Patient Survival by Hsp70 Membrane PhenotypeAssociation With Different Routes of Metastasis

Karin Pfister, MD1

Jurgen Radons, PhD2

Raymonde Busch, PhD3

James G. Tidball, PhD4

Michael Pfeifer, MD5

Lutz Freitag, MD6

Horst-Jurgen Feldmann, MD7

Valeria Milani, MD8

Rolf Issels, MD8

Gabriele Multhoff, PhD9

1 Department for Surgery, University HospitalRegensburg, Regensburg, Germany.

2 Institute of Medical Biochemistry and MolecularBiology, Ernst-Moritz-Arndt-University Greifswald,Greifswald, Germany.

3 Institute of Medical Statistics and Epidemiol-ogy, Technische Universitat Müchen, Munich,Germany.

4 Departments of Physiological Science and Pa-thology and Laboratory Medicine, University ofCalifornia Los Angeles, California.

5 Department for Internal Medicine II, UniversityHospital Regensburg, Regensburg, Germany.

6 Lung Hospital, Hemer, Germany.

7 Departments for Radiooncology and Radiother-apy, Hospital Fulda, Fulda, Germany.

8 Clinical Cooperation Group Hyperthermia, GSF –Munich and University Hospital Grosshadern, LMUMunich, Munich, Germany.

9 Departments for Radiotherapy and Radiooncol-ogy, Technische Universität Munchen Munich andGSF – Institute of Pathology Munich, Munich,Germany.

BACKGROUND. Heat shock proteins (HSPs) play important roles in tumor immu-

nity. 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 metasta-

sis.

METHODS. Hsp70 membrane expression was examined by flow cytometry in 58

colon, 19 gastric, 54 lower rectal carcinoma, and 19 squamous cell carcinoma

specimens and the corresponding normal tissues at time of first diagnosis.

Kaplan-Meier survival curves were analyzed to determine the relation of Hsp70

expression to the patients’ prognosis.

RESULTS. An Hsp70 membrane-positive phenotype was found in 40% (colon),

37% (gastric), 43% (lower rectal), and 42% (squamous cell) of the analyzed tumor

specimens. None of the corresponding normal tissues was found to be Hsp70

membrane-positive. In patients with colon (P 5 .032) and gastric (P 5 .045) carci-

nomas, an Hsp70 membrane expression correlated significantly with an improved

overall survival; a negative association was seen in lower rectal (P 5 .085) and

squamous cell carcinoma (P 5 .048).

CONCLUSIONS. The authors hypothesized that differing relations between surface

expression of Hsp70 on tumor cells and clinical outcomes may reflect differences

in the route of metastases. Colon and gastric carcinomas metastasize into the

liver where hepatic natural killer cells may have the capacity to recognize and kill

Hsp70 membrane-positive tumor cells and thus account for a better overall sur-

vival. Cancer 2007;110:926–35. � 2007 American Cancer Society.

KEYWORDS: Hsp70, clinical trial, prognostic marker, overall survival.

I dentification of early, molecular markers that can predict cancer-

related mortality is an urgent and important goal in the develop-

ment of cancer diagnostics and therapeutics. Ideally, identifying

patients with high-risk carcinomas with a strong metastatic poten-

tial at primary diagnosis would provide a basis for an early and indi-

vidually tailored therapy that could improve clinical outcome. Such

biomarkers would be especially valuable for high-risk tumors of the

gastrointestinal tract or lung because those cancers are the most

The authors thank Dr. Katrin Thelen, Departmentof Surgery, University of Regensburg (Germany),for statistical analyses. The technical assistanceof Lydia Rossacher is gratefully acknowledged.

Address for reprints: Gabriele Multhoff, PhD, Uni-versity Hospital rechts der Isar, Technische Uni-versität Munchen, Department for Radiotherapy &

Radiooncology, Bldg. 557, Room 1.33, Ismaninger-strasse 22, D-81675 Munich, Germany; Fax: (011)49-89-4140-4299; E-mail: gabriele.multhoff@lrz.tu-muenchen.de

Received December 18, 2006; revision receivedApril 30, 2007; accepted May 2, 2007.

