Francesco M. Marincola

Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance.

Publisher Summary It is known for some time that malignant transformation of human cells may be associated with the appearance of tumor associated antigens (TAA). Decades of research have been aimed at the identification of TAA that can serve as targets for the immunotherapy of malignant diseases. The dramatic progress in the understanding of molecular basis of target cell recognition by cytotoxic T lymphocytes (CTL) has provided the background to design effective strategies to identify TAA recognized by CTL on tumor cells. The extensive application of these strategies by a number of investigators has resulted in the identification of various families of TAA on various types of solid tumors. Mouse tumor models have played an important role in elucidating the mechanisms by which the immune system interacts with tumor cells and eradicates cancer. The second line of evidence is represented by the phenomenon of a “mixed response.” A mixed response occurs rather frequently in patients with metastases, although its actual frequency is not documented. Mixed responses are characterized by the different behavior of synchronous metastases in response to T cell-based immunotherapy. This important finding suggests that TAA-specific CTL may be present in some cancer patients but are unable to attack tumor cells due to the presence of inhibitory receptors.

A human memory T cell subset with stem cell-like properties.

Immunological memory is thought to depend on a stem cell–like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T cell population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, specific to multiple viral and self-tumor antigens, were found within a CD45RO−, CCR7+, CD45RA+, CD62L+, CD27+, CD28+ and IL-7Rα+ T cell compartment characteristic of naive T cells. However, they expressed large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity and more efficiently reconstituted immunodeficient hosts, and they mediated superior antitumor responses in a humanized mouse model. The identification of a human stem cell–like memory T cell population is of direct relevance to the design of vaccines and T cell therapies.

High-fidelity mRNA amplification for gene profiling.

The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50–200 μg of total RNA (T-RNA) and 2–5 μg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.

gp100 Peptide Vaccine and Interleukin-2 in Patients with Advanced Melanoma

BACKGROUND Stimulating an immune response against cancer with the use of vaccines remains a challenge. We hypothesized that combining a melanoma vaccine with interleukin-2, an immune activating agent, could improve outcomes. In a previous phase 2 study, patients with metastatic melanoma receiving high-dose interleukin-2 plus the gp100:209-217(210M) peptide vaccine had a higher rate of response than the rate that is expected among patients who are treated with interleukin-2 alone. METHODS We conducted a randomized, phase 3 trial involving 185 patients at 21 centers. Eligibility criteria included stage IV or locally advanced stage III cutaneous melanoma, expression of HLA*A0201, an absence of brain metastases, and suitability for high-dose interleukin-2 therapy. Patients were randomly assigned to receive interleukin-2 alone (720,000 IU per kilogram of body weight per dose) or gp100:209-217(210M) plus incomplete Freunds adjuvant (Montanide ISA-51) once per cycle, followed by interleukin-2. The primary end point was clinical response. Secondary end points included toxic effects and progression-free survival. RESULTS The treatment groups were well balanced with respect to baseline characteristics and received a similar amount of interleukin-2 per cycle. The toxic effects were consistent with those expected with interleukin-2 therapy. The vaccine-interleukin-2 group, as compared with the interleukin-2-only group, had a significant improvement in centrally verified overall clinical response (16% vs. 6%, P=0.03), as well as longer progression-free survival (2.2 months; 95% confidence interval [CI], 1.7 to 3.9 vs. 1.6 months; 95% CI, 1.5 to 1.8; P=0.008). The median overall survival was also longer in the vaccine-interleukin-2 group than in the interleukin-2-only group (17.8 months; 95% CI, 11.9 to 25.8 vs. 11.1 months; 95% CI, 8.7 to 16.3; P=0.06). CONCLUSIONS In patients with advanced melanoma, the response rate was higher and progression-free survival longer with vaccine and interleukin-2 than with interleukin-2 alone. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00019682.).

Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinial relevance

Malignant transformation of melanocytes may be associated with changes in the expression of major histocompatibility complex (MHC) HLA class I antigens. Interest in the characterization of abnormalities in the expression of MHC class I by melanoma cells has been rekindled by the current emphasis on the application of T-cell-based immunotherapy to melanoma. Here, Soldano Ferrone and Francesco Marincola review defects in class I expression as described in melanoma cells, as well as the molecular mechanisms, functional significance and clinical implications of such defects.

Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression‐based stratification. These parameters rely on tumour‐cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named ‘Immunoscore’ has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM‐I (TNM‐Immune).

