Multimetric feature selection for analyzing multicategory outcomes of colorectal cancer: random forest and multinomial logistic regression models.

Colorectal cancer (CRC) is one of the most common cancers worldwide, and a leading cause of cancer deaths. Better classifying multicategory outcomes of CRC with clinical and omic data may help adjust treatment regimens based on individual's risk. Here, we selected the features that were useful for classifying four-category survival outcome of CRC using the clinical and transcriptomic data, or clinical, transcriptomic, microsatellite instability and selected oncogenic-driver data (all data) of TCGA. We also optimized multimetric feature selection to develop the best multinomial logistic regression (MLR) and random forest (RF) models that had the highest accuracy, precision, recall and F1 score, respectively. We identified 2073 differentially expressed genes of the TCGA RNASeq dataset. MLR overall outperformed RF in the multimetric feature selection. In both RF and MLR models, precision, recall and F1 score increased as the feature number increased and peaked at the feature number of 600-1000, while the models' accuracy remained stable. The best model was the MLR one with 825 features based on sum of squared coefficients using all data, and attained the best accuracy of 0.855, F1 of 0.738 and precision of 0.832, which were higher than those using clinical and transcriptomic data. The top-ranked features in the MLR model of the best performance using clinical and transcriptomic data were different from those using all data. However, pathologic staging, HBS1L, TSPYL4, and TP53TG3B were the overlapping top-20 ranked features in the best models using clinical and transcriptomic, or all data. Thus, we developed a multimetric feature-selection based MLR model that outperformed RF models in classifying four-category outcome of CRC patients. Interestingly, adding microsatellite instability and oncogenic-driver data to clinical and transcriptomic data improved models' performances. Precision and recall of tuned algorithms may change significantly as the feature number changes, but accuracy appears not sensitive to these changes.

Breast cancer vaccines for treatment and prevention.

Breast cancer is immunogenic and a variety of vaccines have been designed to boost immunity directed against the disease. The components of a breast cancer vaccine, the antigen, the delivery system, and the adjuvant, can have a significant impact on vaccine immunogenicity. There have been numerous immunogenic proteins identified in all subtypes of breast cancer. The majority of these antigens are weakly immunogenic nonmutated tumor-associated proteins. Mutated proteins and neoantigen epitopes are found only in a small minority of patients and are enriched in the triple negative subtype. Several vaccines have advanced to large randomized Phase II or Phase III clinical trials. None of these trials met their primary endpoint of either progression-free or overall survival. Despite these set-backs investigators have learned important lessons regarding the clinical application of breast cancer vaccines from the type of immune response needed for tumor eradication, Type I T-cell immunity, to the patient populations most likely to benefit from vaccination. Many therapeutic breast cancer vaccines are now being tested in combination with other forms of immune therapy or chemotherapy and radiation. Breast cancer vaccines as single agents are now studied in the context of the prevention of relapse or development of disease. Newer approaches are designing vaccines to prevent breast cancer by intercepting high-risk lesions such as ductal carcinoma in situ to limit the progression of these tumors to invasive cancer. There are also several efforts to develop vaccines for the primary prevention of breast cancer by targeting antigens expressed during breast cancer initiation.

Immuno-Oncology in the Era of Personalized Medicine.

Personalized medicine in oncology utilizes evidence derived from genetic, immune, and proteomic profiling to inform therapeutic options as well as provide prognostic information for each unique individual and their tumor. Our ability to biologically and immunologically define each patient's tumor has been driven by the development of assays characterizing the genomic and proteomic profiles of tumors that in turn have led to the development of large biologic databases and computational tools for the analysis of these large data sets. In Immuno-oncology, the introduction of checkpoint inhibitors and their approval across multiple tumor types has led to the recognition that the majority of patients will not clinically respond to these therapies but will remain at risk for the development of significant immunologic side effects. This challenge highlights the need for the development and validation of both predictive biomarkers for response to such therapies as well as biomarkers prognostic of disease course. Despite extensive investigation into predictive biomarkers using these biologic databases and computational methods, only recently has progress been made in this area. This progress is the first step allowing us to identify patients likely to benefit from these therapies and moving our field closer to a truly personalized approach to the use of immune therapies in oncology.