Deutsche Forschungsgemeinschaft (MU 1238 of 7-2, G. Multhoff), European Union TRANSNET (MRTN-2003-504917, G. Multhoff), SFB-455 (R. Issels, V.Milani), Multimmune GmbH (G. Multhoff).

Karin Pfister and Jurgen Radons contributedequally to this work.

ª 2007 American Cancer SocietyDOI 10.1002/cncr.22864Published online 19 June 2007 in Wiley InterScience (www.interscience.wiley.com).

926

common causes of cancer-related mortality. Current

evidence shows that changes in the expression levels

of numerous molecules including tumor promoter

genes, angiogenic factors, apoptotic and cell cycle

genes could be related to cancer severity and mortal-

ity.1–4 However, many of these relations were based

upon retrospective studies, and the predictive value

of the relations in a clinical setting has frequently

been untested or insignificant. Furthermore, biomar-

kers that predict metastatic potential of primary

tumors or that are predictive for individual patients

remain to be discovered.

Heat shock proteins (HSPs) are potentially bio-

markers for cancer severity or mortality. In particular,

HSPs with molecular weights of 70 and 90 kDa are

attractive candidates because they function in the

cross-presentation of tumor-specific peptides and,

thus, are potent regulators of the adaptive immune

system.5–8 Even in the absence of peptides, the major

stress-inducible Hsp70 can promote the innate

immune responses to tumor cells by stimulating

cytokine production by antigen-presenting cells.9 In

addition, the expression of Hsp70 on the surface of

tumor cells can also play an immunomodulatory role

by increasing tumor cell lysis by natural killer (NK)

cells when in the presence of proinflammatory cyto-

kines.10–12 Nearly all tumor cells actively secrete

Hsp70 in exosomes that stimulate migration and cy-

tolytic activity of NK cells.13 The unusual membrane

expression of Hsp70 occurs only on the surface of tu-

mor cells, but not on normal tissues,14,15 and, there-

fore, can be considered to be a tumor-specific

marker. Furthermore, transplantation of NK cells into

tumor-bearing mice can promote NK cell lysis of

Hsp70-expressing tumor cells in vivo.16 These obser-

vations suggest that surface expression of Hsp70 by

tumor cells could indicate a more favorable progno-

sis because that expression would promote NK-

mediated lysis of these tumor cells. However, an

Hsp70 membrane-positive phenotype has also been

associated with a higher metastatic potential and an

unfavorable prognosis in malignant melanoma17 and

acute myeloid leukemia.15 These latter findings sug-

gest that elevated Hsp70 membrane levels may pre-

dict a more severe cancer pathology, creating a

dilemma for relating Hsp70 surface expression by tu-

mor cells to the severity of the disease.

In the present investigation, we examine the prog-

nostic value of Hsp70 membrane expression in a panel

of colon, gastric, lower rectal, and lung carcinomas by

using an Hsp70-specific antibody that specifically

detects membrane-bound Hsp70 on viable tumor

cells. The tumor entities were chosen because colon,

gastric, lower rectal, and squamous cell lung carcino-

mas differ significantly with respect to their routes of

metastases. In agreement with previous findings on

malignant melanoma and leukemia,15,17 an Hsp70

membrane-positive phenotype was associated with

poorer clinical outcome in lower rectal and in squa-

mous cell carcinomas of the lung. In contrast, patients

with Hsp70 membrane-positive colon and gastric car-

cinomas showed a significantly improved survival.

These apparently contradictory results may be attrib-

utable to differences in the route of metastasis of the

carcinomas that we investigated because the site of

metastasis is one of the most important predicting fac-

tors for survival in cancer. Colon and gastric carcino-

mas, but not squamous cell and lower rectal

carcinomas, preferentially metastasize to the liver

from the mesenteric venous system.18 This route of

metastasis may provide an opportunity for depletion

of tumor cells with Hsp70 surface expression by he-

patic NK cells that would not occur for metastatic cells

from squamous cell or lower rectal carcinomas and

that would not occur for tumor cells lacking an Hsp70

surface expression. Thus, these findings support our

hypothesis that NK cells in the liver may provide an

‘‘immunological filter’’ for tumor cells coming from

the splanchnic system.

