Post-treatment Tumor Specimens Research Articles

Lessons from Post-Immunotherapy Tumor Tissues in Clinical Trials: How Can We Fuel the Tumor Microenvironment in Gliomas?

Despite recent advancements in cancer immunotherapy, many patients with gliomas and glioblastomas have yet to experience substantial therapeutic benefits. Modulating the tumor microenvironment (TME) of gliomas, which is typically "cold", is crucial for improving treatment outcomes. Clinical tumor specimens obtained post-immunotherapy provide invaluable insights. However, access to such post-immunotherapy samples remains limited, even in clinical trials, as tumor tissues are often collected only at tumor relapse. Recent studies of neoadjuvant immunotherapy provided important insights by incorporating surgical resections of post-treatment tumors. Moreover, pre-surgical immunotherapies are increasingly integrated into clinical trial designs to evaluate treatment efficacy. These investigations reveal critical information, particularly regarding the delivery success of therapeutic agents, the expansion and persistence of immune products, and the cellular and molecular changes induced in the TME. In this review, we assess the findings on post-treatment tumor specimens obtained from recent immunotherapy clinical trials on gliomas, highlight the importance of these samples for understanding therapeutic impacts, and discuss proactive investigation approaches for future clinical trials.

Exploratory phase I study of HF1K16 for the treatment of patients with refractory/recurrent advanced glioma: Preliminary efficacy and mechanism as a monotherapy.

e14030 Background: The absence of I.O success in advanced glioma treatment suggests that a deeper understanding of the brain tumor and the immune microenvironment is critical. It is well-documented that refractory/recurrent glioma has an immunosuppressive nature. One frequent observation is the high level of circulating myeloid-derived suppressor cells (MDSCs). We conducted a exploratory study to assess an immunomodulatory approach for relieving immune suppression employing HF1K16, a liposome all-trans retinoic acid (ATRA) suspension. Methods: This Phase I study (NCT05388487) comprise a conventional 3+3 dose escalation plus an advanced glioma-specific expansion arm. Eligible patients had prior confirmed advanced solid tumor and failed standard treatment. HF1K16 infusions were administered in 21-day cycles (q.o.d days 1-14). Prior to and during treatment, peripheral blood mononuclear cells were collected and analyzed with flow cytometry to monitor the changes in myeloid cell phenotype and T cell composition. Survival data were calculated as it is difficult to determine the timing of progression for immunotherapies owing to pseudoprogression. Results: As of Feb 2th, 2024, 14 recurrent/refractory glioma patients including 6 males and 8 females had been enrolled in this study. 2 of them withdrew before finished 1st cycle. 7 had received treatment for at least 3 cycles and 1 had achieved CR after 15 cycles. For the 8 primary diagnosed grade IV glioma patients, the mOS is 7.8m, and 6 (75%) are alive. The mOS for the primary dignosed grade II/III is 13.7m and 100% patients are alive (n=4). There is 1 recurrent GBM patent who had received 5 cycles of treatment. MRI showed an enlargement of tumor lesion while KPS score remained >90. A surgical resection was performed and we subsequently studied the posttreatment tumor specimens (window of opportunity). IHC and flow cytometry analysis revealed prominent perivascular and intratumoral inflammatory infiltrate, containing CD8+CTL cells and HLA-DR expressing cells. Conclusions: While the interpretation of our data is limited by the small sample size, the results provide an encouraging signal and warrants further assessment. Patient recruitment continues with anticipated completion in 2024. Clinical trial information: NCT05388487 . [Table: see text]

Bavituximab decreases immunosuppressive myeloid-derived suppressor cells in newly diagnosed glioblastoma patients.

We evaluated the efficacy of bavituximab - a monoclonal antibody with anti-angiogenic and immunomodulatory properties - in newly diagnosed glioblastoma (GBM) patients who also received radiation and temozolomide. Perfusion MRI and myeloid-related gene transcription and inflammatory infiltrates in pre-and post-treatment tumor specimens were studied to evaluate on-target effects (NCT03139916). Thirty-three adults with isocitrate-dehydrogenase-wild-type GBM received 6 weeks of concurrent chemoradiation, followed by 6 cycles of temozolomide (C1-C6). Bavituximab was given weekly, starting week 1 of chemoradiation, for at least 18 weeks. The primary endpoint was proportion of patients alive at 12 months (OS-12). The null hypothesis would be rejected if OS-12 was ≥72%. Relative cerebral blood flow (rCBF) and vascular permeability (Ktrans) were calculated from perfusion MRIs. Peripheral blood mononuclear cells (PBMCs) and tumor tissue were analyzed pre-treatment and at disease progression using RNA transcriptomics and multispectral immunofluorescence for myeloid-derived suppressor cells (MDSCs) and macrophages. The study met its primary endpoint with an OS-12 of 73% (95% CI 59-90%). Decreased pre-C1 rCBF (HR 4.63, p=0.029) and increased pre-C1 Ktrans were associated with improved OS (HR 0.09, p=0.005). Pre-treatment, overexpression of myeloid-related genes in tumor tissue was associated with longer survival. Post-treatment, tumor specimens contained fewer immunosuppressive MDSCs (p=0.01). Bavituximab has activity in newly diagnosed GBM and resulted in on-target depletion of intratumoral immunosuppressive MDSCs. Elevated pre-treatment expression of myeloid-related transcripts in GBM may predict response to bavituximab.

Dynamic change of immune phenotype assessed by artificial intelligence (AI)-powered analysis of tumor-infiltrating lymphocytes (TILs) during neoadjuvant durvalumab with or without tremelimumab (D+/-T) in head and neck squamous cell carcinoma (HNSCC).