Leveraging premalignant biology for immune-based cancer prevention.

Prevention is an essential component of cancer eradication. Next-generation sequencing of cancer genomes and epigenomes has defined large numbers of driver mutations and molecular subgroups, leading to therapeutic advances. By comparison, there is a relative paucity of such knowledge in premalignant neoplasia, which inherently limits the potential to develop precision prevention strategies. Studies on the interplay between germ-line and somatic events have elucidated genetic processes underlying premalignant progression and preventive targets. Emerging data hint at the immune system's ability to intercept premalignancy and prevent cancer. Genetically engineered mouse models have identified mechanisms by which genetic drivers and other somatic alterations recruit inflammatory cells and induce changes in normal cells to create and interact with the premalignant tumor microenvironment to promote oncogenesis and immune evasion. These studies are currently limited to only a few lesion types and patients. In this Perspective, we advocate a large-scale collaborative effort to systematically map the biology of premalignancy and the surrounding cellular response. By bringing together scientists from diverse disciplines (e.g., biochemistry, omics, and computational biology; microbiology, immunology, and medical genetics; engineering, imaging, and synthetic chemistry; and implementation science), we can drive a concerted effort focused on cancer vaccines to reprogram the immune response to prevent, detect, and reject premalignancy. Lynch syndrome, clonal hematopoiesis, and cervical intraepithelial neoplasia which also serve as models for inherited syndromes, blood, and viral premalignancies, are ideal scenarios in which to launch this initiative.

Immunotherapy of head and neck cancer: Emerging clinical trials from a National Cancer Institute Head and Neck Cancer Steering Committee Planning Meeting.

Recent advances have permitted successful therapeutic targeting of the immune system in head and neck squamous cell carcinoma (HNSCC). These new immunotherapeutic targets and agents are being rapidly adopted by the oncologic community and hold considerable promise. The National Cancer Institute sponsored a Clinical Trials Planning Meeting to address the issue of how to further investigate the use of immunotherapy in patients with HNSCC. The goals of the meeting were to consider phase 2 or 3 trial designs primarily in 3 different patient populations: those with previously untreated, human papillomavirus-initiated oropharyngeal cancers; those with previously untreated, human papillomavirus-negative HNSCC; and those with recurrent/metastatic HNSCC. In addition, a separate committee was formed to develop integrative biomarkers for the clinical trials. The meeting started with an overview of key immune components and principles related to HNSCC, including immunosurveillance and immune escape. Four clinical trial concepts were developed at the meeting integrating different immunotherapies with existing standards of care. These designs were presented for implementation by the head and neck committees of the National Cancer Institute-funded National Clinical Trials Network. This article summarizes the proceedings of this Clinical Trials Planning Meeting, the purpose of which was to facilitate the rigorous development and design of randomized phase 2 and 3 immunotherapeutic trials in patients with HNSCC. Although reviews usually are published immediately after the meeting is held, this report is unique because there are now tangible clinical trial designs that have been funded and put into practice and the studies are being activated to accrual. Cancer 2017;123:1259-1271. © 2016 American Cancer Society.

Phase II trial of albumin-bound paclitaxel and granulocyte macrophage colony-stimulating factor as an immune modulator in recurrent platinum resistant ovarian cancer.

BACKGROUND: Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulates immunity via recruitment of antigen presenting cells and tumor specific T-cell stimulation. Albumin-bound paclitaxel (nab-paclitaxel) followed by GM-CSF may enhance antitumor responses and prolong remissions in ovarian cancer. Immune phenotypes present before treatment may identify responders to chemo-immunotherapy. METHODS: Recurrent platinum-resistant ovarian, peritoneal, or fallopian tube cancer patients received nab-paclitaxel, 100mg/m2 days 1, 8, 15 followed by GM-CSF 250µg days 16-26 every 28days for 6 planned cycles. The primary endpoint was remission duration compared to immediate prior remission. Peripheral blood was evaluated by flow cytometry and interferon-γ ELISPOT. RESULTS: Twenty-one patients were enrolled. Six patients (29%) achieved a biochemical complete response and 9 (43%) a partial response for an overall response rate of 72%. Median time to progression was 4months and 10% of patients achieved longer remissions than the immediate prior regimen. Median overall survival (OS) was 16.8months. Fewer myeloid derived suppressor cells (MDSC) at enrollment significantly associated with complete response (p=0.05). T-cell responses to IGF1R-p1332-1346 (r=0.827, p=0.0003) and IGF1R-p1242-1256 (r=0.850, p=0.0001) during treatment correlated with time to progression. CONCLUSIONS: Nab-paclitaxel combined with GM-CSF demonstrated biochemical responses in a majority of patients, although responses were not sustained. This combination did not demonstrate an advantage in OS over prior studies of nab-paclitaxel monotherapy. Agents that modulate MDSC should be studied as potential adjuvants to therapy. Strategies to expand T cells recognizing tumor-associated antigens biologically significant in ovarian cancer should also continue to be investigated.

