Deciphering molecular and cellular ex vivo responses to bispecific antibodies PD1-TIM3 and PD1-LAG3 in human tumors.

BACKGROUND: Next-generation cancer immunotherapies are designed to broaden the therapeutic repertoire by targeting new immune checkpoints including lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Yet, the molecular and cellular mechanisms by which either receptor functions to mediate its inhibitory effects are still poorly understood. Similarly, little is known on the differential effects of dual, compared with single, checkpoint inhibition. METHODS: We here performed in-depth characterization, including multicolor flow cytometry, single cell RNA sequencing and multiplex supernatant analysis, using tumor single cell suspensions from patients with cancer treated ex vivo with novel bispecific antibodies targeting programmed cell death protein 1 (PD-1) and TIM-3 (PD1-TIM3), PD-1 and LAG-3 (PD1-LAG3), or with anti-PD-1. RESULTS: We identified patient samples which were responsive to PD1-TIM3, PD1-LAG3 or anti-PD-1 using an in vitro approach, validated by the analysis of 659 soluble proteins and enrichment for an anti-PD-1 responder signature. We found increased abundance of an activated (HLA-DR+CD25+GranzymeB+) CD8+ T cell subset and of proliferating CD8+ T cells, in response to bispecific antibody or anti-PD-1 treatment. Bispecific antibodies, but not anti-PD-1, significantly increased the abundance of a proliferating natural killer cell subset, which exhibited enrichment for a tissue-residency signature. Key phenotypic and transcriptional changes occurred in a PD-1+CXCL13+CD4+ T cell subset, in response to all treatments, including increased interleukin-17 secretion and signaling toward plasma cells. Interestingly, LAG-3 protein upregulation was detected as a unique pharmacodynamic effect mediated by PD1-LAG3, but not by PD1-TIM3 or anti-PD-1. CONCLUSIONS: Our in vitro system reliably assessed responses to bispecific antibodies co-targeting PD-1 together with LAG-3 or TIM-3 using patients' tumor infiltrating immune cells and revealed transcriptional and phenotypic imprinting by bispecific antibody formats currently tested in early clinical trials.

Self-associated molecular patterns mediate cancer immune evasion by engaging Siglecs on T cells.

First-generation immune checkpoint inhibitors, including anti-CTLA-4 and anti-programmed death 1 (anti-PD-1) antibodies, have led to major clinical progress, yet resistance frequently leads to treatment failure. Thus, new targets acting on T cells are needed. CD33-related sialic acid-binding immunoglobulin-like lectins (Siglecs) are pattern-recognition immune receptors binding to a range of sialoglycan ligands, which appear to function as self-associated molecular patterns (SAMPs) that suppress autoimmune responses. Siglecs are expressed at very low levels on normal T cells, and these receptors were not until recently considered as interesting targets on T cells for cancer immunotherapy. Here, we show an upregulation of Siglecs, including Siglec-9, on tumor-infiltrating T cells from non-small cell lung cancer (NSCLC), colorectal, and ovarian cancer patients. Siglec-9-expressing T cells coexpressed several inhibitory receptors, including PD-1. Targeting of the sialoglycan-SAMP/Siglec pathway in vitro and in vivo resulted in increased anticancer immunity. T cell expression of Siglec-9 in NSCLC patients correlated with reduced survival, and Siglec-9 polymorphisms showed association with the risk of developing lung and colorectal cancer. Our data identify the sialoglycan-SAMP/Siglec pathway as a potential target for improving T cell activation for immunotherapy.

Ki67 and lymphocytes in the pretherapeutic core biopsy of primary invasive breast cancer: positive markers of therapy response prediction and superior survival.