Tumour immunity : effector response to tumour and role of the microenvironment

Substantial evidence shows that inflammation promotes oncogenesis and, occasionally, participates in cancer rejection. This paradox can be accounted for by a dynamic switch from chronic smouldering inflammation promoting cancer-cell survival to florid, tissue-disruptive inflammatory reactions that trigger cancer-cell destruction. Clinical and experimental observations suggest that the mechanism of this switch recapitulates the events associated with pathogen infection, which stimulate immune cells to recognise danger signals and activate immune effector functions. Generally, cancers do not have danger signals and, therefore, they cannot elicit strong immune reactions. Synthetic molecules have been developed that mimic pathogen invasion at the tumour site. These compounds activate dendritic cells to produce proinflammatory cytokines, which in turn trigger cytotoxic mechanisms leading to cancer death. Simultaneously, dendritic cells capture antigen shed by dying cancer cells, undergo activation, and stimulate antigen-specific T and B cells. This process results in massive amplification of the antineoplastic inflammatory process. Thus, although anti-inflammatory drugs can prevent onset of some malignant diseases, induction of T cells specific for tumour antigen by active immunisation, combined with powerful activation signals within the cancer microenvironment, might yield the best strategy for treatment of established cancers.

Phase I Study of the Intravenous Administration of Attenuated Salmonella typhimurium to Patients With Metastatic Melanoma

PURPOSE A strain of Salmonella typhimurium (VNP20009), attenuated by chromosomal deletion of the purI and msbB genes, was found to target to tumor and inhibit tumor growth in mice. These findings led to the present phase I study of the intravenous infusion of VNP20009 to patients with metastatic cancer. PATIENTS AND METHODS In cohorts consisting of three to six patients, 24 patients with metastatic melanoma and one patient with metastatic renal cell carcinoma received 30-minute intravenous bolus infusions containing 10(6) to 10(9) cfu/m(2) of VNP20009. Patients were evaluated for dose-related toxicities, selective replication within tumors, and antitumor effects. RESULTS The maximum-tolerated dose was 3 x 10(8) cfu/m(2). Dose-limiting toxicity was observed in patients receiving 1 x 10(9) cfu/m(2), which included thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia. VNP20009 induced a dose-related increase in the circulation of proinflammatory cytokines, such as interleukin (IL)-1beta, tumor necrosis factor alpha, IL-6, and IL-12. Focal tumor colonization was observed in two patients receiving 1 x 10(9) cfu/m(2) and in one patient receiving 3 x 10(8) cfu/m(2). None of the patients experienced objective tumor regression, including those patients with colonized tumors. CONCLUSION The VNP20009 strain of Salmonella typhimurium can be safely administered to patients, and at the highest tolerated dose, some tumor colonization was observed. No antitumor effects were seen, and additional studies are required to reduce dose-related toxicity and improve tumor localization.

Cancer classification using the Immunoscore: a worldwide task force

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the ‘Immunoscore’ into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

A Phase I Study of Nonmyeloablative Chemotherapy and Adoptive Transfer of Autologous Tumor Antigen-Specific T Lymphocytes in Patients With Metastatic Melanoma

This report describes a phase I clinical trial using nonmyeloablative, lympho-depleting chemotherapy in combination with adoptive immunotherapy in patients with metastatic melanoma. The chemotherapy-conditioning schedule that induced transient lymphopenia consisted of cyclophosphamide (30 or 60 mg/kg per day for 2 days) followed by fludarabine (25 mg/m2 per day for 5 days). Immunotherapy for all patients consisted of in vitro expanded, tumor-reactive, autologous T-cell clones selected for high avidity recognition of melanoma antigens. Cohorts of three to six patients each received either no interleukin (IL)-2, low-dose IL-2 (72,000 IU/kg intravenously three times a day to a maximum of 15 doses), or high-dose IL-2 (720,000 IU/kg intravenously three times a day for a maximum of 12 doses). The toxicities associated with this treatment were transient and included neutropenia and thrombocytopenia that resolved in all patients. High dose intravenous IL-2 was better tolerated by patients after chemotherapy than during previous immunotherapy cycles without chemotherapy. No patient exhibited an objective clinical response to treatment, although five patients demonstrated mixed responses or transient shrinkage of metastatic deposits. This study established a nonmyeloablative-conditioning regimen that could be safely administered in conjunction with adoptive T-cell transfer and IL-2 in patients with metastatic melanoma.