Epidermal growth factor receptor derived peptide vaccination to prevent lung adenocarcinoma formation: An in vivo study in a murine model of EGFR mutant lung cancer.

The ability to prevent disease is the holy grail of medicine. For decades, efforts have been made to extend the successes seen with vaccination against infectious diseases to cancer. In some instances, preventive vaccination against viruses (prototypically HPV) has successfully prevented tumorigenesis and will make a major impact on public health in the decades to come. However, the majority of cancers that arise are a result of genetic mutation within the host, or non-viral environmental exposures. We present compelling evidence that vaccination against an overexpressed self-tumor oncoprotein has the potential to prevent tumor development. Vaccination against the Epidermal Growth Factor Receptor (EGFR) using a multipeptide vaccine in a preventive setting decreased EGFR-driven lung carcinogenesis by 76.4% in a mouse model of EGFR-driven lung cancer. We also demonstrate that anti-EGFR vaccination primes the development of a robust immune response in vivo. This study provides proof of concept for the first time that targeting tumor drivers in a preventive setting in lung cancer using peptide vaccination can inhibit tumorigenesis and may provide useful clinical insights into the development of strategies to vaccinate against EGFR in populations where EGFR-mutant disease is highly prevalent. © 2015 Wiley Periodicals, Inc.

HER-2/neu vaccine-primed autologous T-cell infusions for the treatment of advanced stage HER-2/neu expressing cancers.

This phase I study evaluated the feasibility of expanding HER-2/neu (HER2) vaccine-primed peripheral blood T-cells ex vivo and assessed the safety of T-cell infusions. Eight patients with HER2(+) treatment refractory metastatic cancers were enrolled. T-cells could be expanded to predefined parameters in seven patients (88%). Ninety-two percent of adverse events were grade 1 or 2. Three of seven patients developed infusion-related inflammatory reactions at their disease sites. HER2-specific T-cells significantly increased in vivo compared to pre-infusion levels (p = 0.010) and persisted in 4/6 patients (66%) over 70 days after the first infusion. Partial clinical responses were observed in 43% of patients. Levels of T-regulatory cells in peripheral blood prior to infusion (p < 0.001), the level of HER2-specific T-cells in vivo (p = 0.030), and development of diverse clonal T-cell populations (p < 0.001) were associated with response. The generation of HER2 vaccine-primed autologous T-cells for therapeutic infusion is feasible and well tolerated. This approach provides a foundation for the application of T-cell therapy to additional solid tumor types.

Natural history of tumor growth and immune modulation in common spontaneous murine mammary tumor models.

Recent studies in patients with breast cancer suggest the immune microenvironment influences response to therapy. We aimed to evaluate the relationship between growth rates of tumors in common spontaneous mammary tumor models and immune biomarkers evaluated in the tumor and blood. TgMMTV-neu and C3(1)-Tag transgenic mice were followed longitudinally from birth, and MPA-DMBA-treated mice from the time of carcinogen administration, for the development of mammary tumors. Tumor-infiltrating CD4(+) and CD8(+) T-cells, FOXP3(+) T-regulatory cells, and myeloid-derived suppressor cells were assessed by flow cytometry. Serum cytokines were evaluated in subsets of mice. Fine needle aspirates of tumors were collected and RNA was isolated to determine levels of immune and proliferation markers. Age of tumor onset and kinetics of tumor growth were significantly different among the models. Mammary tumors from TgMMTV-neu contained a lower CD8/CD4 ratio than that of other models (p < 0.05). MPA-DMBA-induced tumors contained a higher percentage of FOXP3(+) CD4(+) T-cells (p < 0.01) and MDSC (p < 0.001) compared with the other models. Individuals with significantly slower tumor growth demonstrated higher levels of Type I serum cytokines prior to the development of lesions compared to those with rapid tumor growth. Moreover, the tumors of animals with more rapid tumor growth demonstrated a significant increase in the expression of genes associated with Type II immunity than those with slower-progressing tumors. These data provide a foundation for the development of in vivo models to explore the relationship between endogenous immunity and response to standard therapies for breast cancer.

