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e22093 Background: Biomarkers are an integral part of cancer care. A variety of testing methods exist, allowing institutional selection of which technologies to use and in what order to run them. This descriptive study provides a benchmark on methods used in practice for biomarker testing in breast (BC), gastric/esophageal (GC), and non-small cell lung cancers (NSCLC). Methods: We conducted an IRB-approved web survey of the 58 NCI designated cancer centers (pathologists and oncologists) providing adult care in BC, GC, and NSCLC. We developed a survey instrument with 14 BC, 5 GC, and 10 NSCLC items asking about methods used, order of testing, turn around time (TAT) and if tests are internally developed (ID). Results: For BC, 32% (18/57) of sites run concurrent rather than reflex HER2 FISH and IHC testing, citing quality assurance reasons. Average TAT for concurrent vs reflex testing is 6.5 and 9.5 days. Rates of ID tests are 14% (8/57) for PgR, 12% (7/57) for ER and 7% (4/57) for HER2. For GC, 39% (21/54) of sites run concurrent HER2 FISH and IHC tests, citing reasons of quality assurance and evolving guidelines. TAT for concurrent vs reflex testing is 6.3 and 9.5 days. 19% (10/54) of sites use ID HER2 tests. All reporting sites use EGFR and ALK testing in NSCLC and 96% use KRAS. 24% (13/55) of sites run them concurrently with an average TAT of 7.6 days, while the average TAT for sites with sequential testing is 22.8 days. CAP recommends a 10 day TAT for NSCLC tests, and 92% (12/13) of sites with concurrent testing meet this guideline vs only 11% (4/38) of sites with sequential testing (p<.0001). Rates of ID tests are 57% (30/53) for KRAS, 47% (26/55) for EGFR and 35% (19/55) for ALK, for which a kit is listed on the FDA drug label. There is a difference in the use of ID tests between BC and NSCLC, p<.0148. Conclusions: Concurrent testing for BC and GC HER2, and for NSCLC markers is a common practice at NCI cancer centers; and it improves TAT. Concurrent NSCLC testing allows meeting CAP TAT guideline, but sequential testing does not. New markers (GC HER2, EGFR, KRAS and ALK) are commonly adopted, with a higher rate of internally developed tests than for BC HER2. [Table: see text]

1087 Background: EP is a unique topoisomerase 1 inhibitor that provides continuous exposure to SN38. EP demonstrated a 29% overall response rate in patients with mBC, leading to a phase III global BEACON study in patients with mBC. CTCs in patient blood samples provide a minimally invasive approach to detect PD markers of drug activity. We developed quantitative multiplex immunofluorescent assays to measure target-specific PD biomarkers for EP in CTCs isolated pre- and post-treatment. Methods: Assays for Top1, Top2, g-H2Ax, Rad51, ABCG2, and Ki-67 were developed using control (0.1% DMSO) and drug-treated (SN38, 10 uM) tumor cell lines (HCT116, MCF7, A549, SKBr3) and PBMCs from healthy donors. The optimal antibody for each biomarker was then multiplexed in a panel with antibodies against cytokeratin, CD45 and DAPI for phenotypic identification of CTCs. For analysis of BEACON pts, serial 7.5 mL whole blood samples were drawn and shipped ambient to Apocell (Houston, TX) for further processing. PBMCs were separated and CTCs were isolated using ApoStream technology. CTCs were stained for PD markers and analyzed using an iCys laser scanning cytometer equipped with image analysis software. Results: Antibodies bound to tumor cells showed staining confined to the nucleus (Top1, Top2, g-H2AX, Ki-67) or membrane (ABCG2), exhibited defined peak separation from their isotype controls, and signal strength correlated with cellular expression of high and low levels of markers. To date, ~ 80% of BEACON pts consent to participate in the CTC sub-study. As of 30 Oct 2012, data from 167 pre-dose blood samples from BEACON pts were available. 99% of blood samples were successfully processed. 93% had detectable CTCs, yielding a median of 216 CTCs (range: 7.5-14816). Staining was positive for Top1, Top2, g-H2Ax, Rad51, ABCG2, and Ki-67 in 82%, 89%, 16%, 53%, 31%, 52% of samples, respectively. Conclusions: EP target-specific pharmacodynamic biomarkers can be reliably measured in CTCs isolated from patients participating in BEACON. Sample collection and analysis of pre- and post-treatment samples is ongoing. Clinical trial information: NCT01492101.