METHODSPatientsTumor specimens and corresponding normal tissues

were obtained from patients with colon, lower rectal,

and gastric carcinoma who were undergoing surgical

resection at time of primary diagnosis at the University

Hospitals Munich, Regensburg, and at the Hospitals in

Kronach, Hemer, and Fulda in the years 1999–2002

according to the recommendation of the German Soci-

ety of Surgery.19 The study cohort included 112

patients with colon and lower rectal carcinomas (74

men and 38 women ranging in age from 40.9 to 85.4

years; median, 64.3 � 10.1), and 19 gastric cancer

patients (9 men and 10 women ranging in age from

67.5 � 11.5 years; range, 32.1–78.6). Only patients with

a meticulously complete follow-up record were en-

rolled. The median follow-up time for colorectal can-

cer patients was 33.1 � 14.4 months (range, 2.8–59.6

months); that of gastric cancer patients was

21.5 � 11.7 months (range, 4.2–50.4 months).

The study also included 14 men and 5 women

ranging in age from 44.8 to 76.2 years (median,

61.0 � 8.2 months) that were diagnosed for squa-

mous cell carcinomas of the lung in the Hospital

Barmherzige Bruder (Regensburg, Germany) and the

Lung Hospital Hemer (Hemer, Germany). The me-

dian follow-up time was 30.6 � 10.1 months (range,

Hsp70 Membrane Phenotype/Pfister et al. 927

9.3–40.7 months). All patients were censored in sur-

vival analyses according to date last seen. Patients

with tumor recurrence or preoperative therapy were

not included in this study.

Histopathological diagnosis was made according

to the World Health Organization (WHO) classifica-

tion system for tumors of the colorectal, gastrointes-

tinal tract, and the lung. In addition, the tumors

were staged and graded by standard histological

analysis according to guidelines of the International

Union against Cancer (UICC).20 This study was per-

formed according to the Declaration of Helsinki and

approved by the Human Ethics Committee of the

University of Regensburg (Germany). All patients en-

rolled in the study signed an informed consent form.

Flow CytometryTumor and normal tissues derived from the same

patient were minced mechanically in phosphate-buf-

fered saline (PBS). After washing, homogenates were

passed through a sterile mesh in RPMI 1640 medium

supplemented with 10% fetal calf serum (FCS), 1 mM

sodium pyruvate, antibiotics (all from Gibco-BRL,

Eggenstein, Germany), and 2 mM L-glutamine (PAN

Systems, Aidenbach, Germany). For flow cytometry,

0.2–1 3 106 cells were incubated for 30 minutes at 48Cin the dark either with fluorescein 5-isothiocyanate

(FITC)-labeled antihuman Hsp70 mAb (cmHsp70.1;

Multimmune GmbH, Munich, Germany) or with FITC-

labeled IgG1 (BD Pharmingen, Heidelberg, Germany)

as an isotype-matched control antibody, and with a

fibroblast-specific antibody ASO2 (Dianova, Hamburg,

Germany). After washing in PBS conditioned with 2%

FCS, the cells were analyzed directly by flow cytometry

performed on a FACSCalibur instrument (Becton Dick-

inson, Franklin Lakes, NJ) in the presence of 1 lg/mL

propidium iodide (PI; Sigma-Aldrich, Deisenhofen,

Germany). Fluorescence of 10,000 gated events was

measured on a 1024 channel, 4 decades, log scale

through forward light scatter (FSC) and linear scale

through right angle scatter (SSC). The fluorescence

histograms were generated by using gated data. Data

acquisition and analysis were performed automatically

by CellQuest Pro software (Becton Dickinson). Only

viable, PI-negative cells were analyzed.

Statistical AnalysisFor statistical analysis, the SPSS software for Win-

dows Release 12.0 (SPSS, Chicago, Ill) was used. Two

groups were compared by using the multiple propor-

tional hazard model (Cox regression). Differences

were regarded significant at P < .05. The survival

times within each subgroup were estimated from

Kaplan-Meier curves and compared by log-rank

test.21 Continuous variables are given as mean-

s � standard deviation as indicated. A walking cutoff

was run from the lowest to the highest level of Hsp70

expression on the single-cell suspensions of the solid

tumor samples as shown previously for leukemia.22

RESULTSBetween August 1999 and June 2002, this study en-

rolled 112 patients with newly diagnosed colon and

lower rectal carcinoma, 19 patients with gastric carci-

noma, and 19 patients with squamous cell carcinoma

of the lung without any preoperative therapy.

