The Promises of Natural Killer Cell Therapy in Endometriosis.

Endometriosis is a gynaecological disease defined by the growth of endometrium-like tissue outside the uterus. The disease is present in approximately 5-10% of women of reproductive age and causes pelvic pain and infertility. The pathophysiology is not completely understood, but retrograde menstruation and deficiency in natural killer (NK) cells that clear endometriotic cells in the peritoneal cavity play an important role. Nowadays, hormonal therapy and surgery to remove endometriosis lesions are used as treatment. However, these therapies do not work for all patients, and hormonal therapy prevents patients from getting pregnant. Therefore, new treatment strategies should be developed. Since the cytotoxicity of NK cells is decreased in endometriosis, we performed a literature search into the possibility of NK cell therapy. Available treatment options include the inhibition of receptor-ligand interaction for KIR2DL1, NKG2A, LILRB1/2, and PD-1/PD-L1; inhibition of TGF-ß; stimulation of NK cells with IL-2; and mycobacterial treatment with BCG. In preclinical work, these therapies show promising results but unfortunately have side effects, which have not specifically been studied in endometriosis patients. Before NK cell treatment can be used in the clinic, more research is needed.

CD34+ progenitor-derived NK cell and gemcitabine combination therapy increases killing of ovarian cancer cells in NOD/SCID/IL2Rgnull mice.

Combining natural killer (NK) cell adoptive transfer with tumor-sensitizing chemotherapy is an attractive approach against recurrent ovarian cancer (OC), as OC is sensitive to NK cell-mediated immunity. Previously, we showed that CD34+ hematopoietic progenitor cell (HPC)-derived NK cells can kill OC cells in vitro and inhibit OC tumor growth in mice. Here, we investigated the potential of HPC-NK cell therapy combined with chemotherapeutic gemcitabine (used in recurrent OC patients) against OC. We examined the phenotypical, functional, and cytotoxic effects of gemcitabine on HPC-NK cells and/or OC cells in vitro and in OC-bearing mice. To this end, we treated OC cells and/or HPC-NK cells with or without gemcitabine and analyzed the phenotype, cytokine production, and anti-tumor reactivity. We found that gemcitabine did not affect the phenotype and functionality of HPC-NK cells, while on OC cells expression of NK cell activating ligands and death receptors was upregulated. Although gemcitabine pre-treatment of OC cells did not improve the functionality of HPC-NK cells, importantly, HPC-NK cells and gemcitabine additively killed OC cells in vitro. Similarly, combined HPC-NK cell and gemcitabine treatment additively decreased tumor growth in OC-bearing mice. Collectively, our results indicate that combination therapy of HPC-NK cells and gemcitabine results in augmented OC killing in vitro and in vivo. This provides a rationale for exploring this therapeutic strategy in patients with recurrent OC.

TIGIT blockade enhances functionality of peritoneal NK cells with altered expression of DNAM-1/TIGIT/CD96 checkpoint molecules in ovarian cancer.

Advanced ovarian cancer (OC) patients have a poor 5-year survival of only 28%, emphasizing the medical need for improved therapies. Adjuvant immunotherapy could be an attractive approach since OC is an immunogenic disease and the presence of tumor-infiltrating lymphocytes has shown to positively correlate with patient survival. Among these infiltrating lymphocytes are natural killer (NK) cells, key players involved in tumor targeting, initiated by signaling via activating and inhibitory receptors. Here, we investigated the role of the DNAM-1/TIGIT/CD96 axis in the anti-tumor response of NK cells toward OC. Ascites-derived NK cells from advanced OC patients showed lower expression of activating receptor DNAM-1 compared to healthy donor peripheral blood NK cells, while inhibitory receptor TIGIT and CD96 expression was equal or higher, respectively. This shift to a more inhibitory phenotype could also be induced in vitro by co-culturing healthy donor NK cells with OC tumor spheroids, and in vivo on intraperitoneally infused NK cells in SKOV-3 OC bearing NOD/SCID-IL2Rγnull (NSG) mice. Interestingly, TIGIT blockade enhanced degranulation and interferon gamma (IFNγ) production of healthy donor CD56dim NK cells in response to OC tumor cells, especially when DNAM-1/CD155 interactions were in place. Importantly, TIGIT blockade boosted functional responsiveness of CD56dim NK cells of OC patients with a baseline reactivity against SKOV-3 cells. Overall, our data show for the first time that checkpoint molecules TIGIT/DNAM-1/CD96 play an important role in NK cell responsiveness against OC, and provides rationale for incorporating TIGIT interference in NK cell-based immunotherapy in OC patients.

