Search

PDF file - 480K

XLS file 56K, List of called variants re-sequenced by Sanger, their gene identities, protein sequences, and the primers used for Sanger sequencing

PDF file - 105K, Tables describing the somatic SNPs and indels identified by WGS, SV validation results, PCR primers used for validation, and tumor measurements from the patient treated with TAK-733

PDF file - 76K, Includes a description of the patient sample used for WGS in addition to the methods for somatic SNP, indel, SV, and CNV determination by WGS

PDF file 6308K, This includes all supplementary figures except S2 and contains additional clinical data, sequence and copy number analysis as well as immunohistochemical and functional studies

Tumor responses to programmed cell death protein 1 (PD-1) blockade therapy are mediated by T cells, which we characterized in 102 tumor biopsies obtained from 53 patients treated with pembrolizumab, an antibody to PD-1. Biopsies were dissociated, and single-cell infiltrates were analyzed by multicolor flow cytometry using two computational approaches to resolve the leukocyte phenotypes at the single-cell level. There was a statistically significant increase in the frequency of T cells in patients who responded to therapy. The frequency of intratumoral B cells and monocytic myeloid-derived suppressor cells significantly increased in patients' biopsies taken on treatment. The percentage of cells with a regulatory T-cell phenotype, monocytes, and natural killer cells did not change while on PD-1 blockade therapy. CD8+ memory T cells were the most prominent phenotype that expanded intratumorally on therapy. However, the frequency of CD4+ effector memory T cells significantly decreased on treatment, whereas CD4+ effector T cells significantly increased in nonresponding tumors on therapy. In peripheral blood, an unusual population of blood cells expressing CD56 was detected in two patients with regressing melanoma. In conclusion, PD-1 blockade increases the frequency of T cells, B cells, and myeloid-derived suppressor cells in tumors, with the CD8+ effector memory T-cell subset being the major T-cell phenotype expanded in patients with a response to therapy. Cancer Immunol Res; 4(3); 194–203. ©2016 AACR.

PDF file - 57K

XLS file - 364K, Figures summarizing the WGS analysis and validation efforts, immunoblots for BRAF L597Q and K601E, sequence confirmation of mutation in patient treated with TAK-733, and spectrum of melanoma mutations determined at Vanderbilt

Supplementary Figure Legends

Gating strategy to study T cells subpopulations within tumor-infiltrating lymphocytes and PBMC. We represent dot-plots on the top of the figure, and a dendrogram displaying the gating strategy of eight phenotype markers, before applying Boolean gates, and posteriorly analyzed with the exhaustive expansion software. During gating by morphology, and excluding duplex, double positive live and CD3+ T cells were gated on CD8, CD4, CD45RA, CD45RO, CCR7, CD62L, CD27, CD57, CD95 and PD-1.

PDF file 8842K, Supplementary Figure S2 contains Sanger re-sequencing to estimate the specificity of WES

Fluorochromes used on this study

Gating strategy used to analyze immune cell subpopulations within tumor infiltrating lymphocytes. Flow cytometry dot-plots on the left side and a dendrogram displaying the gating strategy of six populations on the right. We gated alive (7AAD negative) singlet cells that were positive for leukocyte antigen (CD45+). Then, the following gating was applied to designate NK cells (CD45+CD56+CD3-), B cells (CD45+CD19/CD20+CD3-), monocytes (CD45+CD14+), and T cells (CD45+CD3+). From the monocytes, a subset of MDSC with low HLA-DR positive cells was used. Finally, T regulatory cells (Treg) were described among CD4+ cells and from those, the CD25+HighCD127Low.

Gating strategy used to study T memory stem and T naïve-like T cell subpopulations within tumor-infiltrating lymphocytes. Dendrogram on the top, and the results of applying Boolean gates on the bottom. X-axis subpopulation of T cells as CD4, CD8 and DN T cells. Y axis, frequency of memory stem cells (MSC) and T naïve-like T cells. B = baseline; P = on therapy; n = 11.