Patients resected with positive margins or with evi-

dence of local recurrence directly after surgery were

excluded from the study.

The clinical course of our patient cohort was an-

alyzed by calculating Kaplan-Meier curves for overall

survival of colorectal carcinoma (n 5 112) and strati-

fying the data for their UICC stages. As expected,

overall survival was associated with the clinicopatho-

logical UICC stage in colorectal (P 5 .001) (Fig. 1).

Tumor invasion, lymph node status, and metastases

also have been found to be consistent with these

observations (Tables 1 and 3).

We also examined the Hsp70 membrane pheno-

type on single-cell suspensions of the different tumor

samples by flow cytometry. For these studies, we

used mAb cmHsp70.1 (Multimmune GmbH, Munich,

Germany), which specifically recognizes the major

FIGURE 1. Overall survival for the period of complete follow-up demon-strated by Kaplan-Meier curves for patients with colon and lower rectal

(n 5 112) carcinomas based on UICC stages 1 to 4. The mean survival

times within each subgroup were compared by log-rank test.

928 CANCER August 15, 2007 / Volume 110 / Number 4

stress-inducible Hsp70 and does not cross-react with

the constitutive Hsc70, as determined by Western

blot analysis, flow cytometry, and dot blot analysis.

By pepscan analysis, the epitope of this antibody was

identified as being TKDNNLLGRFELSG (amino acids

450–463), a sequence located within the C-terminus

of the Hsp70 protein, which is exposed to the extra-

cellular milieu of tumor cells.23 A sample was

regarded as being Hsp70 membrane-positive when

greater than 10% of the cells were stained positively

after subtraction of the control fluorescence. This

cutoff value was evaluated by walking cutoff analysis

(P < .05), as described previously.22 Apart from the

normal connective tissue, which was determined by

using the ASO2 antibody, all tumor cells were found

to be Hsp70 membrane-positive at comparable cell

surface densities. Figure 2 summarizes 4 typical

examples of an Hsp70 membrane-positive phenotype

(gray graph) on colon, gastric, lower rectal, and squa-

mous cell carcinomas compared with corresponding

normal tissue (white graph). The mean fluorescence

intensity (mfi) differed significantly between Hsp70

membrane-positive (46 � 15) and Hsp70 membrane-

negative (22 � 10) samples, but it did not differ sig-

nificantly between the different tumor entities.

Patients’ characteristics, including tumor size

and/or invasion, lymph node status, metastases, grad-

ing, and UICC stage of all tumor types associated with

the Hsp70 status, are summarized in Tables 1–4. In

total, 40% of the colon (23 of 58) and 37% (7 of 19) of

the gastric tumor biopsies were found to be Hsp70

membrane-positive. In colon carcinomas, an Hsp70

membrane-positive phenotype was found to be asso-

ciated with an N0 lymph node status (P 5 .039), lack of

metastases (P 5 .045), and a lower UICC stage

(P 5 .039) at diagnosis (Table 1). Adjuvant chemother-

apy was given only to colon carcinoma patients in

stage pN1; none of the patients received radiotherapy.

In gastric carcinomas (n 5 19), none of the clinico-

pathological parameters correlated significantly with

an Hsp70 membrane-positive phenotype (Table 2);

none of these patients received adjuvant chemother-

apy and/or radiotherapy.

Forty-three percent (23 of 54) of the lower rectal

and 42% (8 of 19) of the squamous cell tumor biop-

sies of the lung were found to be Hsp70 membrane-

positive. It is worth mentioning that in our squa-

mous cell carcinoma panel, 56% were diagnosed as

grade 3 and 44% as grade 2. In lower rectal carcino-

mas (n 5 54), an Hsp70 membrane-positive pheno-

type was found to be associated with a lack of lymph

node metastases (P 5 .044) and lower UICC stage

(P 5 .017) (Table 3) similar to colon carcinomas (Ta-

ble 1). Adjuvant chemo-radiotherapy was given to

lower rectal carcinoma patients in stage pN1. Squa-

mous cell carcinomas of the lung (n 5 19) did not

show any significant correlation between the Hsp70

phenotype and clinicopathological parameters (Table

4). Postoperative squamous cell carcinoma patients

in UICC stages <2 and �2 were treated following

standard adjuvant chemotherapy guidelines.