Background Core needle biopsy plays a crucial role as diagnostic tool for BC. Both Ki67 and likely tumor-infiltrating lymphocytes (TILs) in the near future are determining the kind of systemic therapy. The role of TILs in BC is still an issue for clinical research, albeit preliminary results of neoadjuvant and adjuvant clinical studies already now highlight the crucial impact of TILs on therapy response and survival. Methods Evaluation of related publications (pubmed) and meeting abstracts (ASCO, SABCS). Results The monoclonal antibody Ki67 recognizing a nuclear antigene in proliferating cells is a positive marker of therapy response and superior survival. Endocrine responsive tumors of low proliferation (Ki67 < 14%/11%) respond to tamoxifen, in contrast postmenopausal tumors with higher proliferation respond better to aromatase-inhibitors. Pathological complete response (pCR)-rates increase in tumors with higher proliferation (Ki67 > 19%) vs. tumors with lower proliferation after neoadjuvant chemotherapy (NAC). pCR-rates of up to 60% can be seen in TNBC and HR-, HER2+BC, lower pCR-rates, however, in HR+, HER2- BC. Increased stromal TILs are found in 30% of TNBC and in 19% of HR-, HER2+BC. The percentage of TILs is a significant independent parameter for pCR after NAC. Lymphocyte-predominant BC (LPBC) respond with higher pCR-rates than non-LPBC or tumors without any TILs. Increased TILs in TN and HR-, HER2+ subtypes predict benefit from addition of carboplatin to NAC. TILs are also associated with improved DFS and OS among patients with TNBC and HR-, HER2+ BC. Conversly and interestingly increased TILs in patients with HR+, HER2-(luminal) BC are associated with a 10% higher risk of death per 10% increase of TILs. Interactions between immune system and cancer are complex. The cancer-immunity cycle characterizes these interactions. BC subtypes with higher number of mutations such as TNBC and HR-, HER2+BC are considered to provide a raising number of tumor-associated antigens, thereby capable to build up a higher endogenous immune response. TILs may serve as surrogate marker of both an existing endogenous immune response and the probability to respond to cancer immune therapies. As cancer co-opt immune checkpoint-pathways as a major mechanism of immune resistance, in particular, against cytotoxic T-cells, blockades of checkpoint-pathways by antibodies are one of the goals of the current cancer immunotherapy studies. Therapy studies with antigene-based strategies (vaccines) and antibodies against the immune checkpoints PD-1 and CTLA-4 and their inhibitory pathways in order to enhance cytotoxic T-cell activities against cancer cells with or without chemotherapy are underway. Conclusions It can be suggested that the use of multigene expression testing will increase in order to select more clearly primary HR+, HER2- BC patients with intermediate recurrence risk who likely may benefit from chemotherapy. Furthermore Ki67 and the multigene expression test Oncotype DX can act as dynamic markers to avoid cytostatic overtreatment and endocrine undertreatment. A data-derived optimal Ki67 cut point for pCR and DFS as well as OS is currently not feasible. The integration of stromal TILs into the immunohisto-pathological report after their evaluation has been standardized is likely helpful to determine patients who profit by additional carboplatin chemotherapy. Oncologists need an enlarged information about the tumor-microenvironment in future. The preliminary results of current BC immunotherapy studies are encouraging.

Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations.

BACKGROUND: The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations. METHODS: Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively). RESULTS: There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM. CONCLUSION: Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.

Secretion of fibrinolytic enzymes facilitates human mesenchymal stem cell invasion into fibrin clots.

