Karin Pfister

Treatment of Colon and Lung Cancer Patients with ex Vivo Heat Shock Protein 70-Peptide-Activated, Autologous Natural Killer Cells A Clinical Phase I Trial

Purpose: The 14 amino acid sequence (aa450–463) TKDNNLLGRFELSG (TKD) of heat shock protein 70 (Hsp70) was identified as a tumor-selective recognition structure for natural killer (NK) cells. Incubation of peripheral blood lymphocyte cells with TKD plus low-dose interleukin 2 (IL-2) enhances the cytolytic activity of NK cells against Hsp70 membrane-positive tumors, in vitro and in vivo. These data encouraged us to test tolerability, feasibility, and safety of TKD-activated NK cells in a clinical Phase I trial. Experimental Design: Patients with metastatic colorectal cancer (n = 11) and non-small cell lung cancer (n = 1) who had failed standard therapies were enrolled. After ex vivo stimulation of autologous peripheral blood lymphocytes with Hsp70-peptide TKD (2 μg/ml) plus low-dose IL-2 (100 units/ml), TKD was removed by extensive washing, and activated cells were reinfused i.v. The procedure was repeated for up to six cycles, applying a dose escalation schedule in 4 patients. Results: The percentage of activated NK cells in the reinfused leukapheresis products ranged between 8 and 20% of total lymphocytes, corresponding to total NK cell counts of 0.1 up to 1.5 × 109. Apart from restless feeling in 1 patient and itching in 2 patients, no negative side effects were observed. Concomitant with an enhanced CD94 cell surface density, the cytolytic activity of NK cells against Hsp70 membrane-positive colon carcinoma cells was enhanced after TKD/IL-2 stimulation in 10 of 12 patients. Concerning tumor response, 1 patient was in stable disease during therapy by formal staging criteria and another patient showed stable disease in one metastases and progression in another. Conclusions: Reinfusion of Hsp70-activated autologous NK cells is safe. Immunological results warrant additional studies in patients with lower tumor burden.

A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity

Abstract Compared with normal cells, tumor cell lines exhibit an unusual plasma membrane localization of heat shock protein 70 (Hsp70). This tumor-selective Hsp70 membrane expression has been found to correlate with an increased sensitivity to lysis mediated by human natural killer (NK) cells that transiently adhere to plastic following cytokine stimulation. A human Hsp70-specific monoclonal antibody (mAb) detects membrane-bound Hsp70 on viable tumor cells and blocks the immune response of NK cells against Hsp70-expressing tumor cells. By peptide scanning (pepscan) analysis, the epitope of this mAb was mapped as the C-terminal–localized 8-mer NLLGRFEL (NLL, amino acids [aa] 454–461). Most interestingly, similar to full-length Hsp70 protein, the N-terminal–extended 14-mer peptide TKDNNLLGRFELSG (TKD, aa 450–463) was able to stimulate the cytolytic and proliferative activity of NK cells at concentrations equivalent to full-length Hsp70 protein. Blocking studies revealed that an excess of the 14-mer peptide TKDNNLLGRFELSG inhibits the cytolytic activity of NK cells similar to that of Hsp70 protein. In comparison, other TKD-related peptides, including the 8-mer antibody epitope NLLGRFEL (aa 454–461), the 12-mer TKDNNLLGRFEL (aa 450–461), the 13-mer C-terminal–extended peptide NLLGRFELSGIPP (aa 454–466), the 14-mer TKD-equivalent sequences of Hsp70hom TKDNNLLGRFELTG (aa 450–463), Hsc70 TKDNNLLGKFELTG (aa 450–463), and DnaK AADNKSLGQFNLDG (aa 447–460) failed to activate NK activity.

