Immunotherapy in lung cancer.

More research and new treatment options are needed in all stages of lung cancer. To this end immunotherapy needs a revival in view of recent improved technologies and greater understanding of the underlying biology. In this review we discuss mechanisms of tumour immunotherapy, non-specific, specific and adoptive, with particular reference to a direct therapeutic action on all subtypes of lung cancer.

Summary More research and new treatment options are needed in all stages of lung cancer. To this end immunotherapy needs a revival in view of recent improved technologies and greater understanding of the underlying biology.
In this review we discuss mechanisms of tumour immunotherapy, non-specific, specific and adoptive, with particular reference to a direct therapeutic action on all subtypes of lung cancer.
Keywords: immunotherapy; lung cancer, BCG; vaccine CURRENT TREATMENTS IN LUNG CANCER Lung cancer remains the leading, cause of cancer death in Western countries (Boringa et al. 1993) with more than half a million new cases diagnosed annually worldwide. including, 40 000 in the UK.
About 80% of these tumours are of non-small-cell histological type. including squamous (40%7). adeno-(40%). and large-cell carcinoma (20%). The 5-year sur-vival of patients w ith non-smallcell lung cancer (NSCLC) is stage related and remains poor across all stages at about 12%. The treatment of choice for NSCLC is surgery. but only 20% of tumours are suitable for potentially curative surger- (Hoffman et al. 1980). Small-cell lung cancer accounts for the remaining 2'0% of lung cancer and. despite displaying initial chemosensitivitv. cure is achieved in onlv a minonty of patients.
How can survival be improxved in lung cancer'? Different strategies have been employed to improve the outcome. Despite the suggested benefit of adjuvant chemotherapy (NSCLC Group. 1995). the role of adjuvant therapy in operable disease awaits confirmation in large adjuvant trials. The value of preoperatixe (neoadjuvant) chemotherapy in NSCLC stage I. H and [Ila lung, cancer is currently the focus of large randomized trials. including, the MRC LU22 national study. The interest in this approach comes from the encouraging positive effect of this treatment in two randomized studies (Rosell et al. 1994: Roth et al. 1994. which have shown improved survix al in patients treated with chemotherapy before surgery compared with suryerv in resectable stage IILA disease. In unresectable stage III disease there is accumulatincg evidence to support the use of chemotherapy before local treatment (radiotherapy or surggery). with trials show ing a small survival benefit with the combined approach and improxed quality of life compared with local treatment alone (Sause et al. 1995: Cullen et al. 1997 (Thatcher et al. 1997). This approach is being inx estigated in randomized trials. However. the problem of maintaining a chemotherapy-induced remission remains and needs innoxvatixve approaches.
As in all types of lung cancer current treatment options are limited: there is thus a need to explore new treatments and with improved technology look again at older treatments such as immunotherapy. This systemic anti-tumour approach with low toxicity could form part of a panoply of future treatments in lung cancer with chemotherapy used against micrometastases. radiotherapy or surgery against local disease and possibly immunotherapy for maintenance of remissions.

