locoregional cellular immunotherapy for patients with ... · locoregional cellular immunotherapy...

Locoregional Cellular Immunotherapy for Patients with AdvancedEsophageal Cancer1

Uhi Toh, Hideaki Yamana,2 Susumu Sueyoshi,Toshiaki Tanaka, Fumihiko Niiya,Katsuko Katagiri, Hiromasa Fujita,Kazuo Shirozou, and Kyogo ItohDepartments of Surgery [U. T., H. Y., S. S., T. T., F. N., H. F., K. S.]and Immunology [U. T., K. K., K. I.], Kurume University School ofMedicine, Kurume 830-0011, Fukuoka, Japan

ABSTRACTThe objectives of the present study were to determine

the safety of locoregional administration of autologous lym-phocytes stimulated with autologous tumor cells and inter-leukin (IL) 2 in vitro and to find laboratory markers topredict either clinical toxicity or clinical response. Elevenpatients with advanced (n5 4) or recurrent (n 5 7) esoph-ageal cancers received the locoregional administration ofthese activated lymphocytes every 2 weeks for two to ninetimes (mean, 5.6 times), and mean numbers of the adminis-tered cells were 0.83 109 cells per treatment. The activatedlymphocytes that were pretested for their surface markersand CTL activity were endoscopically injected into primarytumor sites (n5 4) or directly injected into metastatic lymphnodes (n5 2), pleural (n 5 4) or ascitic (n 5 1) regions.Grade 3 hypotension, grade 2 diarrhea, and grade 1 feverwere observed in 1, 1, and 6 patients, respectively, and therewas no adverse effect in the remaining three patients. Theclinical outcome was as follows: one, complete response(CR); three, partial response (PR); two, stable response(SR); and five, progressive disease (PD). CTL activity in theadministered cells was observed in 5 of the 11 patients (1CR, 3 PR, and 1 PD) and was not observed in the remaining6 patients (2 SR and 4 PD). Percentages of CD161 cells inthe peripheral blood of the responder group (CR1PR) sig-nificantly increased when compared with those before treat-ment or with those of the nonresponder group before as wellas after treatment. Because the clinical toxicity was moder-ate and tolerable, this new method of locoregional immuno-therapy will be applicable for use in treatment of patients

with advanced and recurrent esophageal cancers. Both CTLactivity in the administered cells and the percentages ofCD161 cells in the peripheral blood may be useful labora-tory markers for predicting of clinical response.

INTRODUCTIONCancer in the esophagus is one of most common malignant

neoplasms in the world, particularly in the Pacific countries.Surgery remains the standard approach for patients with locore-gional advanced disease that is resectable. Curative resection isfeasible in only 50% of cases, and local or distant failure iscommon after resection (1–3). The 5-year survival is only;30% for stage-III and -IV patients undergoing surgery. Someadjuvant multimodality therapies have been attempted to controlboth local and systemic disease (4–6). However, unresectableand relapsed esophageal cancers are still resistant to the pres-ently available chemotherapy or radiation therapy regimens, andthere is almost no clear advantage from these regimens foroverall survival. Consequently, the development of a new ef-fective therapeutic approach such as immunotherapy could bevaluable to expand treatment modalities (7–9). Recently severalreports presented the clinical efficacy of immunotherapy foradvanced cancer in the digestive tract, but little clinical experi-ence has been reported for advanced esophageal cancer (10–12).We have reported the presence of precursors of HLA classI-restricted and SCC3-specific CTLs in both PBMCs and TILsof patients with esophageal cancer (13–15). In the present study,we investigated the clinical toxicity and clinical response oflocoregional administration of PBMCs stimulated with autolo-gous tumor cells in advanced and recurrent esophageal cancerpatients. Laboratory markers for the prediction of toxicity orresponse were also determined.