1059 Background: Relapsed TNBC is characterized by its poor prognosis. B is a synthetic DNA minor groove binding non-cross resistant agent that is fully active against DNA-mismatch repair deficient tumor cells. C increases GSH/GST in which enhances the efficacy of B. Methods: Phase II study in pts with mTNBC. C Day 1 followed by B Day 2, repeated every 21 days. Aim: Efficacy of C + B in mTNBC. Primary endpoint: PFS at 3 mo. Secondary endpoints include ORR, duration of response (DOR), 6-mo PFS, OS and AE profile. Tertiary endpoints include assessment of baseline GSH levels, prevalence of BCRA1 mutation and pERK, and correlations with outcome. Results: Closed 3/2012. 48 pts accrued (1 ineligible pt). Median f/u 11.4 mo (6.6-14.8); 2 pts still on treatment. 47 pts evaluable for the PFS endpoint and AEs. 49% received therapy as 3rd-5th line. Median number of cycles = 4 (range 1-15). There are currently 9 confirmed responses (8 PR and 1 CR); DOR = 2.6-14.5 mo. The 3-mo PFS = 51%; 6-mo PFS = 28%; the median TTP is 3.2 mo. AE data: 75% G3/4 hematologic AE: febrile neutropenia 19%. Non-hematologic AEs included G3 (47%) and G4 (15%): G3 fatigue 17%; and no G5 non-heme AE. No correlation of GSH/GST levels with outcome was found. Among 35 pts with evaluable data, no association between BRCA1 expression and outcomes was found. pERK expression was negatively associated with 3-mo PFS, but not 6-mo PFS. Cytoplasmic pERK expression was negatively associated with tumor response (p=0.03). No significant effect of BRCA-1 or pERK expression was found on OS. Conclusions: This trial showed B + C is an active regimen, especially in heavily pretreated mTNBC pts. Correlative data demonstrated a negative association between pERK expression and outcomes. A randomized phase II trial is in development. Clinical trial information: NCT01091454.

1050^ Background: In a phase III trial comparing eribulin (E) vs. capecitabine (C) in pts with locally advanced or MBC, a trend for improved OS was observed but a statistically significant superiority was not demonstrated with E vs. C for OS or PFS. The AE profiles were consistent with known side effects. We now report QoL results from this trial. Methods: Pts received eribulin mesylate 1.4 mg/m2 on Days 1 and 8, or C 1.25 g/m2 BID orally on Days 1-14, of a 21-day cycle. Eligible pts had received prior therapy including an anthracycline and taxane, and were receiving study drug as 1st-, 2nd-, or 3rd-line therapy for advanced disease. QoL, a secondary objective, was assessed using EORTC QLQ-C30 and QLQ-BR23 questionnaires at baseline, 6 weeks, 3, 6, 12, 18, and 24 months after starting treatment (or until progressive disease or treatment change), and at unscheduled visits. Longitudinal analyses were carried out using weighted generalized estimating equations adjusted for non-random attrition due to death within 12 months. Model covariates were time (visit), region, and baseline QoL. The primary endpoint was change from baseline for Global Health Status (GHS)/overall QoL; exploratory endpoints were change from baseline for each functional domain, and signs/symptoms. Results: 1,102 pts were randomized (E 554; C 548). GHS/QoL scores were low at baseline for E (56.3) and C (54.7) on a scale of 0 (worse) to 100 (best). GHS/QoL and cognitive functioning improved significantly more in pts receiving E vs. C, (6.5 [p=0.048] and 15.3 [p<0.001], respectively). Emotional functioning improved significantly for pts receiving C vs. E (3.3; p=0.033). Pain was comparable at baseline, and was lower at subsequent visits with both treatments. Patient-reported signs/symptoms in favor of E included nausea and vomiting (E1.9; p=0.043) and diarrhea (-3.7; p=0.001); systemic side effects (5.2; p<0.001) and upset by hair loss (9.3; p=0.023) favored C. Conclusions: GHS/QoL scores improved more in pts receiving E than C. E showed advantages in terms of gastrointestinal effects while C had advantages in relation to hair loss. Clinical trial information: NCT00337103.