Hsp70 plasma membrane expression on primary tumor biopsy material and bone marrow of leukemic patients

Abstract A tumor-selective cell surface localization of heat shock protein 70 (Hsp70), the major heat-inducible member of the Hsp70 group, correlates with an increased sensitivity to lysis mediated by human natural killer (NK) cells and, therefore, might be of clinical relevance. With the exception of mammary carcinomas, an Hsp70 plasma membrane expression was found on freshly isolated human biopsy material of colorectal, lung, neuronal, and pancreas carcinomas, liver metastases, and leukemic blasts of patients with acute myelogenous leukemia. Since normal tissues and bone marrow of healthy human individuals do not express Hsp70 on the cell surface, Hsp70 can be considered as a tumor-selective structure in vivo. Furthermore, we demonstrate that autologous, Hsp70-positive leukemic blasts can be killed by NK cells stimulated with low doses of interleukin 2 plus recombinant Hsp70 protein.

Alert for increased long-term follow-up after carotid artery stenting: results of a prospective, randomized, single-center trial of carotid artery stenting vs carotid endarterectomy.

BACKGROUND Carotid endarterectomy (CEA) has been shown to be effective in stroke prevention for patients with symptomatic or asymptomatic carotid artery stenosis. Although several prospective randomized trials indicate that carotid artery stenting (CAS) is an alternative but not superior treatment modality, there is still a significant lack of long-term data comparing CAS with CEA. This study presents long-term results of a prospective, randomized, single-center trial. METHODS Between August 1999 and April 2002, 87 patients with a symptomatic high-grade internal carotid artery stenosis (>70%) were randomized to CAS or CEA. After a median observation time of 66 +/- 14.2 months (CAS) and 64 +/- 12.1 months (CEA), 42 patients in each group were re-evaluated retrospectively by clinical examination and documentation of neurologic events. Duplex ultrasound imaging was performed in 61 patients (32 CAS, 29 CEA), and patients with restenosis >70% were re-evaluated by angiography. RESULTS During the observation period, 23 patients (25.2%) died (10 CAS, 13 CEA), and three were lost to follow up. The incidence of strokes was higher after CAS, with four strokes in 42 CAS patients vs none in 42 CEA patients. One transient ischemic attack occurred in each group. A significantly higher rate of restenosis >70% (6 of 32 vs 0 of 29) occurred after CAS compared with CEA. Five of 32 CAS patients (15.6%) presented with high-grade (>70%) restenosis as an indication for secondary intervention or surgical stent removal, and three presented with neurologic symptoms. No CEA patients required reintervention (P < .05 vs CAS). A medium-grade (<70%) restenosis was detected in eight of 32 CAS patients (25%) and in one of 29 CEA patients (3.4%). In five of 32 CAS (15.6%) and three of 29 CEA patients (10.3%), a high-grade stenosis of the contralateral carotid artery was observed and treated during the observation period. CONCLUSION The long-term results of this prospective, randomized, single-center study revealed a high incidence of relevant restenosis and neurologic symptoms after CAS. CEA seems to be superior to CAS concerning the development of restenosis and significant prevention of stroke. However, the long-term results of the ongoing multicenter trials have to be awaited for a final conclusion.

Differential up-regulation of cytosolic and membrane-bound heat shock protein 70 in tumor cells by anti-inflammatory drugs.

Purpose: Modulation of the heat shock protein (HSP) response affects sensitivity to therapeutic agents in cancer. Here, drugs with anti-inflammatory potential (cyclooxygenase 1/2 inhibitors) and peroxidase proliferator-activated receptor-γ agonists were analyzed for their capacity to affect Hsp70 expression in human cancer cells with a divergent Hsp70 membrane expression pattern. Experimental Design: In dose kinetics, the nonlethal concentration of acetyl-salicyl acid, celecoxib, rofecoxib, and the insulin-sensitizer pioglitazone was identified for the human adenocarcinoma cell line CX−. With the exception of CLX, which was diluted in DMSO, all reagents were dissolved in water. After treatment with the different compounds at nontoxic concentrations for 6 h, followed by a 1-h recovery period, the cytosolic Hsp70 levels were measured in CX-2 and CX− tumor cells by Western blot analysis. Fold increase was calculated in relation to the housekeeping protein tubulin. Membrane-bound Hsp70 was analyzed by flow cytometry using a FITC-labeled Hsp70-specific monoclonal antibody. Untreated cells and cells incubated with equivalent amounts of the diluting agents served as controls. The immunological function was tested in granzyme B apoptosis assays, standard 51Cr release assays, and antibody blocking studies. Results: Compared with aqua dest, the cytoplasmic amount of Hsp70 was equally enhanced in CX-2 and CX− cells by all compounds. An increase in membrane-bound Hsp70, detected selectively in CX− cells, corresponded to an enhanced sensitivity to granzyme B- and natural killer cell-mediated kill that was blockable by using a Hsp70-specific antibody. Conclusions: Although increase in cytosolic Hsp70 levels conferred resistance to further stress, membrane-bound Hsp70 rendered tumor cells more sensitive to the immunological attack mediated by granzyme B and natural killer cells. Our data provide a biological rational for combining anti-inflammatory drugs with immunotherapy in cancer therapy.