MATERIALS AND METHODSPatients. Patients with unresectable primary or recurrent

metastatic esophageal cancer were eligible to this pilot study.Patients were required to have disease assessable by physical orradiographic examination and life expectancies of at least 2months. Patients’ characteristics are shown in a Table 1. Nopatients had been receiving corticosteroids or any prior immu-notherapy; however, six patients had received prior chemother-apy and radiotherapy. The intervals between these prior treat-ments and immunotherapy were at least 1 month (range, 1–6

Received 5/8/00; revised 7/7/00; accepted 9/7/00.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported in part by Grants-in-Aid from the Ministry of Education,Science, Sport and Culture, Japan, and Grant H10–genome–003 fromthe Ministry of Health and Welfare, Japan.2 To whom requests for reprints should be addressed, at Department ofSurgery, Kurume University School of Medicine, 67 Asahi Machi,Kurume 830-0011, Japan. Phone: 81-942-31-7566; Fax: 81-942-34-0709; E-mail: [email protected].

3 The abbreviations used are: SCC, squamous cell carcinoma; PBMC,peripheral blood mononuclear cell; LN, lymph node; HLA, humanleukocyte antigen; IL, interleukin; CT, computed tomography; CEA,carcinoembryonic antigen; TIL, tumor-infiltrating lymphocyte; CR,complete response; PR, partial response; SR, stable response; PD,progressive disease; LAK, lymphokine-activated killer; NK, naturalkiller.

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months). The protocol was approved by the Institutional ReviewCommittee of the Kurume University. The protocol was ex-plained to each patient, and written informed consent to partic-ipate in the study was obtained from all of the patients whoentered this study. Thirteen patients fulfilled the eligibility cri-teria, but two patients were eliminated before treatment becauseof inadequate growth of cultured PBMCs. Eleven patients withunresectable esophageal cancer (n 5 4) or recurrent metastaticcancer (n5 7) received this treatment (Table 2). These tumorswere histologically confirmed as SCCs by pathological exami-nation. The mean age of the patients was 67.7 years. Six of the11 patients (cases 4–8 and 10) received prior radiotherapy (48,50, 52, 50, 50, and 50 Gy, respectively; mean, 50 Gy) combinedwith two cycles of the following chemotherapy: 110 mg/m2/dayof cisplatin and 700 mg/m2/5 days of 5-fluorouracil. The re-maining five patients (cases 1–3, 9, and 11) did not receive anychemotherapy or radiotherapy before the immunotherapy.

Cells. Tumor samples used for the stimulation of PBMCswere obtained as follows: original esophageal tumors that werebiopsied through an endoscope (n 5 4), resected metastatic leftsupraclavicular LNs (n5 2), carcinomatous pleural effusion(n 5 4), and carcinomatous ascitic fluid (n 5 1; Table 2). Themean weight of the biopsied specimens was 0.296 0.43 g, andeach contained the mean of 0.5 to 2.03 107 viable tumor cells.The tumor cells were irradiated with a dosage of 50 Gy over 10min. Autologous tumor cells were prepared from biopsied sam-ples by mincing and by enzymatic digestion with stirring in 50ml of PBS containing 10 mg of type IV collagenase and 5 mg ofDNase type I (Sigma) for 3–4 h at room temperature, followedby filtration through a layer of 100 nylon mesh and then bywashing twice with PBS. The samples and effusion were appliedon Ficoll-Hypaque solution and were centrifuged to isolate livecells from dead cells and aggregates. Tumor cells were discrim-inated from inflammatory mononuclear cells on the basis of thesize and the other morphological features under the microscope,as reported previously (15). These cells were also cryopreservedin 90% human AB serum (Blood Center of Japanese Red Cross)

plus 10% DMSO (Sigma) at2178°C in liquid nitrogen forrestimulation and subsequent immunological assay. Heparinizedperipheral blood samples (50–100 ml; mean, 78 ml) were col-lected from patients for 2 weeks to prepare cells for the treat-ment. The yield of PBMCs that were obtained from the bloodsamples with the Ficoll-Hypaque gradient method (14) was0.38–1.33 106 cells/ml (mean, 0.54) or 0.5–1.53 106 cells(mean, 1.0) in patients or healthy volunteers, respectively. Thetumor cell lines and normal cell lines used in these studies andtheir HLA class I alleles have been reported previously (13, 14).