1049^ Background: This phase III study, comparing eribulin versus capecitabine, showed a non-significant trend for superior overall survival (OS; hazard ratio [HR] 0.88 [95% confidence interval (CI) 0.77, 1.00]; p = 0.056) but not progression-free survival (PFS; HR 1.08 [95% CI 0.93, 1.25]; p = 0.31). Pre-specified exploratory subgroup analyses previously presented showed that patients with triple-negative, ER-negative or HER2-negative disease may have a greater benefit in OS with eribulin compared with capecitabine. Here we present further pre-specified exploratory analyses of OS and PFS. Methods: Patients (eribulin n=554; capecitabine n=548) with locally advanced or MBC had received ≤3 prior chemotherapy regimens (≤2 for advanced disease), including an anthracycline and a taxane. Patients were randomized (stratified for geographic region and HER2 status) 1:1 to 21-day cycles of eribulin mesylate 1.4 mg/m2 i.v. on days 1 and 8 or capecitabine 1.25 g/m2BID orally on days 1-14. Further pre-specified exploratory subgroups included: age; receptor status; number and setting of prior chemotherapy regimen(s); sites of disease; number of organs involved; and time to progression after last chemotherapy. Results: From analyses for OS, patients with only non-visceral disease (HR 0.51; 95% CI 0.33, 0.80), with >2 organs involved (HR 0.75; 95% CI 0.62, 0.90), who had progressed >6 months after last chemotherapy (HR 0.70; 95% CI 0.52, 0.95), or who had received an anthracycline and/or a taxane in the metastatic setting (HR 0.84; 95% CI 0.72, 0.98), appeared to benefit more from treatment with eribulin compared with capecitabine. For OS, in no subgroup was a trend favoring capecitabine seen. Data for other pre-specified subgroups for both OS and PFS will be presented. Conclusions: In addition to patients with triple-, ER-, or HER2-negative disease, further pre-specified exploratory analyses suggest that other patient subgroups may particularly benefit from treatment with eribulin; further studies are warranted to address these hypotheses. Clinical trial information: NCT00337103.

522 Background: In the adjuvant setting, patients with HER2 breast cancer treated with trastuzumab and chemotherapy have superior survival compared to patients treated with chemotherapy alone. We previously showed that trastuzumab and chemotherapy induces HER2-specific antibodies which correlate with response to therapy in patients with HER2+ metastatic breast cancer. It remained unclear from those studies, however, whether the HER2-specific immunity played a role and if antibody immunity was associated with improved disease free survival in the adjuvant setting. In the present study, we addressed these questions by analyzing sera samples from a subset of patients enrolled in the NCCTG adjuvant trial, N9831, which includes an arm (Arm A) in which trastuzumab was not used. Arms B and C received trastuzumab sequentially or concurrently to chemotherapy, respectively. Methods: Pre-and post-treatment initiation sera were obtained from 50 women enrolled in N9831 (22 Arm A; 14 Arm B, and 14 Arm C). Lambda IgG antibodies (to avoid detection of trastuzumab) to HER2 were measured and presented as an index (>0.2 was considered a positive response). Results: Prior to therapy, across all three arms, N9831 patients had similar mean HER2 IgG levels (0.19 units in Arm A, 0.14 in Arm B, and 0.23 in Arm C, P=0.85). Following treatment, the mean levels of antibodies increased in Arm B to 0.35 units and in Arm C to 0.56 units and were higher (p<0.001) than in Arm A where levels did not increase. The proportion of patients who demonstrated antibody immunity increased by 9% in Arm A, 50% in Arm B and 28% in Arm C (P=0.026). Although the event rate was low in this cohort, Cox modeling suggested that larger increases in antibodies were associated with improved disease free survival (HR=0.23; p=0.04). Conclusions: These results show that the increased antibody immunity observed in adjuvant patients treated with combination trastuzumab and chemotherapy is clinically significant and results from the inclusion of trastuzumab. The findings may have important implications for improving treatment outcomes in patients treated with trastuzumab.