We next analyzed overall survival in Hsp70 mem-

brane-positive and membrane-negative tumor

patients by using the multiple proportional hazard

model (Cox regression). Patients with Hsp70 mem-

brane-positive colon (Fig. 3A; n 5 58, P 5 .032) and

gastric (Fig. 3B; n 5 19, P 5 .045) carcinomas showed

a significantly better overall survival when compared

with their Hsp70 membrane-negative counterparts.

These findings were not expected because previ-

ous results indicated that in malignant melanoma

and leukemia, an Hsp70 membrane-positive pheno-

type was associated with poor prognosis.15,17 In line

with these previous findings, but in contrast to colon

and gastric cancer patients, patients with Hsp70

membrane-positive lower rectal (Fig. 4A, n 5 54;

P 5 .085) and squamous cell carcinomas of the lung

(Fig. 4B, n 5 19; P 5 .048) had a poorer clinical out-

come with respect to overall survival when compared

TABLE 1Correlation of the Hsp70 Plasma Membrane Expression andClinicopathological Parameters in Patients With Colon Carcinoma(n 5 58); Route of Metastasis to the Liver

Factors Category

Total No. of

patients

Hsp70 membrane-

positive tumors

(n 5 23) P

Age Total 58 66.1 � 9.9

Hsp702 35 67.2 � 10.3 NS

Hsp701 23 64.5 � 9.1 NS

Sex Men 38 16 (42%) NS

Women 20 7 (35%)

Tumor size/ pT1 1 1 NS

invasion (T) pT2 9 3

pT3 37 17

pT4 11 2

Lymph node pN0 36 18 .039

Status (N) pN1 22 5

Metastases pM0 42 20 .045

pM1 16 3

Grading 1 0 0 .042

2 35 18

3 21 5

X 2 0

UICC stage 1 6 3 .039

2 26 15

3 10 2

4 16 3

NS indicates not significant.

Hsp70 Membrane Phenotype/Pfister et al. 929

with their Hsp70 membrane-negative counterparts.

The latter data are consistent with previous observa-

tions showing membrane-expressed Hsp70 in 50% of

endometrial carcinomas with poor clinical prognosis

and significantly decreased survival rates.24

Both the Hsp70 phenotype and the UICC stages

could be determined as significant independent neg-

ative prognostic markers for the overall survival in

lower rectal carcinomas (UICC, P 5 .021, hazard rate

6.684; Hsp70, P 5 .012; hazard rate 5.633) in the mul-

tiple proportional hazard model (Fig. 5A). The P-

value for the equality of survival distributions for the

different levels of UICC and Hsp70 was .007.

In colon carcinoma patients, the UICC stage also

served as a significant, independent, negative prognos-

tic marker for overall survival (UICC, P 5 .001; hazard

rate 13.882). In this tumor entity, the Hsp70 membrane

expression was found to be associated with a better

overall survival and, thus, was predictive for a positive

clinical outcome (Fig. 5B). Because of the low patient

number, a detailed analysis of UICC stages and Hsp70

for gastric (n 5 19) and squamous cell carcinomas

(n 5 19) was not performed.

DISCUSSIONThe present investigation findings show that surface

expression of Hsp70 by tumor cells may have clinical

value as a predictive biomarker for metastatic carcino-

mas. However, a surprising outcome of our study is

that an Hsp70 surface-positive phenotype can be asso-

ciated with a positive or negative prognosis, depending

on the cancer entity examined. We found that an

Hsp70 membrane-positive phenotype was associated

with a better overall survival in patients with colon and

gastric carcinomas but predicted worse overall survival

in lower rectal and squamous cell carcinomas of the

lung. Thus, our results emphasize that Hsp70 may

serve multiple roles in the pathophysiology of meta-

static diseases and that specific cancers in individuals

can differ in their surface expression of Hsp70 for rea-

sons that are not yet understood.