2578 Background: The results of a phase II trial evaluating preoperative durvalumab with or without tremelimumab (D+/-T) in resectable HNSCC (NCT03737968) suggest that the neoadjuvant immunotherapy is associated with pathologic tumor regression with manageable safety profiles. Here, we applied AI-powered spatial TIL analyzer, Lunit SCOPE IO, to tumor specimens of the patients included in the trial to assess the impact of spatial TIL density and inflamed immune phenotype (IIP) on immunotherapy responses. Methods: H&E-stained whole-slide images (WSIs) of pre- and post-treatment tumor specimens from HNSCC pts enrolled in the aforementioned D+/-T trial were collected. Patients with locally advanced but operable HNSCC were randomly assigned in 1:1 to receive a single dose of D (1,500mg) or D+T (1,500mg+75mg) followed by surgery, at Severance Hospital in Seoul, Korea. Lunit SCOPE IO was used to segment tumor epithelium and stroma to identify and quantify intratumoral TIL (iTIL) and stromal TIL. IIP was defined as the proportion of high iTIL area more than 33.3% of analyzable area. Clinical data and the combined positive scores (CPS) of PD-L1 were also collected. Results: A total of 39 paired tumor samples of pre- and post-treatment WSI (23 from D+T and 16 from D) were included. Overall, tumor regression grade 2 (TRG2, ≥ 50% of tumor regression) was achieved in 15.4% (6/39) pts, and downstaging after treatment was achieved in 35.9% (14/39) pts. By pre-treatment WSIs, 38.5% (15/39) samples were classified as IIP, and subgroups of D+T and D had 34.8% (8/23) and 43.8% (7/16), respectively. In the D+T arm, the proportions of TRG2 were 25% (2/8) in IIP versus 13.3% (2/15) in non-IIP, whereas D arm had 28.6% (2/7) TRG2 in IIP, but no TRG2 in non-IIP (0/9), respectively. Comparing the WSI of pre- and post- D+/-T samples, the proportion of IIP was significantly increased to 66.7% from 38.5 ( p = 0.015). The increase in IIP proportion was prominent in the D+T arm (34.8% to 69.6%, p = 0.043), but not in the D arm (43.8% to 62.5%, p = 0.37). Similarly, PD-L1 CPS increased after therapy, especially in the D+T group (mean CPS 6.0 to 20.7, p = 0.009). Interestingly, pts who remained non-IIP in both pre- and post-treatment (25.6%, 10/39) had significantly poor prognosis compared to the others, as the 12-month disease-free survival (DFS) rate was 50.0% vs 93.1% (median DFS 13.4m vs not reached, hazard ratio [HR] 4.26, 95% confidence interval [CI] 1.32-13.8, p = 0.009). This prognostic impact was prominent in the D+T arm (HR 6.75, 95% CI 1.49-30.6, p = 0.004). Conclusions: Neoadjuvant durvalumab +/- tremelimumab promotes dynamic change toward inflamed immune phenotype, resulting in favorable clinical outcomes. Resistance mechanism of stand-still-non-inflamed patients even on D+/-T should be further investigated. Clinical trial information: NCT03737968 .

The TRAR gene classifier to predict response to neoadjuvant therapy in HER2-positive and ER-positive breast cancer patients: an explorative analysis from the NeoSphere trial.

As most erb‐b2 receptor tyrosine kinase 2 (HER2)‐positive breast cancer (BC) patients currently receive dual HER2‐targeting added to neoadjuvant chemotherapy, improved methods for identifying individual response, and assisting postsurgical salvage therapy, are needed. Herein, we evaluated the 41‐gene classifier trastuzumab advantage risk model (TRAR) as a predictive marker for patients enrolled in the NeoSphere trial. TRAR scores were computed from RNA of 350 pre‐ and 166 post‐treatment tumor specimens. Overall, TRAR score was significantly associated with pathological complete response (pCR) rate independently of other predictive clinico‐pathological variables. Separate analyses according to estrogen receptor (ER) status showed a significant association between TRAR score and pCR in ER‐positive specimens but not in ER‐negative counterparts. Among ER‐positive BC patients not achieving a pCR, those with TRAR‐low scores in surgical specimens showed a trend for lower distant event‐free survival. In conclusion, in HER2‐positive/ER‐positive BC, TRAR is an independent predictor of pCR and represents a promising tool to select patients responsive to anti‐HER2‐based neoadjuvant therapy and to assist treatment escalation and de‐escalation strategies in this setting.

Abstract PD7-07: Neoadjuvant endocrine therapy helps identify HER2 up-regulation in patients with hormone receptor-positive HER2-negative breast cancer