Supplemental Table 3

Supplemental Figure S1. Mean pharmacokinetic profiles for single agent talazoparib (BMN673 in phase I clinical trial PRP-001) versus combination therapy.

Supplemental Table 2

Supplemental Table 1

Supplemental Table 4

Supplementary Figure 2 from CTLA4 Blockade Induces Frequent Tumor Infiltration by Activated Lymphocytes Regardless of Clinical Responses in Humans

PDF file - 148KB, Supplementary Table 1. Toxicities and response to therapy with the subsequent protocol amendments.

Supplementary Table S1 from Dendritic Cell Vaccination Combined with CTLA4 Blockade in Patients with Metastatic Melanoma

Purpose: This study examined the feasibility, efficacy (abscopal effect), and immune effects of TGFβ blockade during radiotherapy in metastatic breast cancer patients.Experimental Design: Prospective randomized trial comparing two doses of TGFβ blocking antibody fresolimumab. Metastatic breast cancer patients with at least three distinct metastatic sites whose tumor had progressed after at least one line of therapy were randomized to receive 1 or 10 mg/kg of fresolimumab, every 3 weeks for five cycles, with focal radiotherapy to a metastatic site at week 1 (three doses of 7.5 Gy), that could be repeated to a second lesion at week 7. Research bloods were drawn at baseline, week 2, 5, and 15 to isolate PBMCs, plasma, and serum.Results: Twenty-three patients were randomized, median age 57 (range 35–77). Seven grade 3/4 adverse events occurred in 5 of 11 patients in the 1 mg/kg arm and in 2 of 12 patients in the 10 mg/kg arm, respectively. Response was limited to three stable disease. At a median follow up of 12 months, 20 of 23 patients are deceased. Patients receiving the 10 mg/kg had a significantly higher median overall survival than those receiving 1 mg/kg fresolimumab dose [hazard ratio: 2.73 with 95% confidence interval (CI), 1.02–7.30; P = 0.039]. The higher dose correlated with improved peripheral blood mononuclear cell counts and a striking boost in the CD8 central memory pool.Conclusions: TGFβ blockade during radiotherapy was feasible and well tolerated. Patients receiving the higher fresolimumab dose had a favorable systemic immune response and experienced longer median overall survival than the lower dose group. Clin Cancer Res; 24(11); 2493–504. ©2018 AACR.

Background: CTLA4 blocking monoclonal antibodies provide durable clinical benefit in a subset of patients with advanced melanoma mediated by intratumoral lymphocytic infiltrates. A key question is defining whether the intratumoral infiltration (ITI) is a differentiating factor between patients with and without tumor responses.Methods: Paired baseline and postdosing tumor biopsy specimens were prospectively collected from 19 patients with metastatic melanoma, including 3 patients with an objective tumor response, receiving the anti-CTLA4 antibody tremelimumab within a clinical trial with primary endpoint of quantitating CD8+ cytotoxic T-lymphocyte (CTL) infiltration in tumors. Samples were analyzed for cell density by automated imaging capture and further characterized for functional lymphocyte properties by assessing the cell activation markers HLA-DR and CD45RO, the cell proliferation marker Ki67, and the regulatory T-cell marker FOXP3.Results: There was a highly significant increase in ITI by CD8+ cells in biopsy samples taken after tremelimumab treatment. This included increases between 1-fold and 100-fold changes in 14 of 18 evaluable cases regardless of clinical tumor response or progression. There was no difference between the absolute number, location, or cell density of infiltrating cells between clinical responders and patients with nonresponding lesions that showed acquired intratumoral infiltrates. There were similar levels of expression of T-cell activation markers (CD45RO, HLA-DR) in both groups and no difference in markers for cell replication (Ki67) or the suppressor cell marker FOXP3.Conclusion: CTLA4 blockade induces frequent increases in ITI by T cells despite which only a minority of patients have objective tumor responses. Clin Cancer Res; 17(12); 4101–9. ©2011 AACR.

