The conventional treatments for cervical cancer are dependent on the stage of the cancer. For cervical intraepithelial neoplasia (CIN) or dysplasia stage I, routine observation is recommended. For stage II CIN, cryotherapy and laser vaporization is advocated. If it is CIN III or micro-invasive lesion, then loop excision or cone biospy is done to further characterize the cancer lesion following which if it is a micro-invasive lesion or FIGO stage I, simple hysterectomy followed by careful observation after adequate cone is the treatment employed [12]. The 5 year survival rate exceeds 95 % with appropriate treatment. For Stage IB or IIA, radical hysterectomy with or without pelvic node dissection or external beam, intracavitory radiotherapy is employed. Both treatments give a 5 year survival rate of 80–90 %. If tumour is present in the margins of hysterectomy specimen or extends to pelvic nodes, then radiotherapy is given after surgery to decrease recurrence. Presence of pelvic node metastasis and bulky tumour provide poorer prognosis. In a study that analyzed the outcome of chemotherapy with cisplatin in combination with radiotherapy versus that with radiotherapy alone, in patients who underwent subsequent hysterectomy, it was found that chemotherapy with cisplatin halved the risk of disease progression and death [13]. For stage IIB, III or IVA, pelvic radiotherapy with chemotherapy is the strategy employed and for stage IVB, chemotherapy with or without radiotherapy is advised. The 5 year survival rate is 65 %, 40 % and less than 20 % for stage IIB, III and IVA respectively. In general, concurrent chemotherapy with cisplatin or flurouracil improves the prognosis with stages IIB through IVA [12, 14, 15]. However, a meta-analysis reported that though neoadjuvant chemotherapy reduces need for radiotherapy in FIGO stage IB1 to IIA cervical cancer, it did not improve survival compared to patients in whom primary surgical treatment was alone performed [16]. The disadvantages with these conventional treatments include anemia, nausea, vomiting, bleeding disorders, hair loss, fertility problems etc., with chemotherapy and fatigue, diarrhoea, risk of secondary cancer etc., with radiotherapy. With surgery, in addition to the risk of damage to the surrounding organs, it has been indicated that it induces development of distant metastasis [17].

The recent therapies for cervical cancer include the use of chemotherapeutic agents with immunomodulatory effects such as cyclophosphamide, doxorubicin, and paclitaxel. These agents, by virtue of their apoptotic and immunomodulatory properties, help in chemoimmunotherapy. Imiquimod and gemcitabine (GEM) are other two recent agents being employed for chemoimmunotherapy [18]. Inhibition of tumor angiogenesis and epidermal growth factor receptor directed therapies are also being researched upon for cervical cancer [19]. Gene therapy trials that are on-going for cervical cancer are currently aimed at studying the safety, tolerability, and immunogenicity of HPV E6 and E7 oncogenes in combination with immunotherapy and chemotherapeutic drugs. Use of antisense RNA to block the translation of HPV E6 and E7 mRNA and the induction of cancer cell death by administration of specific siRNAs for HPV16/18 E6 and E7 oncogenes are other novel approaches being considered for cervical cancer [18]. The vaccines for HPV will be dealt later in this chapter. Immunotherapy is another major arena in treatment of cervical cancer and the major aim of this chapter is to present an overview of immunotherapy approaches for cervical cancer.