Vaccination with ErbB-2 peptides prevents cancer stem cell expansion and suppresses the development of spontaneous tumors in MMTV-PyMT transgenic mice.

ErbB-2 has been implicated as a target for cancer-initiating cells in breast and other cancers. ErbB-2-directed peptide vaccines have been shown to be effective in prevention of spontaneous tumorigenesis of breast in neu transgenic mouse model, and cellular immunity is proposed as a mechanism for the anti-tumor efficacy. However, there has been no explanation as to how immunity suppresses tumorigenesis from the early stage carcinogenesis, when ErbB-2 expression in breast is low. Here, we investigated a peptide-based vaccine, which consists of two MHC class II epitopes derived from murine ErbB-2, to prevent the occurrence of spontaneous tumors in breast and assess immune impact on breast cancer stem cells. Female MMTV-PyMT transgenic mice were immunized with either ErbB-2 peptide vaccine, or a peptide from tetanus toxoid, or PBS in immune adjuvant. ErbB-2 peptides vaccine completely suppressed spontaneous breast tumors, and the efficacy was correlated with antigen-specific T-cell and antibody responses. In addition, immune serum from the mice of ErbB-2 vaccine group had an inhibitory effect on mammosphere-forming capacity and signaling through ErbB-2 and downstream Akt pathway in ErbB-2 overexpressing mouse mammary cancer cells. We provide evidence that multi-epitope class II peptides vaccine suppresses tumorigenesis of breast potentially by inhibiting the growth of cancer stem cells. We also suggest that a strategy of inducing strong immune responses using multi-epitope ErbB-2-directed helper vaccine might be useful in preventing breast cancer recurrence.

Mining the pre-diagnostic antibody repertoire of TgMMTV-neu mice to identify autoantibodies useful for the early detection of human breast cancer.

BACKGROUND: The use of autoantibodies for the early detection of breast cancer has generated much interest as antibodies can be readily assayed in serum when antigen levels are low. Ideally, diagnostic autoantibodies would be identified in individuals who harbored pre-invasive disease/high risk lesions leading to malignancy. Prospectively collected human serum samples from these individuals are rare and not often available for biomarker discovery. We questioned whether transgenic animals could be used to identify cancer-associated autoantibodies present at the earliest stages of the malignant transformation of breast cancer. METHODS: We collected sera from transgenic mice (TgMMTV-neu) from the time of birth to death by spontaneous mammary tumors. Using sera from a time point prior to the development of tumor, i.e. "pre-diagnostic", we probed cDNA libraries derived from syngeneic tumors to identify proteins recognized by IgG antibodies. Once antigens were identified, selected proteins were evaluated via protein arrays, for autoantibody responses using plasma from women obtained prior to the development of breast cancer and matched controls. The ability of the antigens to discriminate cases from controls was assessed using receiver-operating-characteristic curve analyses and estimates of the area under the curve. RESULTS: We identified 6 autoantibodies that were present in mice prior to the development of mammary cancer: Pdhx, Otud6b, Stk39, Zpf238, Lgals8, and Vps35. In rodent validation cohorts, detecting both IgM and IgG antibody responses against a subset of the identified proteins could discriminate pre-diagnostic sera from non-transgenic control sera with an AUC of 0.924. IgG and IgM autoantibodies, specific for a subset of the identified antigens, could discriminate the samples of women who eventually developed breast cancer from case-matched controls who did not develop disease. The discriminatory potential of the pre-diagnostic autoantibodies was enhanced if plasma samples were collected greater than 5 months prior to a breast cancer diagnosis (AUC 0.68; CI 0.565-0.787, p=0.0025). CONCLUSION: Genetically engineered mouse models of cancer may provide a facile discovery tool for identifying autoantibodies useful for human cancer diagnostics.