Patient survival by Hsp70 membrane phenotype: association with different routes of metastasis.

Heat shock proteins (HSPs) play important roles in tumor immunity. The authors prospectively investigated the correlation between the tumor‐specific Hsp70 membrane expression as an independent clinicopathological marker and overall survival in tumor entities that differ in their route of metastasis.

Effects of antineoplastic agents on cytoplasmic and membrane-bound heat shock protein 70 (Hsp70) levels.

Abstract Here we report on the study of the effects of different antineoplastic agents, including cytarabine, 4-hydroperoxyifosfamide, the activated form of ifosfamide, vincristine, and paclitaxel, with regard to their capacity to modulate the amount of cytoplasmic and membranebound heat shock protein 70 (Hsp70). Hsp70 levels were measured in the myelogenous leukemic cell line K562, in the human colon carcinoma cell line CX2, and in peripheral blood lymphocytes (PBL) under physiological conditions (37C), and following nonlethal heat shock at 41.8C. A concentration of 1 M and an incubation period of 2 h were determined as nonlethal, since none of the different antineoplastic agents induced necrosis or apoptosis in untreated or heatshocked cells under these conditions. Our results show that tubulininteracting agents, including vincristine and paclitaxel, but not DNAinteracting agents, including cytarabine and ifosfamide, selectively increase the amount of cytoplasmic Hsp70 in tumor and normal cells, as measured by semiquantitative Western blot analysis. Mechanistically, a vincristine and paclitaxelinduced tubulin assembly, as demonstrated by immunofluorescence microscopy, might be responsible for the elevated cytoplasmic Hsp70 levels. Interestingly, an increased membrane expression of Hsp70 following treatment with vincristine or paclitaxel was selectively observed on tumor cells, but not on normal cells.

Heat shock protein 70 (Hsp70) membrane expression on head-and-neck cancer biopsy—a target for natural killer (NK) cells☆

PURPOSE Heat shock protein 70 (Hsp70) was detected on the cell membrane of human tumor cell lines, but not on normal cells. Here we studied Hsp70 membrane expression as a target for natural killer (NK) cells on tumor material and control tissues of head-and-neck cancer patients. METHODS AND MATERIALS Membrane-bound Hsp70 was determined by flow cytometry on single-cell suspensions of tumors and the corresponding normal tissues of head-and-neck cancer patients. The cytolytic activity of NK cells against Hsp70-positive tumor cells was measured in a standard cytotoxicity assay. RESULTS In total, 54 of 74 primary tumors were found to be Hsp70 membrane-positive (73%); tongue/mouth, 21 of 24 (88%); oropharynx, 13 of 20 (65%); hypopharynx, 3 of 6 (50%); larynx, 8 of 11 (73%); trachea 1 of 2 (50%); esophagus, 4 of 5 (80%); lymph node metastases, 4 of 6 (67%). The corresponding control tissue was negative for membrane-bound Hsp70. Biopsies (6 of 6) of patients after in vivo gamma-irradiation (fractionated 5 x 2 Gy) were strongly Hsp70 membrane-positive. Irradiated, Hsp70-positive tumor cells are targets for Hsp70-peptide stimulated NK cells. CONCLUSION An irradiation-inducible, tumor-selective Hsp70 membrane localization provides a target structure for Hsp70-peptide stimulated human NK cells.