FIGURE 2. Representative flow cytometric analysis of Hsp70 membrane-positive tumor samples. Single-cell suspensions were freshly prepared from tumorbiopsies from patients with colon, gastric, lower rectal, and squamous cell carcinoma of the lung, and of the corresponding normal tissues. After staining with

the fluorescein 5-isothiocyanate (FITC)-conjugated anti-Hsp70 monoclonal antibody (cmHsp70.1), viable, propidium iodide-negative cells were analyzed by flow

cytometry. A sample was considered as Hsp70 membrane-positive when[10% of the cells were stained positively after subtraction of the isotype-stained con-trol fluorescence. White histograms represent negative control tissues; gray histograms represent Hsp70 membrane-positive tumor cells, respectively.

930 CANCER August 15, 2007 / Volume 110 / Number 4

Our current understanding of the role of NK cells

in cancer immunobiology provides a mechanistic

context to interpret some of our findings because a

growing body of evidence indicates that NK cells can

preferentially target and kill tumor cells that express

Hsp70 on their cell surfaces. Experimental findings,

using both in vitro and in vivo assays, show that

when Hsp70 is administered in combination with

proinflammatory cytokines, it can modulate the

response of NK cells to tumor cells.10,16 Incubation

of NK cells with soluble Hsp70 protein12 or with the

14 mer Hsp70 peptide TKD25 enhances the cytolytic

activity of NK cells and the IFN-c secretion by NK

cells when administered in the presence of IL-2.

Recent finding that these immune modulatory func-

tions of Hsp70 can also be achieved by release of

Hsp70 surface-positive exosomes from tumor cells13

indicated that these exosomes can provide signals to

NK cells remote from tumors, to attract them to

locate and lyse tumors. This has now been confirmed

experimentally in studies that demonstrated that

Hsp70 membrane-positive tumors release Hsp70 sur-

face-positive exosomes that are chemoattractive and

activate NK-cell cytolytic activity. Antibodies to the

Hsp70 peptide TKD have the capacity to block che-

moattraction of NK cells and prevent granzyme B-

mediated killing of tumor cells.13,26,27

Our findings that Hsp70 surface expression by

tumor cells is associated with a positive prognosis in

colon and gastric cancers are consistent with data

that show NK cells selectively target and lyse Hsp70

membrane-positive tumor cells. However, this inter-

pretation is apparently contradicted by findings that

Hsp70 surface expression is not associated with a

positive outcome in lower rectal or squamous cell

carcinomas of the lung. Although experimental data

to interpret these findings are not available, we pro-

pose that differences in routes of metastasis of squa-

mous cell and lower rectal carcinomas to colon and

gastric cancers may underlie the different relations

between Hsp70 expression and clinical outcome.

Whereas colon and gastric carcinomas metastasize

preferentially to the liver,18 metastasis of lower rectal

tumors occurs directly by way of the vena cava to

the lung, bypassing the liver.28 In addition, metasta-

ses of squamous cell carcinomas of the lung do not

pass the liver; instead, they metastasize to regional

lymph nodes and to the brain.29,30 Thus, a potentially

important feature in determining whether tumor

cells with Hsp70 surface expression are correlated

TABLE 2Correlation of the Hsp70 Plasma Membrane Expression andClinicopathological Parameters in Patients With Gastric Carcinoma(n 5 19); Route of Metastasis to the Liver

Factors Category

Total No. of

patients

Hsp70 membrane-

positive tumors

(n 5 7) P

Age Total 19 67.5 � 11.5

Hsp702 12 69.9 � 7.0 NS

Hsp701 7 63.4 � 16.6 NS

Sex Men 9 4 (44%) NS

Women 10 3 (30%)

Tumor size/ pT1 2 1 NS

invasion (T) pT2 11 5

pT3 6 1

pT4 0 0

Lymph node pN0 4 2 NS

Status (N) pN1 15 5

Metastases pM0 15 5 NS

pM1-X 4 2

Grading 1 0 0 NS

2 4 1

3 15 6

X 0 0

UICC stage 1 4 1 NS

2 3 2

3 6 2

4 6 2

NS indicates not significant.