Gamma delta T cells are activated by polysaccharide K (PSK) and contribute to the anti-tumor effect of PSK.

Polysaccharide K (PSK) is a widely used mushroom extract that has shown anti-tumor and immunomodulatory effects in both preclinical and clinical studies. Therefore, it is important to understand the mechanism of actions of PSK. We recently reported that PSK can activate toll-like receptor 2 and enhances the function of NK cells. The current study was undertaken to study the effect of PSK on gamma delta (γδ) T cells, another important arm of the innate immunity. In vitro experiments using mouse splenocytes showed that γδ T cells produce IFN-γ after treatment with PSK and have up-regulated expression of CD25, CD69, and CD107a. To investigate whether the effect of PSK on γδ T cells is direct or indirect, purified γδ T cells were cultured either alone or together with bone marrow-derived DC in a co-culture or trans-well system and then stimulated with PSK. Results showed that direct cell-to-cell contact between γδ T cells and DC is required for optimal activation of γδ T cells. There was also reciprocal activation of DC by PSK-activated γδ T cells, as demonstrated by higher expression of costimulatory molecules and enhanced production of IL-12 by DC in the presence of γδ T cells. PSK can also co-stimulate γδ T cells with anti-TCR and anti-CD3 stimulation, in the absence of DC. Finally, in vivo treatment with PSK activates γδ T cells among the tumor infiltrating lymphocytes, and depleting γδ T cells during PSK treatment attenuated the anti-tumor effect of PSK. All together, these results demonstrated that γδ T cells are activated by PSK and contribute to the anti-tumor effect of PSK.

T-helper I immunity, specific for the breast cancer antigen insulin-like growth factor-I receptor (IGF-IR), is associated with increased adiposity.

Numerous lines of evidence demonstrate that breast cancer is immunogenic; yet, there are few biologically relevant immune targets under investigation restricting the exploration of vaccines to limited breast cancer subtypes. Insulin-like growth factor-I receptor (IGF-IR) is a promising vaccine candidate since it is overexpressed in most breast cancer subtypes, is part of a dominant cancer growth pathway, and has been validated as a therapeutic target. We questioned whether IGF-IR was immunogenic in cancer patients. IGF-IR-specific IgG antibodies were significantly elevated in early-stage breast cancer patients at the time of diagnosis as compared to volunteer donors (p = 0.04). Predicted T-helper epitopes, derived from the IGF-IR extracellular and transmembrane domains, elicited a significantly higher incidence of Th2 immunity in breast cancer patients as compared to controls (p = 0.01). Moreover, the magnitude of Th2 immunity was greater in breast cancer patients compared to controls (p = 0.02). In contrast, both breast cancer patients and volunteer donors demonstrated a similar incidence of Th1 immunity to IGF-IR domains with the predominant response directed against epitopes in the intracellular domain of the protein. As the incidence of IGF-IR type I immunity was not associated with a breast cancer diagnosis, we questioned whether other factors were contributing to the presence of IGF-IR-specific T-cells in both populations. While age was not associated with Th1 immunity, we observed a significantly greater magnitude of IGF-IR IFN-γ-secreting T-cells in obese subjects as compared to overweight (p < 0.001) or healthy-weight (p = 0.006) subjects, regardless of breast cancer diagnosis. No significant difference was observed for Th2 incidence or magnitude when stratified by age (p = 0.174, p = 0.966, respectively) or body mass index (p = 0.137, p = 0.174, respectively). Our data demonstrate that IGF-IR is a tumor antigen and IGF-IR-specific Th1 immunity may be associated with obesity rather than malignancy.

Therapeutic vaccines for ovarian cancer.

While therapeutic vaccines for ovarian cancer represent only a small fraction of active clinical trials, growing interest in this area and the accumulated data supporting the use of vaccines in cancer treatment portend further expansion of trials incorporating these strategies. This review explores the rationale for the use of vaccines for the treatment of ovarian cancer. It examines vaccine platforms that have been investigated and reviews the data from these studies. We also highlight recently reported phase 2 and 3 clinical trials with clinical outcomes as endpoints. Finally, we consider directions for the next generation of vaccines in light of these findings and our emerging understanding of agents that may augment vaccine responses by targeting the immunosuppressive impact of the tumor microenvironment.

