Immunophenotypic and functional characterization of ex vivo expanded natural killer cells for clinical use in acute lymphoblastic leukemia patients.

The management of acute lymphoblastic leukemia (ALL) patients has witnessed profound changes in recent years. Nonetheless, most patients tend to relapse, underlining the need for new therapeutic approaches. The anti-leukemic potential of natural killer (NK) cells has over the years raised considerable interest. In this study, we developed an efficient method for the expansion and activation of NK cells isolated from healthy donors and ALL patients for clinical use. NK cell products were derived from peripheral blood mononuclear cells of 35 healthy donors and 4 B-lineage ALL by immunomagnetic CD3 T cell depletion followed by CD56 cell enrichment. Isolated NK cells were expanded and stimulated in serum-free medium supplemented with irradiated autologous feeder cells and autologous plasma in the presence of clinical grade interleukin (IL)-2 and IL-15 for 14 days. Healthy donor NK cells expanded on average 34.9 ± 10.4 fold and were represented, after expansion, by a highly pure population of CD3(-)CD56(+) cells showing a significant upregulation of natural cytotoxicity receptors, activating receptors and maturation markers. These expanded effectors showed cytolytic activity against K562 cells and, most importantly, against primary adult B-lineage ALL blasts. NK cells could be efficiently isolated and expanded-on average 39.5 ± 20.3 fold-also from primary B-lineage ALL samples of patients in complete remission. The expanded NK cells from these patients showed a significantly increased expression of the NKG2D- and DNAM1-activating receptors and were cytotoxic against K562 cells. These data provide the basis for developing new immunotherapeutic strategies for the management of ALL patients.

Ovarian cancer cytoreduction induces changes in T cell population subsets reducing immunosuppression.

Surgery is the primary therapeutic strategy for most solid tumours; however, modern oncology has established that neoplasms are frequently systemic diseases. Being however a local treatment, the mechanisms through which surgery plays its systemic role remain unknown. We have investigated the influence of cytoreduction on the immune system of primary and recurrent ovarian cancer. All ovarian cancer patients show an increase in CD4+ CD25+ FOXP3+ circulating cells (CD4 T(reg) ). CD4/CD8 ratio is increased in primary tumours, but not in recurrent neoplasms. Primary cytoreduction is able to increase circulating CD4 and CD8 effector cells and decrease CD4 naïve T cells. CD4+ T(reg) cells rapidly decreased after primary tumour debulking, while CD8+ CD25+ FOXP3+ (CD8 T(reg) ) cells are not detectable in peripheral blood. Similar results on CD4 T(reg) were observed with chemical debulking in women subjected to neoadjuvant chemotherapy. CD4 and CD8 T(reg) cells are both present in neoplastic tissue. Interleukin (IL)-10 serum levels decrease after surgery, while no changes are observed in transforming growth factor-ß1 and IL-6 levels. Surgically induced reduction of the immunosuppressive environment results in an increased capacity of CD8+ T cells to respond to the recall antigens. None of these changes was observed in patients previously subjected to chemotherapy or affected by recurrent disease. In conclusion, we demonstrate in ovarian cancer that primary debulking is associated with a reduction of circulating T(reg) and an increase in CD8 T-cell function. Debulking plays a beneficial systemic effect by reverting immunosuppression and restoring immunological fitness.

RFA strongly modulates the immune system and anti-tumor immune responses in metastatic liver patients.

Radiofrequency tumor ablation (RFA) is a therapeutic modality for liver cancer patients inducing localized tumor necrosis with maximal preservation of normal liver parenchyma. We investigated the immunomodulatory effects exerted by RFA treatment in liver cancer patients with metastatic liver lesions (13 patients) or hepatocellular carcinoma (HCC) (4 patients). Analysis of lymphocyte subsets by flow cytometry revealed that after RFA, CD3+ T cells, in particular CD4+, were decreased in metastatic cancer patients, while no change was observed in HCC patients. Moreover, RFA induced trafficking of naïve and memory CD62L+ T cells from circulation to tissues. When characterizing the function of T cells, proliferative response to PHA was strongly increased after 48 h from RFA in metastatic cancer patients. Furthermore, T cells produced IFN-gamma in response to the tumor associated MUC1 antigen. In contrast, humoral immune responses against tumor antigens such as MUC1 and HCV proteins were unaffected by RFA treatment, although increase of circulating B cells was observed only in metastatic cancer patients. These results indicate that RFA application can exert an activating effect on the immune system in metastatic cancer patients, favouring trafficking of lymphocyte subsets and enhancing tumor antigen specific cellular immune responses.

Prognostic and therapeutic role of targetable lesions in B-lineage acute lymphoblastic leukemia without recurrent fusion genes.

To shed light into the molecular bases of B-lineage acute lymphoblastic leukemia lacking known fusion transcripts, i.e. BCR-ABL1, ETV6-RUNX1, E2A-PBX1, and MLL rearrangements (B-NEG ALL) and the differences between children, adolescents/young adults (AYA) and adults, we analyzed 168 B-NEG ALLs by genome-wide technologies. This approach showed that B-NEG cases carry 10.5 mutations and 9.1 copy-number aberrations/sample. The most frequently mutated druggable pathways were those pertaining to RAS/RTK (26.8%) and JAK/STAT (12.5%) signaling. In particular, FLT3 and JAK/STAT mutations were detected mainly in AYA and adults, while KRAS and NRAS mutations were more frequent in children. RAS/RTK mutations negatively affected the outcome of AYA and adults, but not that of children. Furthermore, adult B-NEG ALL carrying JAK/STAT mutations had a shorter survival. In vitro experiments showed that FLT3 inhibitors reduced significantly the proliferation of FLT3-mutated primary B-NEG ALL cells. Likewise, PI3K/mTOR inhibitors reduced the proliferation of primary cells harboring RAS and IL7R mutations. These results refine the genetic landscape of B-NEG ALL and suggest that the different distribution of lesions and their prognostic impact might sustain the diverse outcome between children, adults and partly AYA - whose genomic scenario is similar to adults - and open the way to targeted therapeutic strategies.

A good manufacturing practice method to ex vivo expand natural killer cells for clinical use.

BACKGROUND: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing. RESULTS: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing. DISCUSSION: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.

An update in neoadjuvant chemotherapy in cervical cancer.

More than two decades have passed since neoadjuvant chemotherapy has been introduced in the clinical practice. After several pilot studies, randomized trials and meta-analysis have confirmed the validity of this therapeutic strategy and in particular of neoadjuvant chemotherapy followed by radical surgery. Several open questions still remain unanswered and in particular no standard drug regiment is unanimously accepted. In this paper we describe how this treatment has evolved since its original introduction in the early eighties and describe possible drug regimens with their supporting evidence that can be adopted by physicians worldwide.