Adult human mesenchymal stem cells (hMSC) are involved in wound healing and regeneration of mesodermal tissue, but the underlying homing mechanisms are not well understood. Fibrin clot formation is associated with most wound healing processes and potentially guides the recruitment of hMSC. The objective of this study is the investigation of a fibrinolytic capacity, which is required for hMSC to migrate into a wounded tissue and thus to contribute to tissue regeneration. Using RT-PCR, semiquantitative real-time PCR and ELISA, we detected key components of the fibrinolytic cascade, including the urokinase plasminogen activator (uPA) and its receptor (uPAR), the tissue plasminogen activator (tPA) and the plasminogen activator inhibitor (PAI), suggesting a strong fibrinolytic activity of hMSC. To test this activity in a functional assay, we cultured fibrin-embedded hMSC in vitro for 7 days. The cells efficiently dissolved the surrounding fibrin mesh into the fibrin degradation products, the fibrinopeptides. The fibrinolytic activity of hMSC and human dermal fibroblasts, known to be critically involved in skin wound extracellular matrix remodeling, was similar. Our results suggest that a high intrinsic fibrinolytic capacity of hMSC mediates the invasion into a fibrin clot of a wounded tissue.

Erectile function after non-nerve-sparing radical prostatectomy: fact or fiction?

OBJECTIVE: To evaluate the potency in patients after radical perineal prostatectomy with wide excision of both neurovascular bundles. MATERIAL AND METHODS: In this prospective study, a quality-of-life questionnaire was completed by 128 patients at the preoperative stage, and 6 and 12 months postoperatively. Ten questions concerning the patient's sexuality were included on the pre- and both postoperative questionnaires. In addition, 6 patients who recorded some erectile function were sent a separate questionnaire containing eight more detailed questions. RESULTS: Preoperatively, 74/128 (57.8%) patients reported erections sufficient for sexual intercourse, and of these 74, 6 (8.1%) described having spontaneous erections 1 year postoperatively. These spontaneous erections occurred 1-5 times per week. The reply to the separate mailing made clear that the reported erections were insufficient for intercourse. CONCLUSIONS: Patients undergoing standardized radical prostatectomy with wide excision of the neurovascular bundle have a very small chance of spontaneous erections sufficient for intercourse postoperatively.

Inhibition of gastric cancer cell adhesion in nude mice by inraperitoneal phospholipids.

Adhesion of tumor cells to mesothelial cells or extracellular matrix components is a pivotal step in developing peritoneal dissemination after gastric cancer. As phospholipids were found to reduce adhesion formation, especially at sites of peritoneal lesions, we assessed the inhibition of attachment of NUGC-4 gastric cancer cells by local treatment with phospholipids to the peritoneum in nude mice. Gastric cancer cells (1xl0(6)) suspended in either normal saline (controls) or phospholipid suspension 75 mg/kg body weight (PL75) or 150 mg/kg (PL150) were injected intraperitoneally into 90 female BALB/c nu/nu mice. The treatment groups were subdivided into animals with defined peritoneal lesions and animals without lesions. After 30 days the extent of peritoneal carcinosis and the Peritoneal Cancer Index were evaluated. Statistical analysis was performed with two factorial ANOVAs. The level of significance was adjusted according to Bonferrorni (alpha = 0.00278). During a 90-day observation period the survival rate was determined using the log rank test. After 30 days the intraperitoneal tumor volume was reduced by PL150 up to 0.6 ml (SEM 0.16) and 0.48 ml (SEM 0.09) in mice with peritoneal lesions compared to 0.9 ml (SEM 0.2) and 0.9 ml (SEM 0.1) in the control group (P = 0.04). The mean area of tumor adhesion amounted to 145 mm(2) (SEM 17) (P = 0.08) and 164 mm(2) (SEM 32.8) (P = 0.049) with peritoneal lesions after treatment with PL150 [controls: 216 mm(2) (SEM 28.5) and 245 mm(2) (SEM 29.3)]. The peritoneal cancer index was 16.4 (SEM 1.7) in the control group and 9 (SEM 1.68) with PL150 (P = 0.0002). In the subgroup with peritoneal lesions, the respective values were as follows: controls: 20.8 (SEM 0.85); PL 150:14.3 (SEM 1.07) (P = 0.0001). We found a prolonged survival rate after treatment with PL150. However, this effect was not significantly different to that seen in the control group. Treatment with PL75 had no significant influence. Phospholipids may be an efficacious and economic tool for reducing peritoneal tumor cell adhesion and consequently the development of peritoneal carcinosis after resection of gastric cancer.

