K-ras mutations were detected in the urine of patients with colon adenocarcinomas and pancreatic carcinomas. Conclusions: The data suggest that the kidney barrier in rodents and humans is permeable to DNA molecules large enough to be analyzed by standard genetic methodologies.