Abstract Background: Endocrine therapy provides significant improvement in the long-term outcomes of patients with hormone receptor-positive (HR+) breast cancer (BC). However, metastatic recurrences of endocrine resistant disease develop in about 20-25% of patients and remain a major cause of BC mortality. Up-regulation of the HER2 growth factor receptor represents a common escape strategy used by cancer cells to survive and continue to proliferate in an ER-independent manner. Neoadjuvant endocrine therapy (NET) offers a unique opportunity to identify responsiveness of HR+ BCs and detect tumors that display up-regulation of HER2, an early endocrine resistance mechanism. Methods: A single arm, interventional, exploratory clinical trial evaluating four weeks of NET in early stage HR+/HER2-negative BC patients was conducted at our institution (NCT03219476). The primary objective was to assess changes in HER protein expression (HER1-4) from diagnostic core biopsies to surgically resected tumors treated with NET. Secondary objectives included assessment of other molecular markers, tumor proliferation and volumetric responses. Optimized protocols for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were used to assess HER2 status in the pre- and post-treatment tumor specimens. Chi-square and t-tests were performed. Linear regression multivariate analysis was performed to evaluate association of covariates with the primary outcome with 80% power at a significance level of 0.05. Here we present the changes in HER2 protein expression with NET in this study. Up-regulation was defined as an increase of ≥ 1 in IHC scores (ordinal 0,1,2,3) or gene amplification by FISH. Results: Thirty-seven patients with cT1-T3, cN0 HR+/HER2-negative BC were enrolled. Median age at diagnosis was 64 yrs (42-81) and median BMI was 28.3 (19.3-55.1). Most patients were post-menopausal (83.8%). Median tumor size clinically was 1.3 cm (0.5-7.7). Most tumors were low (42.1%) or intermediate (47.4%) grade and invasive ductal histology was seen in 70%. Median tumor size at surgery was 1.2 cm (0.09-4). One patient had a complete pathologic response (pCR) at surgery. Seven patients had pN1 disease at surgery (six with 1 lymph node involved, one with pN1mi). There was no significant change in ER-positivity between pre and post-treatment specimens. However, a trend towards decrease in PR-positivity was seen in post-treatment tumors consistent with functional ER pathway disruption (p=0.08). On HER2 protein assessment by IHC, most patients had IHC 0 (37.8%) or IHC 1+ (54%) at diagnosis. Significant up-regulation in HER2 protein was seen in 46% (17/37) of patients (p=0.0004), whereas down-regulation was detected in only 5% (2/37) and no change in the remaining 49% (18/37). Three patients converted to HER2-positive status (IHC 3+ or FISH amplified) at surgery and received adjuvant trastuzumab-based treatment. No significant associations were identified between any clinicopathologic covariates and change in HER2 protein expression. Conclusions: HER2 was up regulated in 46% of tumors after short-term NET in patients diagnosed with early stage HR+/HER2-negative BC. Short-term NET is a promising strategy to identify HR+ tumors that up-regulate HER2 as an early escape pathway and endocrine resistance mechanism. Patients with such HER2 up-regulated tumors after NET may benefit from HER2-directed therapies upon disease recurrence or as adjuvant combination therapy. These findings need to be further explored in larger randomized clinical trials. Citation Format: Lubna N Chaudhary, Julie M Jorns, Yee Chung Cheng, Sailaja Kamaraju, Mary Beth Gonyo, Amanda Kong, Caitlin Patten, Tina Yen, Chandler Cortina, Ebony Carson, Nedra Johnson, Carmen Bergom, Anjishnu Banerjee, Yu Wang, Christopher R Chitambar, Hallgeir Rui. Neoadjuvant endocrine therapy helps identify HER2 up-regulation in patients with hormone receptor-positive HER2-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-07.

Abstract P6-04-23: HER2 up-regulation with neoadjuvant endocrine therapy in patients with hormone receptor-positive, HER2-negative breast cancer

Abstract Background Endocrine therapy provides significant disease-free survival (DFS) and overall survival (OS) benefit to patients with early stage hormone receptor positive (HR+) HER2 negative breast cancer. However, metastatic recurrence of endocrine-resistant disease develops in 20-25% of patients and remains a major cause of breast cancer mortality. Several mechanisms play a role in the progression and development of endocrine resistance. Understanding and characterizing the underlying molecular features of resistance for more accurate response prediction and optimal clinical management is of the utmost importance. Methods We designed an interventional study to assess tumor responses, changes in tumor proliferation and molecular biomarkers in early stage HR+ HER2- clinical node-negative breast cancer patients treated with neoadjuvant endocrine therapy for 4 weeks. The primary objective is to assess changes in HER protein expression (HER1-4) with neoadjuvant endocrine therapy; secondary objectives assess other molecular changes such as therapy-resistant CK5+ progenitor cells, iron-related proteins, Bruton’s tyrosine kinase (BTK), PD-L1, PD-L2 protein expressions, association with Ki-67 and tumor responses. Interim data on changes in HER2 protein in the first 16 patients is reported here. Optimized protocols for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were used to assess HER2 status in the pre and post treatment tumor specimens. This is an ongoing investigator-initiated clinical trial funded by Rock River Foundation and the Medical College of Wisconsin Cancer Center (NCT03219476). Results Median age at diagnosis was 65 (42-81) and median BMI was 28.3 (19.3-50.1). Most patients were post-menopausal (81%). Median tumor size clinically was 1.3 cm (0.5-7.7). Most tumors were low (50%) or intermediate (37.5%) grade and invasive ductal histology (62.5%). Median tumor size at surgery was 1.1 cm (0-3.9). One patient had a complete pathologic response (pCR) at surgery. Four patients had pN1 (1 lymph node involved) and one patient had pN1mic disease at surgery. There was no significant change in ER-positivity between pre and post-treatment specimens. However, a significant decrease in PR-positivity was seen in post-treatment tumors consistent with functional ER pathway disruption. On HER2 protein assessment by IHC, most patients had IHC 0 (37.5%) or IHC 1+ (56%) at diagnosis. Up-regulation in HER2 protein was seen in post-treatment tumors with 36% (n=5) IHC 2+, with one case showing Her2 gene amplification by FISH. Conclusions HER2 was up regulated in 36% of tumors after short-term neoadjuvant endocrine treatment in patients with early stage HR+ HER2- breast cancer. Short-term neoadjuvant endocrine treatment holds potential for identifying emergent endocrine resistance mechanisms and pathways. Targeted therapy with HER2-directed antibodies for such HER2 up-regulated tumors after neoadjuvant endocrine treatment may benefit patients upon disease recurrence or possibly as adjuvant combination therapy in patients with high risk of recurrence. Citation Format: Lubna N Chaudhary, Julie Jorns, MaryBeth Gonyo, Amanda Kong, Amy R Peck, Yunguang Sun, Carmen Bergom, Anjishnu Banarjee, Christopher Chitambar, Hallgeir Rui. HER2 up-regulation with neoadjuvant endocrine therapy in patients with hormone receptor-positive, HER2-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-23.