Editor's Choice – Temporary Aneurysm Sac Perfusion as an Adjunct for Prevention of Spinal Cord Ischemia After Branched Endovascular Repair of Thoracoabdominal Aneurysms

OBJECTIVE To report experience with the concept of temporary aneurysm sac perfusion (TASP) and second stage side branch completion to prevent severe spinal cord ischemia (SCI) after branched endovascular aortic repair (bEVAR) for thoracoabdominal aortic aneurysm (TAAA). METHODS Patients were treated for TAAA with bEVAR between January 2009 and September 2012. TASP was performed by non-completion of side branches to one of the reno-visceral arteries, distal aortic or iliac extensions with secondary side branch completion. Primary endpoints of the study were overall technical success, side branch patency, perioperative mortality, and the rate of severe SCI. RESULTS Eighty-three patients were treated for TAAA with branched aortic stent grafts with (n = 40) or without (n = 43) TASP. Overall technical success, including aneurysm exclusion, absence of persistent type I or III endoleak, TASP side branch patency, and secondary side branch completion was 35/40 (88%). Secondary TASP side branch completion was performed after a median of 48 days (range 1-370 days). The rate of early re-interventions for reno-visceral side branch complications was 8/283 (3%) and 6/83 (7%) for perioperative mortality, with three patients in both groups. Severe SCI or paraplegia was observed in 11/83 (13%) of the patients and reduced in the TASP group (2/40) compared with the non-TASP group (9/43; p = .03), especially in Crawford I-III aneurysms (1/29 vs. 7/24; p = .01). However, one TASP patient died 4 months after bEVAR during the TASP interval from suspected aorto-bronchial fistula. CONCLUSION The concept of TASP after bEVAR for TAAA is feasible and seems to reduce the risk of SCI. Early side TASP branch completion within 4 weeks is recommended to reduce the risk of rupture, although, according to the individual clinical presentation, a longer TASP interval might improve neurological rehabilitation from SCI.

Inhibition of tumor growth in mice with severe combined immunodeficiency is mediated by heat shock protein 70 (Hsp70)-peptide–activated, CD94 positive natural killer cells

Abstract Previously, we reported that the major stress-inducible heat shock protein 70 (Hsp70) acts as a recognition structure for natural killer (NK) cells, if localized on the cell surface of tumor cells. Incubation of purified NK cells with low-dose interleukin (IL)-2 (100 IU/mL) plus recombinant Hsp70-protein or the immunogenic 14-mer Hsp70-peptide TKDNNLLGRFELSG450–463, termed TKD (2 μg/mL), enhances the cytolytic activity against Hsp70 membrane-positive (CX+) but not against Hsp70-negative (CX−) tumor cells. Here, we show that the cytolytic activity against Hsp70-positive tumor cells is inducible by incubation of unseparated peripheral blood mononuclear cells (PBMNC) with low-dose IL-2 plus TKD. Cell sorting experiments revealed that within the PBMNC population CD94+/CD3− NK cells, and not CD94−/CD3+ T cells, mediate the cytotoxic activity against Hsp70-positive tumor cells. The antitumoral effect of PBMNC stimulated either with IL-2 plus TKD or with IL-2 alone was assessed in tumor-bearing severe combined immunodeficiency/beige mice. A single intravenous (iv) injection of 40 × 106 IL-2 plus TKD-stimulated PBMNC (containing 5.2 × 106 NK cells) on day 4 results in a 60% reduction in tumor size, from 3.89 g to 1.56 g. In contrast, the adoptive transfer of the identical amount PBMNC stimulated with low-dose IL-2 only (containing 4.4 × 106 NK cells) reduces the tumor size only less than 10% (3.64 g). A phenotypic characterization of the excised tumors revealed that predominantly Hsp70-positive tumor cells were eliminated by TKD-activated PBMNC. Kinetic studies demonstrate that the in vivo cytolytic capacity of TKD-stimulated PBMNC is dependent on the effector to target cell ratio. An iv injection of effector cells on day 1 or 2 after tumor cell inoculation results in significantly smaller tumors (0.77 g or 0.89 g) on day 21 as compared with mice that were immunoreconstituted on day 4 or 8 (1.39 g or 2.23 g). The tumor size of nonimmunoreconstituted control animals was 3.55 g.