Three-dimensional scaffolds to evaluate tumor associated fibroblast-mediated suppression of breast tumor specific T cells.

In the tumor microenvironment, the signals from tumor-associated fibroblasts (TAF) that suppress antitumor immunity remain unclear. Here, we develop and investigate an in vitro three-dimensional (3D) scaffold model for the novel evaluation of TAF interaction with breast tumor cells and breast specific, neu antigen (p98) reactive T cells. Breast cancer cells seeded on 3D chitosan-alginate (CA) scaffolds showed productive growth and formed distinct tumor spheroids. Antigen specific p98 T cells, but not naïve T cells, bound significantly better to tumor cells on scaffolds. The p98 T cells induced potent tumor cell killing but T helper cell cytokine function was impaired in the presence of TAF coseeding on scaffolds. We found that the immunosuppression was mediated, in part, by transforming growth factor beta (TGF-b) and interleukin-10 (IL-10). Therefore, TAF appear capable of inducing potent T cell suppression. CA scaffolds can provide clinically relevant findings prior to preclinical testing of novel immunotherapies.

Safety and Outcomes of a Plasmid DNA Vaccine Encoding the ERBB2 Intracellular Domain in Patients With Advanced-Stage ERBB2-Positive Breast Cancer: A Phase 1 Nonrandomized Clinical Trial.

Importance: High levels of ERBB2 (formerly HER2)-specific type 1 T cells in the peripheral blood are associated with favorable clinical outcomes after trastuzumab therapy; however, only a minority of patients develop measurable ERBB2 immunity after treatment. Vaccines designed to increase ERBB2-specific T-helper cells could induce ERBB2 immunity in a majority of patients. Objective: To determine the safety and immunogenicity of 3 doses (10, 100, and 500 µg) of a plasmid-based vaccine encoding the ERBB2 intracellular domain (ICD). Design, Setting, and Participants: Single-arm phase 1 trial including 66 patients with advanced-stage ERBB2-positive breast cancer treated in an academic medical center between 2001 and 2010 with 10-year postvaccine toxicity assessments. Data analysis was performed over 2 periods: January 2012 to March 2013 and July 2021 to August 2022. Interventions: Patients were sequentially enrolled to the 3 dose arms. The vaccine was administered intradermally once a month with soluble granulocyte-macrophage colony-stimulating factor as an adjuvant for 3 immunizations. Toxicity evaluations occurred at set intervals and yearly. Peripheral blood mononuclear cells were collected for evaluation of immunity. Biopsy of vaccine sites at weeks 16 and 36 measured DNA persistence. Main Outcomes and Measures: Safety was graded by Common Terminology Criteria for Adverse Events, version 3.0, and ERBB2 ICD immune responses were measured by interferon-γ enzyme-linked immunosorbent spot. Secondary objectives determined if vaccine dose was associated with immunity and evaluated persistence of plasmid DNA at the vaccine site. Results: A total of 66 patients (median [range] age, 51 [34-77] years) were enrolled. The majority of vaccine-related toxic effects were grade 1 and 2 and not significantly different between dose arms. Patients in arm 2 (100 µg) and arm 3 (500 µg) had higher magnitude ERBB2 ICD type 1 immune responses at most time points than arm 1 (10 µg) (arm 2 compared with arm 1, coefficient, 181 [95% CI, 60-303]; P = .003; arm 3 compared with arm 1, coefficient, 233 [95% CI, 102-363]; P < .001) after adjusting for baseline factors. ERBB2 ICD immunity at time points after the end of immunizations was significantly lower on average in patients with DNA persistence at week 16 compared with those without persistence. The highest vaccine dose was associated with the greatest incidence of persistent DNA at the injection site. Conclusions and Relevance: In this phase 1 nonrandomized clinical trial, immunization with the 100-µg dose of the ERBB2 ICD plasmid-based vaccine was associated with generation of ERBB2-specific type 1 T cells in most patients with ERBB2-expressing breast cancer, and it is currently being evaluated in randomized phase 2 trials. Trial Registration: ClinicalTrials.gov Identifier: NCT00436254.