Cell Culture. One million PBMCs were incubated in 2ml of the culture medium containing 105 irradiated autologoustumor cells in each well of a 24-well culture plate at 37°C in 5%CO2. The culture medium consisted of RPMI 1640 with 10%heat-inactivated human AB serum, 0.1 mM MEM nonessentialamino acids solution, 100 IU/ml recombinant IL-2, 100 units/mlpenicillin, 100mg/ml streptomycin, 50mg/ml gentamicin, and0.5mg/ml fungizone. PBMCs stimulated with autologous tumorcells were restimulated on day 7 of culture, followed by washingand preparation for clinical use on day 14. PBMCs were alsocultured with IL-2 alone and were used as a control for theinvitro analysis. Contamination in the cultured lymphocytes waschecked by the Department of Laboratory Medicine accordingto the guideline for cellular therapy of our university. Endotoxinwas not checked in this study, although the cells were repeatedlywashed before injection.

Assays. The activated PBMCs were harvested after thesecond stimulation at day 14 of culture and were characterizedfor their phenotypes and cytotoxicity. Autologous tumor cellswere separated from the single cell suspensions of biopsiedtumor samples, pleural effusion, and ascites as mentionedabove. These autologous tumor cells were cryopreserved in 90%human AB serum (Blood Center of Japanese Red Cross) plus10% DMSO (Sigma) at2178°C in liquid nitrogen for restimu-lation and subsequent immunological assay. These cells werethawed, cultured for several days, and used as stimulator ortarget cells. A 6-h51Cr-release assay was used to measure

Table 2 Summary of the cellular immunotherapy

Caseno.

HLA class IA locus

Source of autologoustumor cell

Total no. of injectedcells (total no. of

injections)Method of

administrationCTL

activitya Adverse effectbClinical

Responsec

Period ofremission

(mo)

1 24/31 Primary tumor 2.13 109 (3) Endoscopic Yes Grade 3 (hypotension) CR ;202 2/ Primary tumor 5.33 109 (6) Endoscopic No Grade 1 (fever) PD3 24/26 Primary tumor 183 109 (9) Endoscopic Yes Grade 1 (fever) PR 114 2/11 Pleural effusion 1 3 109 (2) Regional No None PD5 2/ Metastatic lymph node 5.83 109 (7) Regional Yes Grade 1 (fever) PR 96 26/33 Pleural effusion 3.96 109 (5) Regional Yes None PD7 NTd Pleural effusion 1.23 109 (2) Regional No None PD8 24/31 Primary tumor 4.93 109 (6) Endoscopic No Grade 1 (fever) SR 89 24/11 Metastatic lymph node 73 109 (8) Regional Yes Grade 1 (fever) PR 7

10 31/11 Pleural effusion 2 3 109 (3) Regional No Grade 1 (fever) PD11 24/ Ascites 3.63 109 (6) Regional No Grade 2 (nausea, diarrhea) SR ;13

a The term CTL activity was used in this study if the activated PBMCs produced the significantly higher levels of IFN-gor percentage ofcytotoxicity (P, at least 0.05 by a two-tailed Student’st test) by recognition of the autologous tumor cells when compared with those in the allogenictumor cell lines.

b Common toxicity criteria of the National Cancer Institute.c CR, (disappearance of all measurable mass); PR, (.50% reduction in the size of the mass); SR, (,50% reduction in the size of the mass); PD,

(increase in the size of the mass).d Not tested.

4664Immunotherapy for Esophageal Cancer



Fig. 1 Clinical findings of case 1. A barium esophagogram of the lower esophagus in case 1 before treatment (part 1a) showed a bulky irregularfilling defect with destruction of mucosal folds in double contrast and persistent severe stenosis at the esophagogastric junction. Significant reductionin tumor size and significant improvement in the narrowing of the lumen in the same patient were observed after the third treatment (part 1b). Theendoscopic appearance of the lower esophagus before treatment (part 2a) showed typical appearance of an exophytic polypoid carcinoma with asignificant narrowing in the lumen of the esophagus. Significant reduction in the tumor size and improvement in the narrowing of the lumen wereseen at 14 days after the first and second treatments (part 2,b andc, respectively). A feeding tube was observed on the right side of esophagus inpart 2c. Complete tumor regression was observed in the lower esophagus after the third treatment (part 2d). Histology of the tissues in the biopsiedspecimen before treatment showed moderately differentiated SCC cells before treatment (part 3a), whereas the tumor cells were no longer observedand there were some inflammatory changes after the third treatment (part 3b). H&E stain;3500.