PDF file - 190KB, Cytokine production by multiplex assay in plasma from patients F5-12 and F5-14 to study the potential development of a cytokine storm.

Purpose: CTL-associated antigen 4 (CTLA4)-blocking monoclonal antibodies induce long-term regression of metastatic melanoma in some patients, but the exact mechanism is unknown. In this study, biopsies of selected accessible tumor lesions from patients treated with tremelimumab were examined to further elucidate the mechanism of its antitumor activity.Experimental Design: Fifteen tumor biopsies from 7 patients who had been treated with tremelimumab (CP-675,206) were collected. Samples were analyzed for melanoma markers, immune cell subset markers, the presence of the T regulatory-specific transcription factor FoxP3 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO).Results: Clinically responding lesions had diffuse intratumoral infiltrates of CD8+ T cells that were markedly increased in cases where comparison with a baseline biopsy was available. Nonregressing lesions had sparse, patchy CD8+ intratumoral infiltrates. Patients with regressing lesions had an increased frequency of CD8+ cells with or without a concomitant increase in CD4+ cells. Two of 3 responding patients with paired samples showed a slight increase in the number of FoxP3+ cells in the postdosing biopsies. In patients with regressing lesions who had paired samples, the intensity of IDO staining in macrophages and/or melanoma cells showed no clear pattern of change postdosing.Conclusions: Administration of tremelimumab was associated with massive intratumoral infiltrates of CD8+ CTLs in patients with regressing tumors but had varying effects on intratumoral infiltrates of CD4+ and FoxP3+ cells or intratumoral expression of IDO.

Purpose: It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy.Experimental Design: A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation.Results: A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1–specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1–specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells.Conclusion: Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses. Clin Cancer Res; 20(9); 2457–65. ©2014 AACR.

PDF file - 132KB, Recall whole body rash and re-expansion of the TCR transgenic cells in peripheral blood of patient F5-13 with subsequent MART-1/DC vaccination.

PDF file - 127KB, Number and function of TCR transgenic cells infused.

PDF file - 64KB, Comparison of IL-17 production by F5-10 infused TCR transgenic cells stimulated with MART-1-pulsed K562 A2.1 cells with that of patients who did not develop pancytopenia.

PDF file - 470KB, Chest X-rays of patients F5-12 and 14 who developed SAEs of acute respiratory distress.

PDF file - 278KB, Pre- and post-treatment PET scans showing evidence of major tumor response and MART-1-specific TCR transgenic cell levels in patient F5-10.

PDF file - 82KB, TCR transgenic cell therapy manufacture protocol modifications.

Supplementary Figures S1-S2 from Intratumoral Immune Cell Infiltrates, FoxP3, and Indoleamine 2,3-Dioxygenase in Patients with Melanoma Undergoing CTLA4 Blockade

Related Article from Do We Need a Different Set of Response Assessment Criteria for Tumor Immunotherapy?

Supplementary Figure 1 from CTLA4 Blockade Induces Frequent Tumor Infiltration by Activated Lymphocytes Regardless of Clinical Responses in Humans

Tumor shrinkage induced by tumor immunotherapy may be preceded by inflammatory changes. This confounds the assessment of response rates to tumor immunotherapy. In this issue of Clinical Cancer Research, Wolchok et al. attempt to address this peculiarity by proposing a new set of criteria termed immune-related response criteria. (Clin Cancer Res 2009;15(23):7116–8)

PDF file - 243KB, Lack of autoreactivity to bone marrow cells in patient F5-10.