521 Background: The addition of trastuzumab to chemotherapy improves responses to therapy and extends survival among patients with metastatic HER2 breast cancer. Several mechanisms have been proposed for the activity of this combination therapy. Trastuzumab, specifically, is thought to activate NK cells and blunt HER2 signaling. Prior work from us has shown that combination trastuzumab and chemotherapy induces HER2-specific antibodies which correlate with response to therapy. Despite that, it remains unclear whether the immunity that was induced was due to complexing of non-tumor derived HER2 or antigen derived from the tumor site. In the present work, we addressed this question by assessing if combination therapy induced epitope spreading to tumor antigens other than HER2. Methods: Pre-and post-treatment sera were obtained from 56 women enrolled in 2 NCCTG clinical trials, N0337 and 98-32-52. IgG antibodies to HER2 intracellular domain (HER2), p53, IGFBP2, CEA and tetanus toxoid (TT) were examined using ELISAs. Sera from an age-matched group (N=56) of controls and 12 patients treated in the adjuvant setting were also examined. Results: Prior to therapy, metastatic patients had higher IgG levels (≥ 2-fold) to p53 and HER2 but not CEA, IGFBP2 and TT, relative to the controls. Similarly, adjuvant patients had elevated IgGs to multiple tumor antigens prior to therapy, relative to controls. Following therapy, levels of IgG to IGFBP2, HER2, and p53 increased in 81% of metastatic patients, with mean increases of 3.2 (±0.6 sem), 6.2 (±2.7) and 2.7 (±0.7) fold, respectively (p<0.05). Levels of antibodies to TT and CEA were not elevated by treatment. In contrast, IgGs were not increased in adjuvant patients; consistent with the idea that immunity depends on the presence of threshold levels of antigens. Conclusions: These results show that combination treatment induces adaptive immunity to antigens released by tumor and that metastatic patients remain capable of responding immunologically to their cancer. Thus, in metastatic breast cancer patients, combination trastuzumab and chemotherapy may behave as a vaccine.

2015 Background: A prior study of intra-CSF topotecan (TOPO) for unselected pts w/ NM reported PFS 6 mo of 19%, and OS of 15 wks (Groves, NeuroOncol 2008;10:208). We postulated that greater activity might occur in pts w/ malignancies considered sensitive to topoisomerase inhibitors. Methods: We reviewed outcome of women with NM and adenocarcinoma of the breast, ovary or lung receiving IVe TOPO (0.4 mg 2x/wk x 4wk, Q wk x 4, Q 2wk x 2, Q mo x 3, Q 2mo x 3, and Q 3mo x 4) until progression (PROG) or adverse events (AE). All had baseline CSF cytology, and MRI of brain and spine. CSF cytology was obtained at each treatment (Rx), and brain/spine MRI Q 3mo. Neuro-specific PROG was defined as recurrent + CSF cytology; PROG of NM on MRI; all-cause neurologic worsening; pt refusal; or death. PFS/OS were measured from 1st TOPO Rx. All pts signed consent; the study was IRB approved. Results: 17 women (breast -12; lung-3; ovary - 2) were treated via Ommaya reservoirs; 7 (41%) had VP shunts w/ valves, adjusted for Rx. Median (med) age was 53 (41-79), and KPS 70 (50-90). At presentation, 11(65%) had + CSF cytology and 14 (82%) had NM on MRI [brain-11 (65%); spine-10 (59%)]. 15 (88%) had no prior intrathecal Rx. 13 pts (76%) received focal RT for CNS disease, and 8 (47%), chemotherapy for extracranial disease. Med.number of Rx were 13/pt (range, 3-50); med. duration of TOPO Rx was 14 wks (range, 1-109). Med. neuro-specific PFS was 13 wks; PFS6 was 41%, and PFS12, 29 %. Med. OS was 33 wks (range, 5-180), w/ 4 alive at 13+, 30+, 33+, and 180+ wks. 4 pts (24%) lived > 95 wks. Of 11 w/ baseline + CSF cytology, 7 (64%) cleared CSF of malignant cells (med. duration clear = 47 wk (range, 9-104). AE included arachnoiditis (18%), leukoencephalopathy (18%), and Ommaya infections (12%). Rx was stopped for neuro PROG (29%); systemic PROG (23%); refusal (18%); AE (12%); or persistent negative CSF (6%); 12% are still on Rx. Conclusions: Promising activity of IVe TOPO was observed in women with NM from breast, lung and ovarian cancer. PFS 6 and 12, OS and cytologic response were twice that noted in prior studies of NM pts w/ unselected malignancies. This data supports our plan for a phase II study targeting this select cohort.