4665Clinical Cancer Research



cytotoxicity of these activated PBMCs against autologous tumorcells or other tumor cell lines by the methods previously re-ported (14). The PBMCs were also measured for their IFN-gproduction in response to various tumor cells by incubation ofcells for 18 h with target cells at an E:T ratio of 3:1. The

amounts of IFN-gin cell-free supernatants were measured by anIFN-g ELISA kit, and the limit of sensitivity of ELISA was 5pg/ml as reported previously (14). The number of WBCs permm3 was counted by the Department of Laboratory Medicine.Heparinized blood was applied for Ficoll-Hypaque solution and

Fig. 3 A CT scan of the liver in case 5. Therewere metastatic tumors in S2 (a-1,arrow) and S4area (b-2,arrow) before treatment. There wasmarked tumor regression in the measurable mass ofS2 (b-1,arrow) and S4 (b-2,arrow) after the fourthtreatment into the metastatic LN located in the leftsupraclavicular LNs.

Fig. 4 A CT scan of the neck in case 9.There was marked tumor regression in themeasurable mass of the left supraclavicularLN after the seventh treatment (a, beforetreatment;b, after treatment).

Fig. 2 Clinical findings of case 3. A barium esophagogram in double contrast of the lower esophagus of patient 3 showed a bulky, irregular fillingdefect with destruction in the mucosal folds and persistent severe stenosis in the middle thoracic esophagus before treatment (part 1a). There wasmarked regression in tumor size and a significant improvement in the narrowing of the lumen after the sixth treatment (part 1b). The endoscopicappearance of the lower esophagus showed typical appearance of an exophytic polypoid carcinoma with a narrowing in the lumen of the esophagusbefore treatment (part 2,a-1 anda-2). There was a significant improvement both in tumor size and in the narrowing of lumen at day 12 after theseventh treatment (part 2,b-1andb-2). Photomicrographs of the frozen tissue of the biopsied specimen stained by anti-CD3 and anti-CD8 monoclonalantibodies in case 3 before treatment (part 3a) and after the third treatment (part 3,b andc). There was significant infiltration of CD31 (part 3b)and CD81 (part 3c) T cells in the specimen after treatment. Immunohistochemical staining;3200.




was centrifuged to obtain PBMCs, as reported previously (14).Viability of PBMCs was determined by a trypan blue dye-exclusion test. The surface phenotypes of PBMCs were testedby the two-color immunostaining technique with anti-CD3,-CD4, -CD8, and -CD16 monoclonal antibodies and FACScanflow cytometry at 4-week intervals, as reported previously (14).We used the term “CTL activity” in this study if the activatedPBMCs produced significantly higher levels of IFN-g produc-tion or percentage of cytotoxicity (P , 0.05 by a two-tailedStudent’st test) by recognition of the autologous tumor cellscompared with those in response to the allogenic tumor celllines.

Treatment Schedule. The activated PBMCs werewashed in PBS three times, resuspended in 5–10 ml of 0.9%saline, and administered by endoscopic intratumoral injection ordirect regional injection (Table 2). Biopsy for preparation of thetumor cells was carried out before injection of the activatedPBMCs in cases 1–3 and 8. These injections were repeated atleast two times at 2-week intervals and for up to nine times untildisease progression or severe toxicity was seen. The first clinicalevaluation was performed 14 days after the second injection.The clinical outcome was evaluated by the following methods:esophagoscopy, esophagography and ultrasonography at 2-weekintervals; CT scan at 4-week intervals; and serum CEA at4-week intervals. A CR was evaluated as disappearance of all ofthe measurable tumor mass without the appearance of newlesions, and a PR was defined as a reduction of all of themeasurable disease by 50% of the sum products of the twogreatest perpendicular diameters. A SR was defined as,50%reduction in all of the measurable lesions. Adverse effects wereevaluated by history and physical examination and graded ac-cording to the National Cancer Institute common toxicity scale.

Statistical Analysis. Statistical analyses for lymphocytephenotypic markers and IFN-gproduction were performed us-ing the two-tailed Student’st test or the Fisher’s exact test.