Permanent 125I-seed brachytherapy or radical prostatectomy: a prospective comparison considering oncological and quality of life results.

OBJECTIVE: To assess the quality of life in patients with prostate cancer after permanent brachytherapy (BT) or radical perineal prostatectomy (RP). PATIENTS AND METHODS: The American Brachytherapy Society recommends the permanent implantation of radioactive seeds as a monotherapy for patients with T1-T2aN0M0 prostate cancer and a prostate-specific antigen (PSA) level of < or = 10 ng/mL, a Gleason score of <7 and a prostate volume of <60 mL. Using these criteria, 132 patients with low-risk prostate cancer were selected; 52 had BT with 125I-seed implantation, 38 had RP with unilateral nerve-sparing (RP + NS) and 42 extended RP (RP group). Only patients with unilateral tumour on biopsy were considered. Before therapy and 6, 12 and 24 months afterward, patients completed questionnaires to assess perceived health and function. PSA relapse was diagnosed with a PSA of >0.1 ng/mL for patients in the RP groups, and three consecutive PSA increases for those after BT. RESULTS: Extraprostatic tumours were found in 18% of specimens taken during RP, and bilateral tumours in 63% of patients. After a mean follow-up of 27 months, there was PSA relapse in two of the 80 patients in the RP and RP + NS groups, and six of the 52 patients in the BT group; a significant difference, with a hazard ratio of 5.2. The acute morbidity was low in all groups. At 1 year, more than two incontinence pads were used by 5% of patients after RP and by 4% after BT. Similarly, at 1 year 15% of patients after RP and 13% after BT were bothered by urinary incontinence. Newly-developed fecal soiling was reported by 4%, 5% and 11% of the RP, RP + NS and BT groups respectively; none of the patients after RP and 4% after BT were bothered by this symptom. The duration and stiffness of erection was assessed after 1 year and reported to be equal or slightly decreased by a third after RP + NS and 38% after BT. Taking a 5-10 point difference as clinically relevant, role, emotional and social functioning were improved considerably after RP + NS than after BT, but sexual activity was impaired significantly after RP + NS than after BT. CONCLUSIONS: Both therapies showed typical acute and late morbidity; the most bothersome late symptoms were urinary incontinence for patients after RP and fecal soiling after BT. Sexual function was impaired significantly in patients who were potent before RP + NS, whereas after BT men reported only a minor change in sexual performance at 1 year. Tumour control after a median follow-up of 27 months was better after RP but biochemical recurrence may still occur after > or = 5 years; therefore the present results are not mature enough and there were too few patients to provide a more definitive statement. As approximately 18% of patients considered to be appropriate candidates for BT had tumours extending beyond the prostate capsule or invading the seminal vesicles, nomograms are needed for more accurate information before therapy.

Phospholipids reduce the intraperitoneal adhesion of colonic tumor cells in rats and adhesion on extracellular matrix in vitro.