Immunotherapy for cancer has its beginning in the 1950s [20] with the preliminary studies focusing on use of immunization based approach to cancer immunotherapy. A study in 1961 [21] performed in a female patient with metastatic choriocarcinoma involved active immunization using leucocytes from her husband and passive immunization using antibodies generated in rabbits using her husband’s spermatic fluid after hysterectomy and chemotherapy. The combined immunization approach was based on the hypothesis that post-gestational choriocarinoma originates from the placental tissue and thus may act as an antigenic stimuli for the mother’s body to produce antibodies against it. In order to increase the production of antibodies and to enhance their efficacy against the tumour cells, the consort’s leucocytes as active immunization and antibodies generated in rabbits using consort’s spermatic fluid as passive immunization were used. The results showed that the general heath condition of the patient improved with reduction in the size of the metastases [21]. Clinical trials on 21 patients with acute lymphoblastic leukemia using allogenic hematopoietic cells after total body irradiation in 1965 resulted in remission in three patients [22]. Studies similar to this one started the era of adoptive immunotherapy. Immunotherapy for cancer in addition to being explored in western nations can attribute its growth to the huge number of studies done in Japan. Immunotherapy by means of non-specific immunopotentiators like fungal polysaccharides was one of the first approaches in modern immunotherapy for cancer in Japan in the 1970s [23]. The 1970s and early 1980s witnessed the use of recombinant cytokines like interleukins, interferons and tumour necrosis factors in cancer immunotherapy [23, 24]. Transfer factors which were originally described as factors that induce recipients to express cell-mediated immunity [25] in an antigen specific manner gained prominence as immunotherapy agents in this period. When 100 patients with high risk Stage I melanoma were treated with transfer factor after surgery to reduce recurrence, nine patients had a recurrence of disease and in the rest, survival rate was 99 % at 5 years [26]. A randomized double-blind study was done in invasive cervical cancer patients comparing transfer factor administration and placebo after radical surgery and irradiation among whom the patients treated with transfer factor had a significantly lesser recurrence of cancer [27]. However, research into transfer factors did not continue for long owing to the discovery of interleukins and also due to the risk of biological contamination when transfer factors from bovine or other humans were used for immunotherapy. A phase II trial in 48 patients with metastatic renal cell carcinoma with human leukocyte (alpha) interferon demonstrated complete response in 2.5 % of the patients, partial response in 14 % and minimal response or stabilization in 23 % of the patients [28]. In six-bladder cancer patients, who received intralesional injections of interleukin 2 (IL-2), tumour regressions without apparent side-effects were reported [29]. Several studies done during these period demonstrated positive effects with the use of cytokines based immunotherapy [30–32]. However, systemic administration of these recombinant cytokines were associated with significant side-effects including fever, chills, fatigue, anorexia, hepatocellular enzyme elevation and granulocytopenia [23, 31, 32]. These side-effects led to the research on the immune-cell therapy or cell-based immunotherapy.

The earliest approaches in cell-based immunotherapies involved the infusions of lymphokine activated killer (LAK) cells and tumour infiltrating lymphocytes (TIL) along with administration of high dose of Interleukin 2 (IL-2). The team led by Dr. Rosenberg was a pioneer in this form of cell based immunotherapy [33, 34]. The functional definition of LAK cells is that these are lymphocytes, which after culturing in IL2 are capable of lysing fresh tumour cells in vitro. They are lymphocytes consisting mainly of activated T cells with characteristics of larger granular lymphocyte morphology [35]. In Rosenberg et al.’s study on adoptive transfer of autologous LAK cells and recombinant IL-2 in 41 patients with advanced cancer, 14 patients had tumour regression [36]. In another observational study on administration of LAK cells and IL-2 in 25 patients with advanced cancer, objective regression of more than 50 % of tumour volume was observed in 11 patients, 1 patient with metastatic melanoma had complete tumour regression, 9 patients with pulmonary or hepatic metastases from melanoma, colon cancer and renal cell cancer along with a patient with primary lung adenocarcinoma had partial remission [37]. While these studies demonstrated clinical effectiveness, there were other studies which showed that the treatment with LAK in combination with IL-2 was ineffective [38] and the use of IL-2 had severe toxic effects [38, 39]. The side-effects were mainly related to the co-administration of IL-2 and the toxic effects stopped when IL-2 was stopped [39]. TIL are lymphocytes which have migrated from the blood stream into the tumour. The TILs are a lymphocyte population, in which majority of the cells are T cells and few are NK cells or B cells. These TILs were obtained from tumours and used for adoptive immunotherapy [40, 41]. Administration of TILs to 12 patients, of whom 6 had melanoma, 4 had renal cell carcinoma, 1 had breast carcinoma and another had colon cancer resulted in regression of pulmonary and mediastinal masses in one of the melanoma patients, regression of lymph node metastasis in the patient with breast cancer and in a patient with renal cell carcinoma [42]. One study by Rosenberg et al. suggested that TILs are 50–100 times more effective than LAK cells in therapeutic potency [43]. In another study, in which autologous TILs were administered along with IL-2 in 28 patients (13 with malignant melanoma, 7 with renal cell carcinoma, and 8 with non-small-cell lung cancer), objective tumour responses were observed in 29 % of the patients with melanoma and 23 % in patients with renal cell carcinoma [44]. Though these studies demonstrated positive response, the toxic effects associated with use of IL2 were observed when they were given in combination with TILs too [42, 45]. Further, it has been indicated that TILs obtained from fresh isolated TILs had impaired cytotoxic responses which were hypothesized to be due to production of immunosuppressive factors by tumours and absence of adequate co-stimulatory signals on tumour cells resulting in T cell anergy [46]. A study by Schöndorf et al. revealed that TIL contains a slightly increased CD4/CD8 ratio compared to peripheral blood lymphocytes (PBL). Also IL-4 production is predominant in TIL while IFN gamma production is predominant in PBL thus indicating a downregulated cellular immunity in TIL and an increased cytotoxic immune response in PBL. This study attributed this to be the reason for the decreased clinical effectiveness observed in few studies on TIL in cancer patients [47]. After these preliminary approaches, several types of cell-based immunotherapies have been advocated using dendritic Cells (DCs), γδ T cells (gamma delta T cells) natural killer (NK) cells, NKT cells, activated T lymphocytes, cytotoxic T lymphocytes (CTLs) and lymphokine activated killer (LAK) Cells. We will consider the application of these cell-based immune therapies in cervical cancer.