Purpose: To determine whether altering the dietary content of ω-6 (n-6) and ω-3 (n-3) polyunsaturated fatty acids affects the growth of androgen-sensitive prostate cancer xenografts, tumor membrane fatty acid composition, and tumor cyclooxygenase-2 and prostaglandin E2 (PGE2) levels.Experimental Design: Individually caged male severe combined immunodeficiency mice were fed isocaloric 20% kcal fat diets with the fat derived either primarily from n-6 fatty acids (n-6 group) or with the fat consisting of n-6 and n-3 fatty acids in a ratio of 1:1 (n-3 group), and injected s.c. with Los Angeles Prostate Cancer 4 (LAPC-4) cells. Tumor volumes and mouse weights were measured weekly, caloric intake was measured 3 days per week, and tumors and serum were harvested at 8 weeks postinjection.Results: Tumor growth rates, final tumor volumes, and serum prostate-specific antigen levels were reduced in the n-3 group relative to the n-6 group. The n-3 group tumors had decreased proliferation (Ki67 staining) and increased apoptosis (terminal nucleotidyl transferase–mediated nick end labeling staining). In vitro proliferation of LAPC-4 cells in medium containing n-3 group serum was reduced by 22% relative to LAPC-4 cells cultured in medium containing serum from the n-6 group. The n-6/n-3 fatty acid ratios in serum and tumor membranes were lower in the n-3 group relative to the n-6 group. In addition, n-3 group tumors had decreased cyclooxygenase-2 protein and mRNA levels, an 83% reduction in PGE2 levels, and decreased vascular endothelial growth factor expression.Conclusion: These results provide a sound basis for clinical trials evaluating the effect of dietary n-3 fatty acids from fish oil on tumor PGE2 and membrane fatty acid composition, and serum and tumor biomarkers of progression in men with prostate cancer.

Supplementary Table S1. Antibodies and dextramers used for flow cytometric analysis Supplementary Table S2. Patients with one or more treatment positively-related (maximum grade possible, probable, definite) AEs by treatment arm Supplementary Table S3. Levels of circulating tumor-specific CD8+ T cells with reactivity for JARID1B, Muc1 or Her2 in breast cancer patients with HLA-A*0201 status Supplementary Table S4. Humoral immune responses were not significantly affected following TGFβ blockade and radiation Supplementary Figure S1: Study schema Supplementary Figure S2. Gating strategy in panel 1. Supplementary Figure S3. Gating strategy in panel 2. Supplementary Figure S4. Changes in PBMC, survivin- and JARID1B-reactive CD8 T cells in response to TGFβ blockade and radiation. Supplementary Figure S5. Diametrically opposing dynamics in the myeloid suppressor compartment versus the suppressor T cell pool in response to TGFβ blockade and radiation. Supplementary Figure S6. Changes in plasma levels of tryptophan and kynurenine in response to TGFβ blockade and radiation.

Purpose: Tumor antigen–loaded dendritic cells (DC) are believed to activate antitumor immunity by stimulating T cells, and CTL-associated antigen 4 (CTLA4)–blocking antibodies should release a key negative regulatory pathway on T cells. The combination was tested in a phase I clinical trial in patients with advanced melanoma.Experimental Design: Autologous DC were pulsed with MART-126-35 peptide and administered with a dose escalation of the CTLA4-blocking antibody tremelimumab. Sixteen patients were accrued to five dose levels. Primary end points were safety and immune effects; clinical efficacy was a secondary end point.Results: Dose-limiting toxicities of grade 3 diarrhea and grade 2 hypophysitis developed in two of three patients receiving tremelimumab at 10 mg/kg monthly. Four patients had an objective tumor response, two partial responses and two complete responses, all melanoma free between 2 and 4 years after study initiation. There was no difference in immune monitoring results between patients with an objective tumor response and those without a response. Exploratory gene expression analysis suggested that immune-related gene signatures, in particular for B-cell function, may be important in predicting response.Conclusion: The combination of MART-1 peptide–pulsed DC and tremelimumab results in objective and durable tumor responses at the higher range of the expected response rate with either agent alone. (Clin Cancer Res 2009;15(19):6267–76)