OBJECTIVE: To evaluate the influence of intraperitoneal treatment with phospholipids on the formation of peritoneal carcinosis after inoculation of colonic tumor cells in rats. The presence of tumor cells in the peritoneal cavity serves as a prognostic marker for postoperative survival after resection of gastrointestinal cancer. Intraperitoneal tumor cell attachment is a pivotal step in developing peritoneal carcinosis. Intraabdominal application of phospholipids resulted in a significant decrease of adhesion formation, especially at sites of peritoneal lesions. METHODS: 2x10(6) colonic tumor cells (DHD/K12/TRb) were injected intraperitonely in female BD-IX rats. A total of 90 rats were divided into three groups with treatments of phospholipids at 75 mg/kg or 150 mg/kg bodyweight or sodium chloride at 0.9% in the control group. The treatment groups were subdivided into animals with defined peritoneal lesions and animals without lesions. After 30 days, the extent of peritoneal carcinosis was determined by measuring the tumor volume, the area of tumor attachment and the Peritoneal Cancer Index. Over a 90-day observation period, the survival rate was analyzed. In vitro, we examined the reduction of tumor cell adhesion on extracellular matrix components after treatment with phospholipids. Microtiter plates were coated with laminin, fibronectin or collagen IV for adhesion experiments. RESULTS: In our study, we found a significant reduction of peritoneal dissemination with respect to all evaluation methods after treatment with phospholipids at 150 mg/kg in animals without peritoneal lesions. This could not be achieved using the lower concentration of phospholipids (75 mg/kg). In vitro, the maximum reductions of tumor cell adhesion by phospholipids compared with the control values for laminin and fibronectin were 46% and 37%, respectively, whereas for collagen IV the reduction was only 24% ( p<0.0001). CONCLUSIONS: A new method of prevention of intraperitoneal tumor cell adhesion, possibly leading to a reduced incidence of peritoneal carcinosis after surgery of gastrointestinal tumors, is introduced.

Functional expression of HGF and HGF receptor/c-met in adult human mesenchymal stem cells suggests a role in cell mobilization, tissue repair, and wound healing.

Human mesenchymal stem cells (hMSC) are adult stem cells with multipotent capacities. The ability of mesenchymal stem cells to differentiate into many cell types, as well as their high ex vivo expansion potential, makes these cells an attractive therapeutic tool for cell transplantation and tissue engineering. hMSC are thought to contribute to tissue regeneration, but the signals governing their mobilization, diapedesis into the bloodstream, and migration into the target tissue are largely unknown. Here we report that hepatocyte growth factor (HGF) and the cognate receptor HGFR/c-met are expressed in hMSC, on both the RNA and the protein levels. The expression of HGF was downregulated by transforming growth factor beta. HGF stimulated chemotactic migration but not proliferation of hMSC. Therefore the HGF/c-met signaling system may have an important role in hMSC recruitment sites of tissue regeneration. The controlled regulation of HGF/c-met expression may be beneficial in tissue engineering and cell therapy employing hMSC.

Effect of intraperitoneal antiadhesive fluids in a rat peritonitis model.

HYPOTHESIS: Phospholipids and icodextrin reduce peritoneal adhesions resulting from general peritonitis without promoting abscess formation. DESIGN: Evaluation of adhesion reduction fluids in a randomized animal study using a standardized peritonitis model. SETTING: Experimental animal model in a university laboratory. INTERVENTIONS: In 60 rats, experimental peritonitis was induced using the cecal ligation and puncture model. On day 1, the abdominal cavity was rinsed with 10 mL of isotonic sodium chloride solution and the cecum was resected. Animals were randomly assigned to 3 groups: the RL group, which received Ringer lactate intraperitoneally; the PL group, which received phospholipids intraperitoneally; and the ID group, which received icodextrin intraperitoneally. In each group, 50% of the animals were humanely killed at day 11 and 50% at day 21. MAIN OUTCOME MEASURES: The areas of adhesions were measured and the abscess formation was scored according to location and size. Abscesses, abdominal fluid, and blood were sampled for microbiologic workup. RESULTS: The median area of adhesions was significantly lower in the PL groups (PL(11), 43.7 mm(2); PL(21), 20.4 mm( 2)) than in the RL groups (RL(11), 163.8 mm(2); RL( 21), 120.9 mm(2)) and ID groups (ID(11), 418.5 mm( 2); ID(21), 218.6 mm(2)). Abscess formation was increased by icodextrin but not influenced by phospholipids, whereas microbiologic investigations did not reveal any differences among these 3 groups. CONCLUSIONS: In this model of general peritonitis, phospholipids significantly reduced adhesion formation without promoting septic complications. Icodextrin enhanced adhesion and abscess formation in this peritonitis model. Phospholipids may be beneficial for adhesion control in general peritonitis.