TABLE 3Correlation of the Hsp70 Plasma Membrane Expression andClinicopathological Parameters in Patients With Lower RectalCarcinoma (n 5 54); Route of Metastasis to the Lung

Factors Category

Total No. of

patients

Hsp70 membrane-

positive tumors

(n 5 23) P

Age Total 54 62.4 � 10.0

Hsp702 31 60.4 � 8.6 NS

Hsp701 23 65.0 � 11.3 NS

Sex Men 36 16 (44%) NS

Women 18 7 (38%)

Tumor size/ pT1 2 2 NS

invasion (T) pT2 12 8

pT3 37 12

pT4 3 1

Lymph node pN0 29 16 .044

Status (N) pN1 25 7

Metastases pM0 43 19 NS

pM1 11 4

Grading 1 0 0 NS

2 39 19

3 12 3

X 3 0

UICC stage 1 11 9 .017

2 14 6

3 18 4

4 11 4

NS indicates not significant.

Hsp70 Membrane Phenotype/Pfister et al. 931

with a positive or negative prognosis may be the

route of their metastases.

The passage of metastasizing tumor cells through

the liver may be important in determining whether

these cells are eliminated because the liver functions

as an ‘‘immunological filter’’ for circulating tumor cells

and infectious organisms in portal venous blood that

are constantly drained from the splanchnic venous

system.18 The liver also plays a key role in the innate

immune response by influencing the homing and dif-

ferentiation of NK cells.31–34 Up to 65% of all lympho-

cytes present in the normal liver consists of NK cells,

NKT cells, and cd T cells, and the frequency of NK cells

among human intrahepatic lymphocytes ranges

between 25% and 30%.31,32 Furthermore, NK cells are

located in the liver sinusoids, adherent to the liver si-

nusoidal endothelial cells and, thus, are well situated

to eliminate arriving metastasizing tumor cells.35 He-

patic NK cells have a higher cytolytic capacity against

tumor cells than blood or spleen NK cells. They also

express high levels of the C-type lectin receptor

CD94,36 which is involved in the interaction of NK cells

with tumor membrane-bound Hsp70.37

Hsp70-specific immune responses in NK cells

require proinflammatory cytokines (unpublished ob-

servation). Among these cytokines, IL-15 has emerged

as a key regulator for activation, recruitment, prolifera-

tion, differentiation, and survival of NK cells.30,38,39

This may be important in the modulation of the

immune response by the liver because Kupffer cells are

a rich source of IL-15.40,41 Thus, the liver can provide

an environment for NK cells in which they can selec-

tively eliminate colon and gastric carcinoma cells that

express Hsp70 on their surfaces. The better overall sur-

vival demonstrated by our Kaplan-Meier analyses may

reflect NK cell-mediated killing of tumor cells as they

transit the liver.

Although our hypothetical model is sufficient to

explain the positive relation of an Hsp70 membrane-

positive phenotype in metastasizing colon and gas-

tric carcinomas, the model does not explain why

Hsp70 surface expression reflects a worse outcome

in lower rectal and squamous cell carcinomas. Previ-

ous investigators have also shown that Hsp70 expres-

sion was found to correlate significantly with poorer

clinical outcome in endometrial carcinomas,24 in

which the route of metastasis is entirely extrahepatic.

Presumably, the predictive value of an Hsp70 surface

expression for a worse clinical outcome in cancers

that have an extrahepatic route of metastasis reflects

the diverse and complex roles played by Hsp70.

HSPs are over-expressed in a broad range of

human tumors and play crucial roles in tumor inva-

sion, metastasis, cell proliferation, differentiation,

and cell death (for review see ref. 8).8 Depending on

their location, HSPs either mediate protection against

lethal damage induced by exogenous stress or exert

immune activation as danger signals in cancer im-

munity. In the cytosol, HSPs function as molecular

chaperones supporting folding and transport of a

great variety of polypeptides and proteins under both

physiological conditions and after chemical or physi-

cal stress stimuli. Hsp70, the major stress-inducible

HSP, is able to protect cells from a wide range of ap-

optotic and necrotic stimuli.42–45 In the absence of

Hsp70 and cytokine-activated NK cells, any of these

numerous Hsp70 functions may dominate the immu-

nostimulatory role by Hsp70 in tumor cells. Unpub-

lished data from our own laboratory further indicate

that tumor cells exhibiting an Hsp70 surface-positive

phenotype are significantly more resistant to chemi-

cally and physically induced stress stimuli when

compared with their Hsp70 surface-negative counter-

parts. We hypothesize that, in the absence of cyto-

kine/TKD-activated NK cells, membrane-bound

Hsp70 helps the tumor to be protected from lethal

damage induced by chemotherapy and/ or radiother-

apy and, thus, results in a worse clinical outcome.