TUMOUR IMMUNOLOGY
Cancer cells differ from normal cells both qualitatively and quantitativelv. These differences are due to abnormal alycosxlation of surface proteins. expression of viral. mutated or overexpressed oncogene products or differentiation anticens (Boon. 1997: Wevnants. 1997). Both the innate (natural killer (NK) cells. macrophages and granulocytes). and the specific arms (T and B cells) of the immune system can recognize these tumour-specific or -associated antigens (TS/AA). NK cells that detect abnormal gylvcosylated proteins are efficient at clearing low tumour load. especially blood-borne micrometastases. and kill cells that express a low level of HLA class I molecules. On the other hand. T cells only recognize and are stimulated by a high level of HLA molecules. They interact via their T-cell receptor with a specific peptide antigen presented on a groove of an HLA molecule. This is the first signal delivered to T cells. For T-cell activation to take place. a second signal has to be delivered via lvmphokines such as interleukin (IL-2) or an interaction betwxeen the T-cell molecules (e.g. CD28) and co-stimulatory molecules (B7.1 ) on the antigen presenting cell (APC) (Schwartz et al. 1992). Usually. these sianals are delivered via professional APC-like dendritic cells.
The fact that tumour cells are different from normal cells is not enough for efficient tumour control and. during the past few years. progress has been made in understandinc the immunological escape mechanisms of tumour growth. T cells. which has-e the capacity of immunological memory (their response is amplified at a second antigen encounter). are pixotal for any specific immune response either because they mediate the killing of the tumour cells as in the case of cxtotoxic T lymphocytes CTLs) or because thev secrete cvtokines. as both T helper and CTLs do. and regulate NK and CTL actix-ation and antibody production by B lymphocxtes. T-cell anergy to tumour cells could occur from the absence of tumour-specific antigens. defective antigen presentation or lack of co-stimulator-signals (Pardoll et al. 1993 (Huang et al. 199$).
Two main approaches are used to target TS/AA for tumour killing. The first is active immunotherapy. which aims to boost the anti-tumour immune response of the patient. using for example a therapeutic tumour v accine. The second is passixve immunotherapy. x-hich bypasses the patient's immune system bv administration of tumour-specific antibodies or T cells. The tx o approaches are not mutually exclusive and can be syner2istic. In addition. a complex network of cvtokines and cells regulate the immune response and any immune therapx that can influence any part of it (antigen presentation. T-cell or antibody response. cvtokine production) could in theorv have an effect on tumour g-rowth. Cvtokines are arbitrarily diVided into type 1 [IL-2. interferon gamma (IFN-y). IL-12]. w-hich promotes T-cell response. and type 2 (IL-4. 5. 6 and 10). which promotes antibody response (Romagnani et al. 1997). It is thought that tiltinc the balance towards a type 1 response is beneficial in the context of solid tumours but this rule is too simple to fit all situations. Therefore.
non-specific immunomodulators that could modifx the quality and the intensity of an immune response could help boost an antitumour effect.

IMMUNOTHERAPY IN LUNG CANCER Non-specific immunostimulants
There hax e been sex eral randomized clinical trials using the bacille Calmette-Guerin (BCG) -Xaccination in NSCLC Awith various administration schedules (Table 1). These trials reported mixed but mainly negatixve results. Although the initial tnrals by McKneally et al 1981 ) showed a statistical survival benefit for the X accinated arm. subsequent trials failed to shox-anx sur ix al adxantage. Similarly in SCLC. BCG x-accination following, four cycles of chemotherapy shoxed no benefit in terms of complete response. disease-free survival or surnival (Maurer et al. 1985).
We are at present testing in lung cancer patients the use of MvNcobacterium *vaccae (MV). a heat-killed preparation dex oid of toxicity. with a particular interest in combininc this approach wxith chemotherapythe rationale being that specific tumour activity may be seen after release of tumour antiaens by chemotherapy combined xxith non-specific immunostimulation by MV (O'Brien et al. 1997). The Ludwia Lung Cancer Group (1985) studied the administration of intrapleural Corvnebactenium parnum in a randomized phase III trial of 475 patients with resectable lunc cancer. The treated group had a significant decrease in survival. Lexramisole is used in association w-ith 5-fluorouracil (5-FUI in colon cancer but appears. ox erall. to make the outcome w orse in lung cancer. It has been administered in different settings as shox-n in Table 2. EL-2 used alone or in combination xxith other cvtokines or ly mphokine-actixated killer (LAK) cells in phase II trials in NSCLC has induced some responses (Table 3). In the Eastern Co-operative Oncologx group trials. IL-2 xxas used alone or with IFN'-P: only 3 out of 73 patients showed a response. x ith a median sunrixal of 35.6 w-eeks and no added adxantage xxith IFN-f (Kriegel et al. 1991). Lissoni et al (1994) randomized 60 patients xith advanced cancer to receixe low--dose IL-2 and melatonin (pineal immunomodulating hormone) or cisplatin and etoposide chemotherapy. Although the response rates were not significantly different (24% and 19%7 respectixely). the mean progression-free period and percentage survival xxere significantly different at 1 X ear in favour of the immunotherapy arm.
The use of LFN alone has not demonstrated activitagainst NSCLC. but synergy has been proposed between interferon and chemotherapy (Bou-man et al. 1990). Phase II studies of interferon and chemotherapy show ed response rates comparable wvith chemotherapy alone wxith acceptable toxicity (Table 4A. Phase III trials usin2 LFN alone or IFN and chemotherapy in NSCLC are shown in Table 5. These studies showed no statistically significant difference in time to progression or surnix al. Randomized trials have examined the use of recombinant IFN-a as maintenance therapy following response to chemotherapy in SCLC (Table 6). All these studies shoxxed no surnixal improxement for the IFN arm except for one study by Mattson et al ( 1992). In this study. 237 patients were randomized following chemotherapx and radiotherapy treatment to no treatment or maintenance treatment x ith lEN-ax. A statistically significant difference in long-term sun-ix al and surnixval in limited stage disease xxas found in faxour of the immunotherapy group. In conclusion. the concept of merely boosting the immune svstem without presentation of British Joumal of Cancer (1998) Silva (1996) IFN-a Cisplatin. mitomycin C.