Harnessing natural killer cells for the treatment of ovarian cancer.

INTRODUCTION: Adoptive cellular immunotherapy could be an interesting new treatment option for ovarian carcinoma (OC), as research has demonstrated that OC is an immunogenic disease. In particular, natural killer (NK) cells have attracted attention due to their ability to kill tumor cells without prior sensitization. The therapeutic value of allogeneic NK cells has been first observed in hematological cancers and is increasingly being explored in solid tumors. METHODS: To substantiate the rationale for NK cell therapy in OC we performed a literature search in the Pubmed database and in the international trial register clinicaltrials.gov with attention for the effect of OC on NK cell function, the effect of current treatment on NK cell biology and the evidence on the therapeutic value of NK cell therapy against OC. RESULTS: In six clinical trials only 31 OC patients have been reported that received NK cell adoptive transfer. The majority of patients reached stable disease after NK cell therapy, with a mild pattern of side effects. In patients who received repeated infusions, more complete responses are described. All reported studies investigated the intravenous infusion of NK cells. Whereas the studies that are currently recruiting, investigate intraperitoneal infusion of allogeneic NK cells. CONCLUSION: In this review the pre-clinical evidence and current trials on NK cell immunotherapy in OC patients are summarized. Furthermore, challenges that have to be overcome for NK cell adoptive therapy to have a significant impact on disease outcome are discussed.

Intraperitoneal infusion of ex vivo-cultured allogeneic NK cells in recurrent ovarian carcinoma patients (a phase I study).

INTRODUCTION: Recurrent ovarian carcinoma has dismal prognosis, but control of disease and prolonged survival are possible in some patients. The estimated 5-year survival is 46% for all stages of ovarian cancer, and only 28% for metastasized disease. Notably, the majority of women with ovarian cancer are diagnosed with stage III or IV disease with a high recurrence rate. As most women with relapsed or metastatic cancer will die of progressive disease, there is an urgent need for novel therapeutic strategies. The primary aim of our study is to evaluate safety and toxicity of intraperitoneal infusion of ex vivo-expanded natural killer cells (NK), generated from CD34+ umbilical cord blood (UCB) progenitor cells, with and without a preceding non-myeloablative immunosuppressive conditioning regimen in patients suffering from recurrent ovarian cancer. The secondary objectives are to compare the in vivo lifespan, expansion, and biological activity of intraperitoneally infused NK cell products with or without preparative chemotherapy, as well as evaluate effects on disease load. METHODS: In this phase I safety trial, 12 patients who are suffering from recurrent ovarian cancer, detected by a significant rise in serum level of CA-125 on two successive time points, will be included. Prior to UCB-NK cell infusion, a laparoscopy is performed to place a catheter in the peritoneal cavity. The first cohort of three patients will receive a single intraperitoneal infusion of 1.5-3×10 UCB-NK cells, generated ex vivo from CD34+ hematopoietic progenitor cells obtained from an allogeneic UCB unit, without a preparative chemotherapy regimen. The second group of three patients will be treated with a similar dose of UCB-NK cells following a preparative four days non-myeloablative immunosuppressive conditioning regimen with cyclophosphamide and fludarabine (Cy/Flu). If no severe toxicity is seen in these 6 patients, an extension cohort of 6 patients will be included to answer the secondary objectives. DISCUSSION: This study investigates the safety of a promising new cellular therapy in a group of patients with a poor prognosis. Demonstration of safety and in vivo expansion capacity of allogeneic UCB-NK cells in the absence of Cy/Flu pretreatment will provide rationale for UCB-NK cell infusion after regular second-line chemotherapy.

Peritoneal NK cells are responsive to IL-15 and percentages are correlated with outcome in advanced ovarian cancer patients.