Influence of intraperitoneal therapy with mitomycin C adsorbed on activated carbon on anastomotic and wound healing in rats.

In an effort to prevent intraperitoneal dissemination of gastric carcinoma, local chemotherapy with mitomycin C adsorbed to activated carbon (MMC-CH) has been implemented. Results of clinical studies showed improved survival and a reduced systemic toxicity after the use of prophylactic treatment with MMC-CH. A significantly higher rate of intraperitoneal septic complications following MMC-CH therapy was found. The aim of this study was to assess whether intraperitoneal MMC-CH affects wound healing or healing of intestinal anastomoses. Standardized laparotomy was performed in 77 rats. The examinations were performed in 27 animals in the control group, 24 animals in the charcoal group, and 26 animals in the MMC-CH group. The animals and groups were distributed randomly. After an ileal anastomosis was performed, MMC-CH, charcoal, or sodium chloride 0.9% was administered intraperitoneally. After 10 days, collagen content as well as bursting strength/pressure of the fasciotomy and the anastomotic site was examined. Body weight and blood parameters analyzed included hemoglobin level, white blood cell count, platelet count, and total protein. Concerning body weight and hematology, no significant changes were observed. Three of 26 animals in the MMC-CH group, 2/24 in the charcoal group and 1/27 in the control group developed an anastomotic leakage. The bursting pressure of the anastomoses and the bursting strength of the fasciotomy as well as the relative collagen content did not differ significantly after treatment with charcoal or mitomycin C compared to the control group. Local inflammation consisting of charcoal-laden granulomas was detected histologically in the MMC-CH group and to a lesser extent in the charcoal group. In conclusion, no significant influence of intraperitoneal mitomycin C adsorbed on activated charcoal, in terms of its effect systemically or its effect on wound healing, could be demonstrated as a result of slow release. Histological changes seen with the use of activated charcoal suggest that perhaps a more ideal absorbable carrier should be sought.

Influence of early drainage of intraperitoneal phospholipids on efficacy of adhesion prevention.

Postoperative peritoneal adhesions impose a long-term risk of morbidity and mortality. Adjunctive means are needed to prevent these complications. In previous studies we could demonstrate the efficacy and safety of intraperitoneally applied phospholipids with regard to adhesion prevention and wound healing, respectively. The assumption is that phospholipids rapidly adhere to the peritoneal surface and to the mesothelial lesions. This study was designed to evaluate the influence of early drainage of the administered fluid volume on the control of adhesion formation. Forty chinchilla rabbits underwent median laparotomy and standardized abrasion of circumscript areas of the ventral abdominal wall, the cecum, and the ileum. The animals randomly received either 75 mg/kg body weight of phospholipids in a volume of 5.0 mL/kg body weight (n = 20) or the same volume of Ringer's lactate solution (n = 20) prior to closing the laparotomy wounds. In 50% of the rabbits with either medication, 80% of the volume was recovered after 30 min before final closure of the abdominal wall ("drainage"). In the remaining animals the intraabdominal fluid load was not evacuated ("no drainage"). At day 10 after surgery all rabbits were sacrificed for evaluation of adhesion areas by computer-aided planimetry and histopathologic examination. The mean areas of adhesion in both Ringer's lactate groups were significantly larger than in the comparable phospholipid groups (p < .05). In the Ringer's lactate groups, adhesions averaged 341.7 (318.6) mm2 without and 263.3 (275.5) mm2 with drainage. In the phospholipid groups the respective mean areas reached only 24.6 (36.7) mm2 without drainage and 27.0 (49.7) mm2 following evacuation of the fluid 30 min after administration (median, mean in parentheses). These results prove the efficacy of phospholipids after a limited contact period of 30 min. The frequent use of drains in abdominal surgery will not impair the beneficial effect of phospholipids on prevention of adhesions.