RESULTSClinical Results. Eleven patients with advanced (n5 4)

or recurrent (n5 7) esophageal cancer received the locoregionalcellular immunotherapy of the PBMC-activated autologous tu-mor cells and IL-2 every 2 weeks for 2–9 times (mean, 5.6times). The mean number of administered cells per treatmentwas 0.83 109 cells (range, 0.5–23 109 cells). The patients’profiles are summarized in a Table 1, and the treatment sched-ule, CTL activity of injected cells, adverse effects, and clinicalresponse are summarized in Table 2. Six of the 11 patientsdeveloped a transient febrile reaction (,38°C) within 72 h afterthe treatment. One patient (case 1) developed grade III hypo-tension that required a Neo-Synephrine pressure drip for recov-ery, and the other patient (case 11) had transient grade II nauseaand diarrhea. No adverse effect was observed in the remainingthree patients (Table 2). No toxicity was associated with theintrapleural administration (cases 6, 7, and 10) of activated cells.Transient grade II nausea and diarrhea were observed by theintra-abdominal administration of the cells in case 11. Theclinical outcome was as follows: 1 CR, 3 PR, 2 SR, and 5 PD.Clinical findings of the four cases that showed major tumorregression (1 CR and 3 PR) are shown in the next paragraphs.

Fig. 5 Kinetic study of serum CEA levels in cases 9 and 11, accordingto the clinical course. The levels of serum CEA significantly decreasedin case 9 after the fifth treatment and case 11 after the fourth treatmentof cellular therapy.

Fig. 6 A CT scan of the abdomen in case 11. There was marked tumorregression in the measurable mass of the para-aortoarterial LN (arrow)after the fifth treatment (a, before treatment;b, after treatment).




One patient (case 1) had a CR with no evidence of diseasefor .20 months after the last treatment. Esophagography of thelower esophagus before treatment (Fig. 1,part 1a) and after thethird treatment (Fig. 1,part 1b) showed a marked tumor regres-sion for tumor size. Endoscopic appearance before treatment

(Fig. 1, ,part 2a), after the first treatment (Fig. 1,part 2, b andc), and after the third treatment (Fig. 1,part 2d) also clearlyindicated a significant reduction in tumor size followed by thecomplete disappearance of tumor. Moderately differentiatedSCC cells observed in the biopsied sample before treatment

Fig. 7 CTL activity of the administered cells. Ina, PBMCs, stimulated with autologous tumor cells, and IL-2 were measured on day 14 of culturefor their activity to produce IFN-gin response to the autologous tumor cells or to the cells of KE3 (A2/A24), TE10 (A24/A26) esophageal SCC cellline, and K562 erythroleukemia cell line. PBMCs cultured with IL-2 alone, taken as the control, were also measured for their activity. Values representthe mean of IFN-gproduction of different effector cells (n5 2–9). Each sample was measured in triplicate determinants at an E:T ratio of 3:1.Background IFN-gproduction (50–100 pg/ml) was subtracted from the values. Two-tailed Student’st test was used for statistical analysis. Inb,PBMCs, stimulated with autologous tumor cells, and IL-2 in cases 1 and 2 were measured for their cytotoxicity by a 6-h51Cr-release assay againstfive different targets [autologous tumor cells, KE3, TE10, K562, and MKN28 (A31/) gastric adenocarcinoma cell line]. PBMCs cultured with IL-2alone, taken as the control, were also measured for cytotoxicity. Values represent the mean of the percentage of specific lysis of the different effectorsamples of the different experiments (n5 3 or 4). Each sample was measured in triplicate at an E:T ratio of 20:1.Bars,6SD.




(Fig. 1,part 3a) were no longer observed in the biopsied sampleafter the third treatment (Fig. 1,part 3b). Instead, the markedinfiltration of mononuclear cells was seen in the later sample.Similar histological changes were also observed in the samplesafter the treatment in the other responders (data not shown).