The demonstration that ovarian carcinoma (OC) is an immunogenic disease, opens opportunities to explore immunotherapeutic interventions to improve clinical outcome. In this regard, NK cell based immunotherapy could be promising as it has been demonstrated that OC cells are susceptible to killing by cytokine-stimulated NK cells. Here, we evaluated whether percentage, phenotype, function and IL-15 responsiveness of ascites-derived natural killer (NK) cells is related to progression-free survival (PFS) and overall survival (OS) of advanced stage OC patients. Generally, a lower percentage of NK cells within the lymphocyte fraction was seen in OC ascites (mean 17.4 ± 2.7%) versus benign peritoneal fluids (48.1 ± 6.8%; p < 0.0001). Importantly, a higher CD56+ NK cell percentage in ascites was associated with a better PFS (p = 0.01) and OS (p = 0.002) in OC patients. Furthermore, the functionality of ascites-derived NK cells in terms of CD107a/IFN-γ activity was comparable to that of healthy donor peripheral blood NK cells, and stimulation with monomeric IL-15 or IL-15 superagonist ALT-803 potently improved their reactivity towards tumor cells. By showing that a higher NK cell percentage is related to better outcome in OC patients and NK cell functionality can be boosted by IL-15 receptor stimulation, a part of NK cell immunity in OC is further deciphered to exploit NK cell based immunotherapy.

The effect of subcellular localization on the efficiency of EGFR-targeted VHH photosensitizer conjugates.

Photodynamic therapy (PDT) is an emerging method to treat light-accessible malignancies. To increase specificity and allow dose reduction, conjugates of photosensitizers (PS) with antibodies against tumor-associated antigens have been developed for photoimmunotherapy (PIT). However, so far it is unclear whether cellular internalization of these conjugates after binding affects PIT efficacy. The use of low molecular weight llama single domain antibodies (VHHs, nanobodies) for PIT is preferred above full size antibodies because of better tumor penetration. Therefore, we functionalized the VHH 7D12, directed against the epidermal growth factor receptor (EGFR), with a PS (IRDye700DX). To assess the impact of cellular internalization on activity, the VHHs were additionally conjugated to a cell-penetrating peptide (VHH[PS]-CPP). Here we show that upon illumination with near-infrared (NIR) light, both VHH[PS] and VHH[PS]-CPP conjugates specifically induce cell death of EGFR expressing cancer cell lines and of EGFR-expressing cells derived from surgically obtained ascites from patients with high-grade serous ovarian cancer. However, VHH[PS] conjugates were significantly more effective compared to internalizing VHH[PS]-CPP suggesting that cell surface association is required for optimal therapeutic activity.

Decitabine enhances targeting of AML cells by CD34+ progenitor-derived NK cells in NOD/SCID/IL2Rgnull mice.

Combining natural killer (NK) cell adoptive transfer with hypomethylating agents (HMAs) is an attractive therapeutic approach for patients with acute myeloid leukemia (AML). However, data regarding the impact of HMAs on NK cell functionality are mostly derived from in vitro studies with high nonclinical relevant drug concentrations. In the present study, we report a comparative study of azacitidine (AZA) and decitabine (DAC) in combination with allogeneic NK cells generated from CD34+ hematopoietic stem and progenitor cells (HSPC-NK cells) in in vitro and in vivo AML models. In vitro, low-dose HMAs did not impair viability of HSPC-NK cells. Furthermore, low-dose DAC preserved HSPC-NK killing, proliferation, and interferon gamma production capacity, whereas AZA diminished their proliferation and reactivity. Importantly, we showed HMAs and HSPC-NK cells could potently work together to target AML cell lines and patient AML blasts. In vivo, both agents exerted a significant delay in AML progression in NOD/SCID/IL2Rgnull mice, but the persistence of adoptively transferred HSPC-NK cells was not affected. Infused NK cells showed sustained expression of most activating receptors, upregulated NKp44 expression, and remarkable killer cell immunoglobulin-like receptor acquisition. Most importantly, only DAC potentiated HSPC-NK cell anti-leukemic activity in vivo. Besides upregulation of NKG2D- and DNAM-1-activating ligands on AML cells, DAC enhanced messenger RNA expression of inflammatory cytokines, perforin, and TRAIL by HSPC-NK cells. In addition, treatment resulted in increased numbers of HSPC-NK cells in the bone marrow compartment, suggesting that DAC could positively modulate NK cell activity, trafficking, and tumor targeting. These data provide a rationale to explore combination therapy of adoptive HSPC-NK cells and DAC in patients with AML.