Supplementary Table S1 from Effect of Altering Dietary ω-6/ω-3 Fatty Acid Ratios on Prostate Cancer Membrane Composition, Cyclooxygenase-2, and Prostaglandin E2

PURPOSE: Guidelines support early integration of palliative care (PC) into standard oncology practice; however, little is known as to whether outcomes can be improved by modifying health care delivery in a real-world setting. METHODS: We report our 6-year experience of embedding a nurse practitioner in an oncology clinic (March 2014-March 2020) to integrate early, concurrent advance care planning and PC. RESULTS: Compared with patients with advanced cancer not enrolled in the palliative care nurse practitioner program, in March 2020, patients who are enrolled are more likely to have higher quality of PC (eg, goals of care note documentation [82% v 15%; P < .01], referral to the psychosocial oncology program [67% v 37%; P < .01], and referral to hospice [61% v 34%; P < .01]) and less inpatient utilization in the last 6 months of life (eg, hospital days [12 v 18; P < .01] and intensive care unit days [1.2 v 2.3; P < .01]). The program expanded over time with the support of faculty skills training for advance care planning and PC, supporting a shared mental model of PC delivery within the oncology clinic. CONCLUSION: Embedding a trained palliative care nurse practitioner in oncology clinics to deliver early integrated PC can lead to improved quality of care for patients with advanced cancer.

Background In patients with melanoma and asymptomatic brain metastases (MBM), nivolumab plus ipilimumab provided an intracranial response rate of 55%. Here, we present the first report for patients who were symptomatic and/or required corticosteroids and updated data for asymptomatic patients. Methods Patients with measurable MBM, 0.5-3.0 cm, were enrolled into Cohort A (asymptomatic) or Cohort B (stable neurologic symptoms and/or receiving corticosteroids). Nivolumab, 1 mg/kg, and ipilimumab, 3 mg/kg, were given intravenously every 3 weeks ×4, followed by nivolumab, 3 mg/kg, every 2 weeks until progression, unacceptable toxicity, or 24 months. The primary endpoint was intracranial clinical benefit rate (CBR; complete response [CR], partial response [PR], or stable disease ≥6 months). Results Symptomatic patients (N = 18) received a median of one nivolumab and ipilimumab combination dose and had an intracranial CBR of 22.2%. Two of 12 patients on corticosteroids had CR; 2 responded among the 6 not on corticosteroids. Median intracranial progression-free survival (PFS) and overall survival (OS) were 1.2 and 8.7 months, respectively. In contrast, with 20.6 months of follow-up, we confirmed an intracranial CBR of 58.4% in asymptomatic patients (N = 101); median duration of response, PFS, and OS were not reached. No new safety signals were observed. Conclusions Nivolumab plus ipilimumab provides durable clinical benefit for asymptomatic patients with MBM and should be considered for first-line therapy. This regimen has limited activity in MBM patients with neurologic symptoms and/or requiring corticosteroids, supporting the need for alternative approaches and methods to reduce the dependency on corticosteroids. Clinical trial registration. ClinicalTrials.gov, NCT02320058.

Physicians are encouraged to communicate with their patients about financial concerns, but are infrequently taught skills necessary to do so. This study describes a curriculum for oncology fellows aimed to improve skills of cost-health literacy, and provides assessment of the curriculum impact on self-perceived cost communication practices. Oncology fellows at a large academic program in 2019 participated in a cost-health literacy curriculum over 3 months. The curriculum consisted of a didactic on financial toxicity (45 min), a problem-based learning case highlighting financial toxicity risk factors and areas for intervention (30 min), and a group discussion (30 min) to review and consolidate strategies to navigate financial toxicity in direct patient care. A cost-health literacy survey was administered at baseline and at the conclusion of the curriculum to evaluate the impact of the program. Of 19 participants, 16 completed both the pre-survey and post-survey and were included in the analysis. After the intervention, participants were more likely to report comfort discussing out-of-pocket costs (50% vs. 19%, p = 0.002) and to feel they could help a patient experiencing financial toxicity (62% vs. 6%, p = 0.005). There was no improvement in the subjective assessment of patient financial distress (57% v 50%, p = 0.759). Oncology fellows can improve self-reported cost-health literacy skills through participation in a targeted, brief curriculum. Further studies are warranted to determine how this approach can be applied in other settings and if it objectively impacts cost communication practices.