Three patients (cases 3, 5, and 9) had PRs. Esophagographybefore (Fig. 2,part 1a) and after treatment (Fig. 2,part 1b) incase 3 demonstrated a marked reduction in tumor size. Simi-larly, endoscopic appearance before (Fig. 2,part 2,a-1anda-2)and after treatment (Fig. 2,part 2, b-1 and b-2) also clearlyshowed a marked reduction in tumor size. Marked infiltration ofCD31CD81 T cells was observed in the biopsied sample of case3 after treatment (Fig. 2,part 3, a–c). Injection of the activatedcells into metastatic tumors at the left supraclavicular LN re-sulted in complete disappearance after the second treatment(data not shown). Furthermore, the tumor size of liver metasta-ses in the S2 (Fig. 3a-1) and S4 (Fig. 3a-2) areas of case 5 wasmarkedly reduced after the fourth treatment (Fig. 3,b-1 andb-2). This patient received regional injections of the activatedcells into metastatic LNs in the left supraclavicular area but notinto the liver. A significant decrease in size of the metastatictumor on the neck in the supraclavicular LN was observed incase 9 after the seventh treatment (Fig. 4,a andb). The durationof PR was 11, 9, and 7 months in cases 3, 5, and 9, respectively.No additional or subsequent treatment was given to these re-sponders while they were in response.

In addition, the serum CEA levels largely decreased from21.9 ng/ml before treatment to 9.8 ng/ml after the fifth treatmentin case 9, who showed PR (Fig. 5). The levels also significantlydecreased from 334 ng/ml before treatment to 170 ng/ml afterthe fourth treatment in case 11, who showed SR (Fig. 5). Thedecrease in the size of the para-aortoarterial LN after the fifthtreatment of case 11 as measured by CT is shown in Fig. 6.

Laboratory Markers. PBMCs stimulated with autolo-gous tumor cells and IL-2 were measured for their activity toproduce IFN-gin response to autologous tumor cells and threeallogeneic tumor cells (KE3,A2/A24; TE10,A24/A26; K562).PBMCs cultured with IL-2 alone that were not used for thetreatment were also measured for their activity as the control.The PBMCs stimulated with autologous tumor cells and IL-2produced a significantly higher level of IFN-g in response to theautologous tumor cells than those in response to any of the otherthree target cells (case 1) or to those in response to K562 targetcells (cases 3, 5, 6, and 9; Fig. 7a). The stimulated PBMCs ofcase 3 (HLA-A24/A26), case 5 (HLA-A2/), and case 9 (HLA-A24/A11) also produced significantly higher levels of IFN-g byrecognition of allogeneic but HLA-A locus-matched tumor cellsthan those produced by recognition of K562 target cells. Four(cases 1, 3, 5, and 9) of these five patients responded to thetreatment, whereas the remaining patient (case 6) had PD. Therewas no significant difference among the levels of IFN-g pro-duction by the stimulated PBMCs in response to the four targetcells tested in the remaining six patients who had either SR (n52) or PD (n 5 4; data not shown). Similarly, there was nosignificant difference among the levels of IFN-g production bythe control PBMCs by recognition of the four target cells testedin any of the 11 patients. The results of PBMCs of the respond-ers are shown in Fig. 7a.

These results suggested that the CTL activity was observed

in the stimulated PBMCs of 5 cases (cases 1, 3, 5, 6, and 9),whereas LAK cell activity was observed both in the stimulatedPBMCs of the remaining 6 cases and in the control PBMCscultured with IL-2 alone in all of the 11 cases. A 6-h51Cr-release assay was used in cases 1 and 2, in which a relativelylarge number of cells were available for the study (Fig. 7b). ThePBMCs of case 1 (HLA-A24/A26) showed significantly higherlevels of cytotoxicity against HLA-A241 esophageal cancercells than those against MKN28 (HLA-A31/) stomach cancercells or K562 target cells. In contrast, there was no significantdifference among the levels of cytotoxicity either by thestimulated PBMCs in case 2 or by the control PBMCs in cases1 and 2.