Comparative analysis of the lipid composition of

TABLE 4Correlation of the Hsp70 Plasma Membrane Expression andClinicopathological Parameters in Patients With SquamousCell Carcinoma of the Lung (n 5 19); Route of Metastasis tothe Lymph Nodes and to Brain

Factors Category

Total No. of

patients

Hsp70 membrane-

positive tumors

(n 5 8) P

Age Total 19 61.9 � 8.2

Hsp702 11 61.6 � 9.0 NS

Hsp701 8 62.3 � 7.5 NS

Sex Men 14 7 (50%) NS

Women 5 1 (20%)

Tumor size/ pT1 3 1 NS

invasion (T) pT2 14 6

pT3 1 1

pT4 1 0

Lymph node pN0 11 5 NS

Status (N) pN1 8 3

Metastases pM0 16 7 NS

pMX 3 1

Grading 1 0 0 NS

2 7 4

3 9 4

X 2 0

UICC stage 1 11 5 NS

2 4 1

3 4 2

4 0 0

NS indicates not significant.

932 CANCER August 15, 2007 / Volume 110 / Number 4

tumors with a differential Hsp70 membrane expres-

sion revealed a higher membrane rigidity in Hsp70

membrane-positive tumors, which may cause better

chemotherapy resistance and/or radiotherapy resist-

ance. In summary, we hypothesize that chemother-

apy resistance and/or radiotherapy resistance may

not correlate with resistance to an NK cell-mediated

immunity,

Although many questions remain concerning the

immunobiology of NK cell interactions with tumor

cells that express Hsp70 on their cell surface, recent

and continuing studies indicate that these interactions

can be productively exploited for diagnostic and thera-

peutic goals. The observation that the adoptive trans-

fer into tumor-bearing mice of Hsp70-stimulated NK

cells caused the eradication of primary tumors and

FIGURE 3. Kaplan-Meier analysis shows overall survival after surgery of colon (A, n 5 58) and gastric (B, n 5 19) carcinoma patients based on their Hsp70

membrane phenotype. The mean survival times within each subgroup were compared by using the Cox regression test; P-values are indicated in the lower right

part of each graph; significant differences were observed between groups.

FIGURE 4. Kaplan-Meier analysis shows overall survival after surgery of patients with lower rectal carcinoma (A, n 5 54) and squamous cell carcinoma of

the lung (B, n 5 19) based on their Hsp70 membrane phenotype. The mean survival times within each subgroup were compared by using the Cox regression

test; P-values are indicated in the lower right part of each graph; significant differences between groups were detected for squamous cell carcinoma.

Hsp70 Membrane Phenotype/Pfister et al. 933

metastases16,46,47 provides strong support for the ther-

apeutic potential of this approach.

Cytokine-activated NK cells can induce regression

of established lung and liver tumors.48–51 On the basis

of these findings in mouse models, we tested the

effects of delivering ex vivo cytokine and Hsp70 preac-

tivated NK cells to patients with therapy-refractory,

multiple, metastasized colon and nonsmall cell lung

carcinomas. As demonstrated in this phase I clinical

trial, reinfusion of ex vivo-stimulated, autologous NK

cells is safe, feasible, and well tolerated.52 Further-

more, we showed that it is possible to stimulate an

Hsp70-reactivity in NK cells in multiple metastasized

and pretreated patients. Although refractory to stand-

ard chemotherapy, 2 of 5 patients who received more

than 4 NK cell reinfusions showed clinical responses.

These promising immunological results and clinical

responses justify additional studies in patients with

lower tumor burden and an established Hsp70 mem-

brane-positive phenotype.

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