The MAPK signaling pathway is the most commonly mutated and/or dysregulated pathway in cancer. Strategies to target it have yielded some success with inhibitors against KRASG12C and BRAF, and to a lesser extent, MEK1/2. However, the impact of these molecules is often limited by toxicity and rapid and diverse mechanisms of resistance; both adaptive and/or acquired. For example, treatment with MAPK pathway targeting agents results in compensatory activation of the downstream mediator ERK1/2 and enables tumors to subvert the targeted therapy. Thus, targeting ERK1/2 provides promising potential advantages in overcoming and/or preventing adaptive and acquired resistance. We evaluated multiple preclinical and clinically staged ERK1/2 inhibitors—including ERAS-007 and BVD-523—in a 500+ cell line screening platform and identified cancers with subpopulations that are sensitive to this class of inhibitor. KiNativ analyses helped to inform the differences in sensitivity/selectivity that we observed between each ERK1/2 inhibitor. Comprehensive molecular profiling of the cell lines at baseline allowed us to screen for potential molecular markers of sensitivity/resistance to these compounds. Using this platform, we have developed a novel, potent ERK1/2 small molecule inhibitor, UCT-01-097, with improved selectivity over other clinically staged inhibitors. These data, coupled with the broad spectrum of in vitro responses, suggests an improved therapeutic index with this molecule. UCT-01-097 shows kinase selectivity in both cell free and in-cell assays and robust efficacy in panel of pancreatic PDX models. Inhibition of xenograft tumor growth was achieved using both daily dosing and intermittent dosing schedules. We have successfully submitted a regulatory IND and are currently enrolling a Phase 1 clinical trial in advanced solid tumors for treatment with UCT-01-097 (NCT04761601). Citation Format: Neil A. O'Brien, Martina S. McDermott, Brendan M. O'Boyle, Corey M. Reeves, Michael Bartberger, Oliver Loson, Kevin Chau, Jenny J. Hong, Weiping Jia, Naeimeh Kamranpour, Tong Luo, Raul Ayala, Athena M. Madrid, John A. Glaspy, Brian M. Stoltz, Dennis J. Slamon. Development of UCT-01-097, a novel orally available ERK1/2 inhibitor for the treatment of ERK1/2 dependent cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4044.

Introduction: Erythropoietin stimulating agents (ESAs) have been established both to correct anemia and provide the clinical benefits of increased exercise capacity, reduced transfusion requirements, and improved quality of life. An increase in physician and patient adoption of biosimilars, as well as changes to healthcare reimbursement policies, have driven market competitors to innovate and expand the range of biosimilar products. While erythropoietin biosimilars have been approved by the EMA since 2007, the FDA's approval of epoetin alfa-epbx in 2018 marks the first erythropoietin biosimilar approved in the United States. Areas covered: In this article, we critically review the biology, clinical use, manufacturing, safety, and efficacy of ESAs and erythropoietin biosimilars. We then review the regulatory framework and potential impact on healthcare costs offered by erythropoietin biosimilars. Expert opinion: Due to the complex nature of manufacturing large-molecule biologics, it is important to recognize the challenges to quality assurance and overall safety posed by the introduction of biosimilars, which undergo much more limited clinical testing than their reference biologic product before coming to market. With many biologic therapies nearing patent expiration, biosimilars will become increasingly common in clinical practice. Ensuring patient safety with these products will require increased post-marketing surveillance and awareness from prescribers.