A phase II randomized control trial of erlotinib in combination with celecoxib in patients with operable oral squamous cell carcinoma (OSCC): Erlo-Xib Study.

6054 Background: When combined with COX-2 inhibitors, the EGFR tyrosine kinase inhibitor Erlotinib has shown a better antitumor response in preclinical studies. Since high volume hospitals in many countries usually have a longer waiting period for surgery, neoadjuvant targeted therapy may be helpful in reducing disease progression and downstaging oral squamous cell cancers. Methods: Sixty-four treatment-naïve operable oral cancer patients were randomized into a four-arm window of opportunity study consisting of treatment with erlotinib 150mg daily, celecoxib 200mg twice daily, the combination of both or observation alone (NCT02748707). Since the regular wait period for surgery at our hospital was four to five weeks, we planned a 21-day drug treatment versus observation followed by definitive surgery in the fourth week. MRI scans and biopsies were done before and after drug treatment. Post-operative adjuvant treatments were given as per the standard guidelines used for regular patients. Results: There were 10 females and 54 males with a mean and median age of 44 and 45 years respectively. Taking a 20% reduction in the maximum tumor dimension after drug treatment (assessed clinically and radiologically) as partial response, the combination arm had a 60% partial response and a 25% stable disease. Whereas, 60% in the control arm had disease progression. The ratio of the longest tumor dimension at day 21 versus day 0 (Clinical & MRI assessment) also showed a significant difference between the observation vs erlotinib arms (p < 0.001) using Mann-Whitney Test. Grade II/III rashes was the commonly observed toxicity predominantly in the combination arm. Though not powered for survival analysis, a significant difference (p = 0.048) was observed for two-year overall survival for celecoxib + control (60%) versus erlotinib + combination groups (86%) using Kaplan Meier estimator. Biomarker analysis (transcriptome sequencing and IHC) is being done on pre and post-treatment tumor specimens and the final results will be presented. Conclusion: Preoperative targeted therapy with erlotinib and celecoxib combination can arrest disease progression and downstage tumors with possible impact on survival. The identified biomarkers can further refine a future cohort for effective neoadjuvant targeted therapy in oral cancers. Clinical trial information: CTRI/2012/07/002828.

Molecular and imaging correlates of exceptional pathologic response to neoadjuvant ADT plus enzalutamide.

61 Background: AR directed therapies are standard of care for metastatic prostate cancer, and their use for high-risk localized disease may improve survival. Previous trials showed that suppression of AR activity resulting in greater pathologic responses improved survival rates. However, the underlying biology and molecular features of exceptionally responding tumors remain unknown. Methods: Men with locally advanced prostate cancer (median Gleason score 8, median PSA 9.58 ng/dl) received mpMRI and MRI/US-fusion targeted biopsies at baseline. Patients received 6 months of ADT plus enzalutamide, then received a second mpMRI, and underwent radical prostatectomy. RP specimens were whole mounted in the same plane as MRI. The location of each tumor on biopsy was mapped to prostatectomy specimens using pathologic and imaging hallmarks. FFPE sections of baseline and post-treatment tumor were stained, laser capture microdissected, and analyzed using whole exome sequencing. Amongst nonresponding patients (residual tumor burden > 0.05 cc) comparison of pre- and post-treatment tumor specimens identified evolutionary phylogenies over space and time representing intrinsic or clonal selection of pre-existing cells. Amongst responding patients, clonal extinction was confirmed by sequencing and immunostaining of surgical specimens. Results: Gleason score at baseline did not differentiate responding and nonresponding patients. Focal ERG staining in biopsies was absent in 100% of responders and present in 60% of responders. Intraductal carcinoma architecture was also absent in all responding tumors. Focal PTEN loss was observed in all nonresponders at baseline. Responding tumor foci were enriched for deletion of chromosome 6q. Comparison of baseline mpMRI sequences indicated a trend towards lower variance on dynamic contract enhanced (DCE) MR and lower local texture heterogeneity metrics in responding lesions. Conclusions: Response to neoadjuvant intense ADT correlates with distinctive pathologic, molecular, and imaging features that can be observed prior to treatment. Selection of patients based on these parameters may improve overall responses to treatment in subsequent clinical trials. Clinical trial information: NCT02430480.