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Overall response rate 3 x 106 unit D,-D vindesine 51o comparable with chemotherapy TIW, three times a week: IFN-a. interferon alpha; MU, million unit.
antioens is probably the reason for the overall lack of success of these approaches.

SPECIFIC IMMUNOTHERAPY
Giving lung tumour-specific or -associated antigens (TS/AAs) has been tested using either irradiated autologous or allogenic tumour cells, tumour lysates and soluble tumour antigens. usually with an immunological adjuvant such as BCG. Studies of active specific immunization trials in lung cancer are shown in   Ardizzoni (1993) Cisplatinum/epirub.cin/cyckophosphamide 182 Increase response rate but no improvement or CEP + IFN-a in DFS or OS Cinaco (1995) Preoperative (mitomycin, vinblastine, 110 No significant difference in DFS or OS cisplatinum) alone or cisplatinum. etoposide. alpha thymosin and IFN-a Ardizzoni (1995) Mitomycin C, ifosfamide, cisplatinum 93 No significant difference in DFS or OS alone (MIP) or MIP and IFN-a cisplatinum and carboplatinum Salvati (1996) Ifosfamide alone or ifosfamide followed by 22 No improvement in DFS or OS thymosin alpha and low dose IFN-a DFS. disease-free survival; OS, overall survival.  Souter (1981) No Rx vs intradermal injecbon of 80 No survival difference autologous tumour cells and C. parvum Stack (1982) No Rx vs Autologous tumour cells 83 No survival difference and BCG Hollinshead (1987) No Rx, CFA alone. CFA + TAA 243 Survival advantage for immunotherapy arm (see text) Price-Evans (1987) No Rx vs irradiated autologous 120 No survival difference cells and BCG Takita (1991) No Rx, CFA alone 85 Survival advantage in immunotherapy group TM + CFA TM. tumour-associated antigen; MTX, methotrexate: CFA, complete Freund's adjuvant. immunotherapy groups (38 months. 71 months. 106 months respectively) (Takita et al. 1991). More recently. Carbone and his colleagues (Gabrilovich et al. 1997) have vaccinated lung cancer patients with peptides encoding mutated ras and p53 oncogene products. They are using the dendritic cell vaccination approach: dendritic cells are purified from cancer patients loaded with the specific peptide antigens and reinfused intravenously to the patient. The rational behind this approach is that dendritic cells are professional APCs. w%hich express high levels of co-stimulatorv molecules and HLA molecules and so an efficient T stimulation should follow after dendritic cell vaccination. Rosenberg et al (1986). pioneered the use of tumour-infiltrating lymphocytes (TILs) and showed that adoptively transferred TILs exerted anti-tumour activity in patients with cancer. The ability of IL-2 to expand these cells in vitro made such an approach feasible. The initial few small trials that used adoptive immunotherapy alone or in combination wvith IL-2 in advanced lung cancer. demonstrated the feasibility of such an approach (Bemstein et al. 1989: Kradin et al. 1989: Faradji et al. 1991 ). A more recent study (Kimura et al. 1996)  following curative resection. The patients were randomized to receive IL-2 and LAKs following two courses of combination chemotherapy (cisplatin, vindesine and mitomycin) or chemotherapy alone. The 5-and 7-year survival rates of the chemo-immunotherapy group and chemotherapy group were 58.2% and 31.5% respectively in stage H and IIIA patients. This difference was statistically significant (P=0.0038). In patients undergoing non-curative resection, Kimura et al (1995) reported a survival benefit for the immunotherapy arm (IL-2 and LAK) following randomization of 105 patients to chemotherapy, radiotherapy or immunotherapy. The 7-year survival rate was greater in the immunotherapy group compared with the chemotherapy and chemo-radiotherapy groups (39.1%. 12.7%, P < 0.01).