Umbilical cord blood CD34+ progenitor-derived NK cells efficiently kill ovarian cancer spheroids and intraperitoneal tumors in NOD/SCID/IL2Rgnull mice.

Adoptive transfer of allogeneic natural killer (NK) cells is an attractive therapy approach against ovarian carcinoma. Here, we evaluated the potency of highly active NK cells derived from human CD34+ haematopoietic stem and progenitor cells (HSPC) to infiltrate and mediate killing of human ovarian cancer spheroids using an in vivo-like model system and mouse xenograft model. These CD56+Perforin+ HSPC-NK cells were generated under stroma-free conditions in the presence of StemRegenin-1, IL-15, and IL-12, and exerted efficient cytolytic activity and IFNγ production toward ovarian cancer monolayer cultures. Live-imaging confocal microscopy demonstrated that these HSPC-NK cells actively migrate, infiltrate, and mediate tumor cell killing in a three-dimensional multicellular ovarian cancer spheroid. Infiltration of up to 30% of total HSPC-NK cells within 8 h resulted in robust tumor spheroid destruction. Furthermore, intraperitoneal HSPC-NK cell infusions in NOD/SCID-IL2Rγnull (NSG) mice bearing ovarian carcinoma significantly reduced tumor progression. These findings demonstrate that highly functional HSPC-NK cells efficiently destruct ovarian carcinoma spheroids in vitro and kill intraperitoneal ovarian tumors in vivo, providing great promise for effective immunotherapy through intraperitoneal HSPC-NK cell adoptive transfer in ovarian carcinoma patients.

[Hemorrhage after Implanon replacement when using oral anticoagulants]. / Bloeding na Implanon-vervanging bij gebruik orale anticoagulantia.

INTRODUCTION: Implanon is a widely used contraceptive. It is a progestagen-containing implant which is inserted subcutaneously on the inside of the upper arm. This procedure is relatively simple. CASE DESCRIPTION: A 26-year-old woman had her contraceptive implant replaced in the outpatient clinic. Because of a mechanical heart valve, she used phenprocoumon, which she did not discontinue prior to the procedure. Three days after the Implanon replacement, the patient presented with persistent blood loss from the insertion site and a large hematoma around the new implant. The placement of a new pressure bandage was not sufficient. The concentration of Hb fell from 8.1 to 5.0 mmol/l with an INR of 3.5; therefore, the patient received a blood transfusion. The use of phenprocoumon was temporarily discontinued and the INR was corrected with prothrombin complex. Good hemostasis was ultimately achieved. CONCLUSION: Although the replacement of a subcutaneous hormone implant is a relatively simple procedure, a severe complication such as hemorrhaging may occur, particularly when there are risk factors.

The correlation between clinical and urodynamic diagnosis in classifying the type of urinary incontinence in women. A systematic review of the literature.

AIMS: To determine the reclassification rate of clinically diagnosed stress, mixed, and urge urinary incontinence after urodynamic investigation. METHODS: A systematic review of the published literature in MEDLINE and EMBASE of clinical trials among women with urinary incontinence. Studies were included in case the diagnosis based on symptoms and/or signs was compared with the diagnosis after urodynamic investigation. RESULTS: Twenty-three articles involving 6,282 women with urinary incontinence met the inclusion criteria. A clinical diagnosis of stress urinary incontinence was reclassified into mixed urinary incontinence in 9% of women and into detrusor overactivity (DO) in 7% of cases. The pooled reclassification rate was highest among patients with symptoms of mixed urinary incontinence, where 46% of the patients had stress urinary incontinence and 21% had DO on urodynamic investigation. The available literature does not allow the identification of the additional value of non-invasive test, such as stress test and voiding diary, accessory to symptoms. None of the studies had therapeutic effects as an outcome measure. CONCLUSIONS: This review of clinical studies shows that the level of agreement between classification based on clinical evaluation and based on urodynamic investigation is poor. Urodynamic observations are regarded as gold standard, but based on the poor correlation, this assumption should be questioned.