Increased immune infiltration and chemokine receptor expression in head and neck epithelial tumors after neoadjuvant immunotherapy with the IRX-2 regimen

ABSTRACTIRX-2 is an injectable cancer immunotherapy composed of cytokines purified from stimulated normal-donor peripheral blood mononuclear cells. In a phase 2a trial (n = 27), neoadjuvant IRX-2 significantly increased lymphocyte infiltration (LI) into resected head and neck tumors and was associated with changes in fibrosis and necrosis. Event-free survival was 65% at 2 years, and overall survival 65% at 5 years. Overall survival was longer for patients with LI greater versus lower than the median. This substudy of the mechanisms responsible for the increase in LI with neoadjuvant IRX-2 employed multiplex immunohistochemistry (IHC) and transcriptome analysis to interrogate matched pre- and post-treatment tumor specimens from 7 available phase 2a trial patients. Multiplex IHC showed substantial increases in CD68-expressing cells (5 patients), T-cell density (4 patients), and PDL1 mean fluorescent intensity (4 patients). Consistent with IRX-2 activation of multiple immune cells, transcriptome analysis showed mean increases in expression of genes associated with NK cells, B cells, CD4+ T cells, CD8+ T cells, and dendritic cells, but not of genes associated with neutrophils. There were increases in mean expression of genes for most immune subsets, most markedly (2- to 3-fold) for B cells and dendritic cells. Mean increases in gene expression for chemokines suggest that tumor LI may be driven in part by IRX-2-induced production of chemo-attractants. Upregulation of checkpoint genes including PDL1 and CTLA4 along with increased T-cell infiltration suggests a functional antitumor immune response such that the efficacy of IRX-2 may be enhanced by combination with immune checkpoint inhibitors.

A Phase II Study of Anti PD-1 Antibody Pembrolizumab, Pomalidomide and Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

BACKGROUNDSeveral recent studies have linked the interactions of programmed death 1 (PD-1) receptor and its ligand (PD-L1) to immunologic control of MM. Expression of PD-L1 on myeloma cells and the abundance of PD-1 on various bone marrow microenvironment components contribute to tumor-mediated immune suppression. We hypothesized that pembrolizumab, a PD-1-blocking antibody, will activate myeloma specific cytotoxic T cells that can be enhanced by pomalidomide in RRMM patients to induce clinical responses. METHODSIn this ongoing single arm, phase II study, 24 patients with RRMM received 28-day cycles of pembrolizumab (at a dose of 200 mg IV) every 2 weeks (in a run off phase, first 6 patients received 200 mg IV every 4 weeks) plus pomalidomide (4 mg daily x 21 days) and dexamethasone 40 mg weekly. Study objectives were measurements of safety and efficacy and assessment of the PD-1 and PD-L1 protein expression in bone marrow samples. RESULTSThe median age was 65 years (range: 41-75); 35% were African American and 71% were men. Of the 24 patients, 75% had prior autologous transplantation and 96% were refractory to last therapy. All patients had received both IMids and Proteosome inhibitors; 75% were double refractory to both IMids and Proteosome inhibitors and additional 21% were refractory to lenalidomide alone. Patients had received a median of 3 lines of prior therapy (range: 1-6). The median time from MM diagnosis to study entry was 4 years (range: 1.2-15). All patients had abnormal cytogenetics: most common were 1q+ (72%) and high-risk FISH (40%) [del 17p, t(4:14) and/or t(14:16)]. There were no infusion-related reactions. Hematologic toxicities (≥ grade 3) were neutropenia (29%), lymphopenia (17%) and thrombocytopenia (8%). Non-hematologic adverse events included (Grade ≤2; ≥3): fatigue (n=12; 1), constipation (n=10; 0), dyspnea (n=9; 2), itching (n=6; 0), muscle spasms (n=6; 0), infection (n=4; 3), hyperglycemia (n=5; 0), edema (n= 4; 0), fever (n=3; 0), palpitation (n=2; 1), rash (n=3; 1) and hypotension (n=3; 0). Events of clinical significance, autoimmune mediated, included hypothyroidism (n=2), transaminitis (n=2), and pneumonitis (n=1). Four patients had pomalidomide dose reductions due to rash, neutropenia, palpitations and fatigue. Two patients died; one after cycle 1 (progressive disease) and one during cycle 2 (sepsis). Objective responses (modified IMWG criteria) were observed in 11 of 22 evaluable patients (50%) including: near complete response (n=3), very good partial response (n=2), partial response (n=6); additionally, 3 patients had minimal response, 6 had stable disease and 2 progressed. At a median follow up of 16 weeks; 17 of 22 patients continued on the study. Reasons for discontinuation included disease progression (n= 4) and protocol violation (n=1). Analysis of pretreatment and post-treatment tumor specimens for PD-1 and PD-L1 is in progress. CONCLUSIONSPembrolizumab in combination with pomalidomide and dexamethasone has promising therapeutic activity and an acceptable safety profile in heavily treated RRMM patients.ClinicalTrials.gov number, NCT02289222. DisclosuresOff Label Use: Pembrolizumab.

The predictive value of ARv7 expression in localized prostate caner treated with abiraterone, degarelix, and bicalutamide.

71 Background: Androgen receptor splice variants (ARvs) are upregulated in response to castration, and can activate transcription in the absence of ligand. Recent evidence indicates that CTC ARv7 expression is an accurate predictor of absence of response to abiraterone and enzalutamide, providing strong clinical support for ARv7 upregulation as potential overarching mechanism of castration resistance. There is however limited data regarding the expression of ARv7 in clinically localized prostate cancer (CaP), and it is unknown if expression in the primary tumor is similarly predictive. We were therefore interested to determine the expression of ARv7 in high-risk disease, and correlate this with response to an abiraterone-containing castrating regimen. Methods: We performed an open label non-randomized Phase II neoadjuvant study of ‘supercastration’ in men with high-risk clinically localized CaP using an optimal 2-stage design (ACTRN12612000772842). Treatment consisted of degarelix 240/80mg q 1/12, abiraterone 1000mg OD, bicalutamide 50mg OD and prednisolone 5mg OD for 24 weeks. The primary endpoints were safety/tolerability and pT0 response rate. ARv7 expression was determined by immunohistochemistry and qRT-PCR in both pre- and post treatment tumor specimens, and correlated with pathological response. Fresh specimens from resistant tumors are currently being profiled by RNA-seq. Results: 17 patients were recruited to the study. The combination treatment was well tolerated, with hot flushes and fatigue being the most commonly reported side effects, and all patients completed six months of treatment. Six patients with asymptomatic elevation of liver transaminases required Abiraterone dose reductions, and there were no unexpected toxicities. 16 patients proceeded to prostatectomy, and a pT0 response observed in one patient, with minimal residual disease present in a further three patients. ARv7 expression was uniformly present in all pre-treatment specimens, and neither level nor localization of expression predicted tumor response. Conclusions: ARv7 expression in primary CaP does not predict response to abiraterone-based castration. Clinical trial information: ACTRN12612000772842.