Eleven patients were divided into the three groups asfollows: responder group (n5 4, 1 CR 13 PR; cases 1, 3, 5,and 9), SR group (n5 2; cases 8 and 11), and PD group (n55; cases 2, 4, 6, 7, and 10) to find laboratory markers useful forpredicting the clinical response. Freshly isolated PBMCs (n 557 from 11 cases) consisted of 716 4% CD31 T cells, 446 3%CD41 T cells, 186 5% CD81 T cells, and 56 2% CD161 NKcells (Table 3). The PBMCs stimulated with autologous tumorcells and IL-2 (n5 57 from 11 cases) consisted of 866 5%CD31 T cells (P 5 0.0007versusfreshly isolated PBMCs),28 6 3% CD41 T cells (P, 0.0001), 426 4% CD81 T cells(P , 0.0001), and 186 4% CD161 NK cells (P5 0.0031). Thepercentages of surface markers of the control PBMCs (n 5 30from 11 cases) cultured with IL-2 alone were similar to those ofstimulated PBMCs, although the percentages of CD81 T cellsand of CD161 NK cells in the stimulated PBMCs were slightlyhigher than those in the control PBMCs. In the responder group,the percentage of CD81 T cells in the stimulated PBMCs (55620%) was significantly higher than that of freshly isolatedPBMCs (226 3%), whereas that in the stimulated PBMCs ofthe SR group or the PD group was not significantly differentfrom that of fresh isolated PBMCs. The percentage of CD161

Table 3 Surface markers of PBMCs

PBMCscultured with

% positive (mean6 SD)

CD3 CD4 CD8 CD16

None (fresh)(n 5 57)

716 4a 446 3 186 5 56 2

GroupResponder 726 5 456 3 226 3 76 1SR 756 20 416 14 146 7 46 2PD 716 5 446 4 196 5 66 3

Tumor and IL-2(n 5 57)

866 5 286 3 426 4 186 4

GroupResponder 826 5 306 2 556 20 176 4SR 826 23 286 9 286 22 176 5PD 836 7 306 8 336 12 156 8

IL-2 (n 5 30) 886 4 316 4 376 5 146 5Group

Responder 836 4 326 6 386 3 136 2SR 766 23 296 9 346 11 136 4PD 806 7 326 7 326 16 136 7a Brackets indicate the two groups in which the values were sta-

tistically significant (P, 0.05 by a two-tailed Student’st test).




NK cells in stimulated PBMCs of the responder group (1764%) and SR group (176 5%) was significantly higher than thatin the freshly isolated PBMCs of the responder group (76 1%)and of the SR group (46 2%), respectively, whereas that inthe stimulated PBMCs of the PD group (156 8%) was notsignificantly different from that of the freshly isolated PBMCs(6 6 3%).

The mean number of administrated lymphocytes percase was 8.23 109 cells in the responder group, 4.23 109

cells in the SR group, and 2.73 109 cells in the PD group.The mean number of administrated lymphocytes per injection

was 1.23 109 cells in the responder group, 0.73 109 cellsin the SR group, and 0.73 109 cells in the PD group. Akinetic study showed no significant difference in the meannumber of WBCs, PBMCs, or CD31, CD41, or CD81 cellcounts that were measured before treatment and after thesecond and fourth treatments in these three groups (Fig. 8). Incontrast, the mean number of CD161 lymphocytes in theperipheral blood after the second and fourth treatment in theresponder group significantly increased compared with thatbefore treatment in the responder group (Fig. 8). Thenumber of CD161 lymphocytes was also significantly higher

Fig. 8 Kinetic study of cell numbersand their phenotypes in peripheralblood. Number of WBCs; PBMCs inWBCs; and CD31, CD41, and CD81

cells in PBMCs were measured beforetreatment and after the second andfourth treatments. The 11 patients weredivided into the three groups as fol-lows: responder group (n5 4, 1 CR13 PR); SR group (n5 2); and PD group(n 5 5). p, #, two-tailed Student’st testwas used for statistical analysis.




than those after the second and fourth treatments in the PDgroup.

DISCUSSIONA number of clinical studies of adoptive immunotherapy

using mostly LAK cells and TILs have shown a significantresponse rate in patients with melanoma, renal cell carcinoma,and some other cancers (16–21). However, there has been noreport on the clinical studies demonstrating the obvious tumorregression of advanced esophageal cancers, to the extent that wesearched in the literature. This study showed that locoregionalcellular immunotherapy resulted in marked tumor regression in4 of 11 patients with advanced or recurrent esophageal cancer.Our report seems to be the first report showing clear evidencefor the potential of the application of immunotherapy for esoph-ageal cancer patients.