FURTHER AVENUES FOR IMMUNOTHERAPY
The recent advances in tumour antigen characterization will encourage the development of more standardized anti-tumour vaccines. For example, the identification of a series of melanomaspecific gene products termed MAGEs has raised the hopes that similar specific antigens can be found in other tumours. Indeed. some of the MAGE antigens are expressed in about 40% of NSCLCs (Weynants et al, 1994) Another approach is to provide the TS/AA via irradiated wholecell tumour vaccines. A multitude of preclinical studies have shown that ex vivo transfection of cytokine genes [e.g. IL-2. granulocyte-macrophage colony-stimulating factor (GM-CSF)] and co-stimulatory molecule genes can augnent the immunogenicity of the cell vaccine in vivo. This is improved by gene combination. e.g. B7.J and IL-2 genes (Gaken et al. 1997) or GM-CSF and IL4 (Wakimoto et al, 1996).
The IL-2 gene has been introduced into TILs via a retroviral vector to improve IL-2 delivery into the tumour. A recent phase I study used this approach in ten patients with advanced NSCLC with pleural effusion who showed some improvement in the pleural effusions (Tan et al. 1996).
Targeting the tumour by in vivo gene therapy is another option that at present is only feasible by local intratumoral delivery. It is likely that in the next 10 years progress in gene delivery systems will allow in vivo gene targeting after i.v. injection of the vector. One option is to deliver genes coding for immunostimulatory molecules such as IL-2 (Tursz et al. 1996). Another option is to correct genetic abnormalities in tumour cells. Roth et al. ( 1996) have delivered a retroviral vector containing the wild-type p53 gene directly into p53-mutated NSCLC tumours in nine patients with advanced disease. Wild type p53 regulates the progression of cells in the cell cycle from GI to the S-phase. Mutation ofp53 is usualy a late event in lung cancer and leads to uncontrolled growth of cancer cells. Reintroduction of the dominant wild-type (unmutated gene) can revert this process. Roth et al (1996) observed tumour regression and apoptosis in the tumours of some treated patients. Another option is to introduce a gene whose product converts a non-toxic pro-drug to a toxic compound. Herpes simplex virus thymidine kinase (HSV-TK) in combination with endogenous TK phosphorylates the pro-drug gancyclovir (GCV) to toxic gancyclovir triphosphate (GCV-PPP). Interestingly, GCV-PPP can enter untransfected neighbouring tumour cells through communicating gap junctions. and this leads to death of non-expressing HSV-TK cells (local bystander effect). This is important as only a small proportion (20%) of the cells in a tumour need to express HSV-TK to bring about 100% of tumour cell death. An inflammatory raction in response to the cell death with accumulation of type-I cytokines fiuther increases the bystander effect by boosting local and systemic immunological recognition of the tumour cells (Freeman et al. 1997). Recently, this approach has been used in the treatment of pleural mesothelioma in rats. HSV-TK expressing adenoviral vectors were injected directly intrapleurally with significant reduction in tumour burden (Elshami et al. 1996). Human studies are on-going (Treat et al. 1996).
Another approach is to use anti-idiotypic antibodies. These antibodies are raised against monoclonal antibodies recognizing cellsurface tumour antigen and have a similar shape to the tumour antigen. This approach is currently the focus of an EORTC trial (SIILVA study) that uses an anti-idiotype BEC2 (anti-idiotype to ganglioside GD3) combined with BCG adjuvant in SCLC. A pilot study (Grant et al, 1996) using BEC2/BCG in patients with SCLC showed minimal toxicity, with median survival not reached after 15 months. which compares favourably with historic controls.

CONCLUSION
Overall outcome from standard treatments for lung cancer remains poor. Immunotherapy could have an important role to play in the treatment of lung cancer. Active specific vaccination is safe to administer and available data suggest beneficial effect in the adjuvant setting; recent advances in tumour antigen characterization and gene therapy will aid the design of more effective vaccines. randomised trial in patients With localised. inoperable disease Iabstact). Lng 18Suppl.