Association of CD133 expression levels with the k-ras mutation status in rectal cancers before and after preoperative radiochemotherapy.

400 Background: The role of the potential stem cell marker CD133 as a predictive or prognostic marker in multimodal rectal cancer treatment is currently under debate. While CD133 has been identified as a prognostic marker in rectal cancers after preoperative radiochemotherapy (RCT) it was recently characterized as a very unspecific feature for colorectal cancer stem cells. We therefore analyzed the association between CD133 expression and mutations in the proto-oncogenes K-Ras and PI3K in rectal cancer patients receiving neoadjuvant RCT. Methods: CD133 expression was evaluated immunhistochemically in pre-treatment biopsies and surgical specimens of 128 patients with locally advanced rectal cancers (cUICC II/III) treated with preoperative RCT within the phase-III German Rectal Cancer Trials. K-Ras mutations were analyzed by sequencing of exons 1, 2, and 3. PI3K mutations were detected by sequencing the p110α subunit (PIK3CA) and correlated with histopathologic parameters, tumor regression and survival. Results: CD133 expression was significantly associated with mutations in the K-Ras gene in both pre-treatment biopsies and post-treatment tumor specimens in uni- and multivariate analyses. However, no significant correlation was observed between CD133 and PI3K mutations. Post-treatment CD133 levels were correlated with neoadjuvant RCT (50.4 Gy/5-FU vs. 50.4 Gy/5-FU+Ox) and tumor regression grading. Anyway, there was no significant association between pre- and post-treatment CD133 expression and disease-free survival. Conclusions: CD133 expression levels are strongly associated with mutations in the K-Ras proto-oncogene in rectal cancers before and after preoperative RCT. Our results strengthen the hypothesis that CD133 is not a specific marker for colorectal stem cells but might be integrated in proliferation pathways like the ras-raf axis.

Phase I Clinical Trial of Systemically Administered TUSC2(FUS1)-Nanoparticles Mediating Functional Gene Transfer in Humans

BackgroundTumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2.MethodsPatients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks.ResultsThirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in post-treatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high post-treatment levels of TUSC2 mRNA and protein showed significant post-treatment changes in the intrinsic apoptotic pathway. Twenty-nine genes of the 82 tested in the apoptosis array were identified by Igenuity Pathway Analysis to be significantly altered post-treatment in both patients (Pearson correlation coefficient 0.519; p<0.01).ConclusionsDOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in uptake of the gene by human primary and metastatic tumors, transgene and gene product expression, specific alterations in TUSC2-regulated pathways, and anti-tumor effects (to our knowledge for the first time for systemic DOTAP:cholesterol nanoparticle gene therapy).Trial RegistrationClinicalTrials.gov NCT00059605

Association of the expression of CD133 in locally advanced rectal cancer with tumor regression and survival after neoadjuvant radiochemotherapy.

479 Background: CD133 has been described as a stem cell marker in colorectal cancer and is associated with higher tumorigenic potential and resistance to radiochemotherapy (RCT). In this study the expression of CD133 was evaluated in pre-treatment tumor biopsies and corresponding post-treatment surgical specimens of locally advanced rectal cancer patients undergoing multimodal therapy and was correlated to histopathological features and clinical follow-up. Methods: 97 patients with UICC II/III rectal cancer treated with preoperative 5-FU based RCT within the German Rectal Cancer Trials were investigated. Pre- and post-treatment CD133 expression levels were determined using immunhistochemistry and correlated with histopathologic parameters, tumor regression, tumor recurrence and disease-free survival (DFS). Results: As compared to pre-treatment biopsies we observed a significantly higher CD133 expression in post-treatment tumor specimens (p=0.01). There was, however, no correlation for both biopsies and tumor specimens between CD133 expression levels, pathological characteristics and survival. In matched analyses of corresponding biopsy/tumor pairs, patients with a decreased number of CD133 positive cells after preoperative RCT showed significantly lower post-treatment tumor stages (p=0.03) and higher histopathological tumor regression (p&lt;0.01). Moreover, these patients had a significantly improved DFS in uni- (p&lt;0.001) and multivariate analyses (p=0.001). Conclusions: CD133 expression displays a marker with prognostic and monitoring validity in rectal cancer patients. A decrease of the CD133 positive cell fraction in post-treatment tumor tissue is associated with a more favorable outcome after preoperative RCT.