The adverse events in all of the 11 cases were moderate andtolerable. There was no relationship between the number ofadministered cells and adverse effects. Grade 1 fever was oftenobserved and might have been attributable in part to productionof cytokines by the administered lymphocytes (16). Other ad-verse effects included grade 3 hypotension in one case and grade2 nausea and diarrhea in one other case. These adverse effectsmay also have been attributable to cytokine production frominjected activated lymphocytes. These results suggest that thisregimen of cellular immunotherapy was safe.

We provided the activated lymphocytes by stimulation ofPBMCs with autologous tumor cells and IL-2in vitro. Theseactivated lymphocytes consisted of 42% CD81 and 28% CD41

cells. CTL activity was observed in these PBMCs from 5 (cases1, 3, 5, 6, and 9) of the 11 cases, and 4 of them had significanttumor regression (1 CR and 3 PR), although the detailed studiesof CTL activity, including the51Cr-release assay against varioustarget cells at different E:T ratios, were not carried out, mainlybecause of the limited number of cells for thein vitro analyses.LAK cell activity, instead of CTL activity, was observed in thePBMCs of the remaining six cases, and none of them showedmajor tumor regression (2 SR and 4 PD). LAK cell activity wasobserved in the control PBMCs cultured with IL-2 alone in allof the 11 patients tested. These results suggested that CTLs butnot LAK cells were needed to achieve the tumor regression inthis regimen of locoregional cellular immunotherapy. Conse-quently, the CTL activity of administered cells could be anappropriate laboratory marker to predict the clinical response.This phenomenon was supported by the fact that the meanpercentage of CD81 T cells (55 6 20%) of the stimulatedPBMCs of the responder group was highest among those of thedifferent groups tested, and it was significantly higher than thatof fresh PBMCs. The other laboratory marker for prediction ofclinical response would be the percentage of CD161 NK cells inthe peripheral blood, which significantly increased in PBMCs ofthe responder group after treatment. These results suggest thatboth CTLs at the tumor sites and NK cells in peripheral bloodwere needed for tumor regression in this regimen. Therefore,both CTL activity in the injected cells and percentage of CD161

cells in the peripheral blood might be useful laboratory markersfor monitoring the clinical response to this locoregional cellularimmunotherapy. These results, however, must be confirmed by

a large-scale clinical study. The magnitude of CTL activity ofthe activated TILs was well correlated with clinical efficacy inmetastatic melanoma patients in the regimens of adoptive cel-lular therapy with high-dose IL-2 (22). In contrast, no reliablelaboratory markers for PBMCs, including the percentage ofCD161 cells, were found in the past decade, regardless ofnumerous trials in the field of immunotherapy using LAK cells,TILs, or cytokines.

It is of note that the injection of cells into the left supra-clavicular LN in case 5 resulted in both the disappearance oftumors at the injection site and the decrease in tumor sizes at theuninjected site (liver). Some element of systemic immunitymight been achieved by local therapy in this case, but this typeof tumor regression was not observed in any other cases tested.It is also of note that regional treatment of pleural effusions andascites was ineffective in 5 of the 11 patients. Subsequently, theapplication of this locoregional immunotherapy to the control ofcancer cells in pleural or abdominal regions is not recom-mended. In addition, prior chemoradiotherapy is not recom-mended for this immunotherapy. Three of five patients whoreceived no prior chemoradiotherapy responded to the immuno-therapy, whereas only one of six patients who received priorchemoradiotherapy responded to the immunotherapy. Thiscould be in part attributable to the number of cells available forthe treatment. The mean number of administered cells per in-jection was 1.23 109 cells in the responders, whereas it was0.7 3 109 cells in the other groups. Prior chemoradiotherapyshould suppress T-cell proliferation in response to IL-2 andautologous tumor cells.

In conclusion, we have shown that this treatment regimenwas safe and resulted in tumor regression in 4 of 11 patientswith advanced or recurrent esophageal cancer. A large-scaleclinical trial is recommended to confirm the evidence from thisPhase I clinical study.

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2000;6:4663-4673. Clin Cancer Res Uhi Toh, Hideaki Yamana, Susumu Sueyoshi, et al. Advanced Esophageal CancerLocoregional Cellular Immunotherapy for Patients with

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