1083 Background: Pixantrone (Pix) is a novel aza-anthracenedione with structural similarities to mitoxantrone and promising activity against non-Hodgkin’s lymphoma. Due to the lack of iron binding it is theorized to exhibit less cardiotoxicity than the anthracyclines. Methods: N1031 is a phase II RCT of 2 schedules of Pix, in pts with MBC. Group A pts received 180mg/m2 IV q3 wks, and group B pts 85mg/m2 IV on days 1, 8, 15 q4 wks. Eligibility included prior exposure to anthracyclines and/or taxanes, and 1 to 3 regiments in the metastatic setting (minimum of 2 if no prior adjuvant therapy given). Due to lack of long term cardiac safety data no more than 12 cycles were allowed. Frequent cardiac imaging was performed per protocol. Primary endpoint was RR and secondary endpoints included PFS, OS, safety, and QOL. Planned sample size was 25 pts per group. Results: In total 46 pts were evaluable (23 per group), mean age 55.5 yrs (range 38-79), 37% PS 0, 52% PS 1, and 11% PS 2. 80% of pts had prior exposure to doxorubicin, 72% had prior (neo)-adjuvant therapy, 76% were ER+ and 57% received prior HT. Number of prior metastatic regiments was: 1 (28%), 2 (61%) and 3 (11%). Most common adverse events (%) of any grade were: alopecia (74), anemia (74), fatigue (85), nausea (67), ANC decrease (87), and skin disorder (41). Grade 3-4 adverse events (%) at least possibly attributed to Pix and occurring in at least 2 pts were: ANC decrease (57), fatigue (9), increased AST (4%). One pt from each group (4%) had a grade 3 decrease in EF. There were no major differences between the two groups except for more oral mucositis in group A (35% vs 4%). Median number of cycles was 3 in group A (range 1-12) and 2 in group B (range 1-8). There was only 1 confirmed tumor response per group (4%,95% CI: 0.1-22%) prompting early termination of the trial. The median PFS was 2.7 mo (95% CI: 1.8-3.8), and the median OS was 8.9 mo (95% CI: 7.5-N/A). Conclusions: Pixantrone has insufficient activity in patients with MBC exposed to prior anthracyclines and/or taxanes. Adverse events were similar to prior experience with Pix. There were no major differences between the 2 schedules of administration. There was no significant cardiac toxicity seen in this trial. Correlative studies are underway.

CRA1002 The full, final text of this abstract will be available at abstract.asco.org at 12:01 AM (EDT) on Monday, June 4, 2012, and in the Annual Meeting Proceedings online supplement to the June 20, 2012, issue of Journal of Clinical Oncology. Onsite at the Meeting, this abstract will be printed in the Monday edition of ASCO Daily News.