Abstract 5391: Systemic gene therapy with tumor suppressor TUSC2/FUS1 nanoparticles for recurrent/metastatic lung cancer

Abstract Background: The tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the Apaf-1-dependent apoptotic pathway. DOTAP:cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) showed preferential uptake by cancer cells and prolonged survival in mouse xenograft metastatic lung cancer models. Methods: Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous DOTAP:chol-TUSC2 every 3 weeks. The primary end point was assessment of DOTAP:chol-TUSC2 toxicity and determination of the MTD. TUSC2 plasmid expression in pretreatment and 24 hour posttreatment tumor specimens from subjects consenting to tumor biopsies was analyzed with quantitative RT-PCR analysis using a TUSC2 plasmid sequence-specific probe, in situ proximity ligation assay for TUSC2 protein, and apoptosis signaling nanoscale PCR array. Results: Thirty-one patients were treated at 6 dose levels ranging from 0.01 to 0.09 mg/kg and 7 had paired pre- and posttreatment biopsies. RT-PCR analysis detected high TUSC2 plasmid expression in 6 of 7 posttreatment tumor specimens but not in pretreatment specimens and negative controls. Immunohistochemical staining has been performed on 1 paired specimen, demonstrating low background TUSC2 protein staining in the pretreatment tissue compared with high intense TUSC2 protein staining in the posttreatment tissue. RT-PCR gene expression profiling analysis of apoptotic pathway genes in one paired specimen demonstrated significant upregulation and downregulation of genes involved in both the intrinsic and extrinsic apoptotic pathways. Among 4 patients treated without premedications, all 4 developed grade 2 or 3 fever. Among the 27 patients premedicated with dexamethasone and diphenhydramine, the highest fever was grade 2, which occurred in 2 subjects. The only dose-limiting toxicities were 2 episodes of transient grade 3 hypophosphatemia, resulting in an MTD of 0.06 mg/kg. Twenty-three patients who received 2 or more doses were evaluable for response, with 5 achieving stable disease (2.6-10.8 months) and 18 progressing. One patient with stable disease had evidence of a durable metabolic response on positron emission tomography imaging. The pretreatment apoptotic index was predictive of disease stability. Median survival time was 9.1 months. Conclusions: DOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in demonstrable gene and protein expression in tumors. It appears to regulate apoptotic pathway genes with evidence of antitumor activity. The MTD for phase II testing is 0.06 mg/kg every 3 weeks. Based on promising preclinical data, a trial in combination with erlotinib is planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5391. doi:10.1158/1538-7445.AM2011-5391

A pilot study evaluating surrogates of response to short-term vorinostat in women with newly diagnosed breast cancer

e14508 Background: Epigenetic modifications contribute to breast cancer initiation and progression and may be reversible, thus representing an attractive area for new drug investigation. In preclinical breast cancer models, the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA, vorinostat) induces cell cycle arrest, apoptosis and differentiation. Methods: We evaluated the safety and tolerability of short term vorinostat in women with a primary clinical stage I-III histologically confirmed carcinoma of the breast on a core needle biopsy. Participants received vorinostat 300 mg PO bid for a total of 6 doses, the last dose 2 hours prior to surgery or biopsy. Peripheral blood mononuclear cells were collected at baseline and following the last vorinostat dose to determine histone acetylation. Baseline and post-treatment tumor specimens were collected for analysis of histone acetylation, candidate gene methylation and expression. Tissues were also collected from untreated controls. Paired t-tests and Fisher's exact tests were used to evaluate changes from baseline for continuous and categorical data, respectively. Results: From March 2006 to October 2008, 25 women signed an informed consent and initiated study drug. Median age was 55 and 80% had hormone receptor positive tumors. Twenty-two women took all 6 prescribed doses. One participant took 4 and one 5 doses due to insurance clearance delay; one received a single dose due to fatigue and abdominal pain. Study-related surgical delays did not occur. Grade 1 toxicities included diarrhea (28%), low white blood cell count (24%), and fatigue, taste alterations and nausea (16% each). Tissue and blood samples were successfully collected. Tissue was also collected from 25 untreated controls. No significant change was observed in the proliferation marker Ki67 following 3 days of agent administration. Modulation of apoptosis, histone acetylation and gene methylation will be presented. Conclusions: Short-term administration of vorinostat is feasible, safe, and allows for studies of biomarker modulation. The results will be used to design future studies in which vorinostat will be administered in combination with other targeted therapies or with other epigenetic modifiers. [Table: see text]

Phase II trial of neoadjuvant temozolomide in advanced melanoma

9058 Background: Neoadjuvant treatment provides the potential for upfront systemic therapy of micrometastases, an in vivo read-out of drug sensitivity, and tumor samples pre- and post-treatment for analysis. We treated melanoma patients with extended dose temozolomide (TMZ) in the neoadjuvant setting and collected tumor pre- and post-treatment for methylguanine methyltransferase (MGMT) promoter methylation analysis and for oligonucleotide microarray analysis. Methods: Chemotherapy-naive patients who were surgical resection candidates with palpable, measurable stage IIIB, IIIC or IV (M1a) cutaneous melanoma were accrued to this phase II trial from October, 2005 to November, 2007. Prior treatment with interferon, IL-2 or vaccine was allowed. TMZ was administered orally at 75 mg/m2/d for 6 weeks of every 8 week cycle. Cycles were repeated until complete response (CR), progressive disease (PD) or stable disease (SD) x 2 cycles. The primary endpoint was radiographic response rate as determined by WHO criteria. Secondary goals included 1) assessment of MGMT promoter methylation status by pyrosequencing of bisulfite-treated DNA, 2) tumor microarray analysis. Results: 23 patients were consented of whom 20 began treatment. Five of 20 were inevaluable because of withdrawal of consent (2), early progression (2), or mucosal primary (1). Two patients are still on study. Of the 13 treated and evaluable patients, the median number of cycles administered was 1 (range 1–3). There have been 2 CRs and 1 PR (RR 23%). One patient had SD and 9 progressed. Nine patients went on to surgery; 2 with PD were deemed surgically unresectable. The MGMT promoter was unmethylated in all 5 tumor samples analyzed to date including in 2 patients with CR. Pre-treatment tumor microarray results in 8 non-responders and 3 responders revealed significantly increased expression of S100-calcium-binding protein A8 (S100A8) and matrix metallopeptidase-1 (MMP-1) in responders. Conclusions: TMZ can induce CRs in the neoadjuvant setting. MGMT promoter was unmethylated in all tumors tested and did not correlate with response to TMZ. The observation that S100A8 and MMP-1 expression is correlated with response to TMZ merits further testing. We are analyzing the remaining pre- and post-treatment tumor specimens. Supported by Schering-Plough Corporation Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Schering-Plough Corporation

Phase I–II trial of ONYX-015 in combination with MAP chemotherapy in patients with advanced sarcomas

ONYX-015 is a provisionally replication competent adenovirus with oncolytic activity in cells with malfunctioning p53. Sarcomas represent a rational target for this approach given the high frequency of p53 mutations (40-75%) and MDM-2 amplification (10-30%). We, therefore, undertook a phase I/II study of ONYX-015, days 1-5 every month administered intratumorally under radiographic guidance, in combination with MAP (mitomycin-C, doxorubicin, cisplatin) chemotherapy in patients with advanced sarcoma. Six patients were treated. Injected lesions included liver metastases in four patients and chest wall metastases in two patients. Sarcoma histologies were gastrointestinal stromal tumors (GIST, two patients), leiomyosarcoma (two patients), liposarcoma (one patient), and malignant peripheral nerve sheath tumor (1 patient). Dose escalation was performed from 10(9) plaque forming units (PFU)/dose (total dose of 5 x 10(9) PFU/cycle) to 10(10) PFU/dose (total dose of 5 x 10(10) PFU/cycle) without dose-limiting toxicity being encountered. Immunohistochemistry of the metastatic lesions prior to treatment showed that five out of six patients were positive for p53, while two patients also had mdm-2 overexpression. Adenoviral replication was detected in two out of six patient biopsies on day 5 of the first cycle, by in situ hybridization (ISH). Both patients were treated at the highest dose level. ONYX-015 viral DNA was detected by quantitative PCR in the plasma of 5/6 patients on day 5 of the first cycle, and up to day 12 (7 days after the last viral dose) in one patient who had extended sampling for viral kinetics performed, suggesting viral replication in sarcoma tissue. One patient with p53 mutation and MDM-2 amplification achieved a partial response to treatment that lasted 11 months. In conclusion, intratumoral administration of ONYX-015 in combination with MAP chemotherapy is well tolerated with no significant toxicity due to ONYX-015 being encountered. Detection of viral DNA in post treatment tumor specimens by ISH and detection of the ONYX-015 genome in the peripheral blood by quantitative PCR, up to 7 days after the last viral dose provide evidence for adenoviral replication. There was evidence of antitumor activity in one out of six patients. Further investigation of this approach in patients with recurrent sarcomas is warranted.

Cyclin D1 repression as a marker of erlotinib response in aerodigestive tract cancers

3093 Background: Erlotinib (OSI-774, Tarceva™) is an epidermal growth factor receptor (HER1) tyrosine kinase inhibitor (TKI) with clinical activity in lung cancer. Mechanisms engaged during clinical response to erlotinib need to be determined. Methods: Using cultured human bronchial epithelial cells, in vitro effects of erlotinib were investigated. Cells were treated with erlotinib and effects on HER1 and cell cycle regulatory proteins were determined by immunoblot analyses. To evaluate further molecular changes in patient tumor specimens, a phase II pharmacokinetic and pharmacodynamic trial was performed in patients with aerodigestive tract cancers, who were candidates for serial tumor sampling. Five patients were enrolled and 4 patients were evaluable (3 NSCLC, 1 laryngeal SCC). Each received erlotinib at an oral dose of 150mg daily. Detailed plasma pharmacokinetics were performed on days 1 and 8. Tissue and plasma concentrations were determined at the second tissue sampling on day 9. Changes in the described markers were assessed immunohistochemically by comparing pre- and post-treatment biopsies. Results: In vitro studies revealed that erlotinib caused a dose-dependent increase in total HER1 and p27 and a prominent decrease in cyclin D1 expression. In the clinical trial, no treatment related deaths or serious adverse events were observed. Of 4 evaluable patients, 3 had symptomatic benefit and 2 of those 3 had pathologic evidence of activity (necrosis). Pre- and post-treatment tumor specimens revealed that cyclin D1 and Ki-67 decreased in the 2 patients with both clinical and pathologic responses while the remaining 2 patients, one of whom had symptomatic benefit, had no change in cyclin D1 or Ki-67 expression. Assessment of pharmacokinetics revealed that the 24 hour AUC increased by day 8 with a median accumulation index of 1.62. Tissue pharmacokinetics revealed appreciable tissue penetration with a median tissue:plasma ratio of 0.434. Conclusions: Erlotinib was well tolerated in this trial with appreciable tissue penetration in aerodigestive tract tumors. This study strongly suggests cyclin D1 as a potential surrogate marker of erlotinib response in vivo. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration OSI Pharmaceuticals OSI Pharmaceuticals OSI Pharmaceuticals Abbott; Amgen; Aventis; Bristol-Myers Squibb; Genentech; GlaxoSmithKline; Ligand; Merck; Ortho Biotech; OSI; Pfizer; Roche