DCs are antigen presenting cells of the immune system, which process the antigen and initiate several immune responses like sensitization of MHC-restricted T cells to antigens, the rejection of transplanted organs, and the formation of T-dependent antibodies [48]. Immature DCs which are monocytes cultured for 5–7 days in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 lack the full stimulatory activity on T cells and also can be suppressed by factors like transforming growth factor beta (TGF-β) produced by tumours. However, mature DCs have higher T cell stimulatory activity, decreased sensitivity to immunosuppressive factors like TGF-β and the expression of selected chemokine receptors are upregulated in these mature DCs and guiding their migration to secondary lymphoid organs for priming antigen specific T cells [49]. Immunotherapy with DCs has been referred to as vaccination which has shown effectiveness in various malignancies [50–52]. The same has been applied in higher proportions for prostate cancers wherein the DCs pulsed with prostate-specific antigen peptides have been administered [53–55]. Sipuleucel-T (Trade name: Provenge), which has shown effectiveness for prostate cancer in randomized clinical trials [56, 57] has been approved by FDA to treat asymptomatic or minimally symptomatic metastatic hormone-refractory prostate cancer (HRPC) [58]. With reference to DC therapy for cervical cancer, in a phase I dose escalation trial on administration of mature autologous DC pulsed with full-length HPV16/18 E7 oncoprotein and keyhole limpet hemocyanin (KLH) in 10 patients of HPV16/18-infected stage IB or IIA cervical cancer after radical surgery, the results showed that the DC vaccine was well tolerated and generated an immunogenic response in these patients which was inferred from the CD4+ T cell and antibody responses detected by enzyme-linked immunosorbent spot (ELISpot) and enzyme-linked immunosorbent (ELISA) assays, respectively [49]. In another study on 15 stage IV cervical cancer patients in whom HPV E7 antigen-loaded autologous DCs were administrated, the results showed immunologic response in these patients, but no clinical response [59]. A study by Ye et al. indicated that the percentages of CD11c + (DC1) and CD123+ (DC2) sub-sets were decreased in the peripheral blood of the cervical cancer patients and there is accumulation of immature DCs in the peripheral blood of the cervical cancer patients which are impaired in their stimulatory function. Thus, the study suggested that it might not be appropriate to use peripheral blood derived DCs for immunotherapy in cervical cancer patients [60]. Cathelin et al.’s study [61] in 2011, which reviewed the clinical trials on DC-based vaccines, reported that spectacular clinical results have not been observed either with DC vaccines or with DC loading with tumor antigens and therefore their differentiation and activation still requires optimization [61].

As explained earlier, LAK cells were the first forms of cell-based immunotherapies. However, there are not many clinical studies reported on use of LAK cells in cervical cancer. Berezhnaya et al.’s study showed that LAK cells possess more anti-tumour potential than PBL in chemoresistant epithelial tumours including cervical cancers [62]. Another study reported that peripheral blood mononuclear cells from cervical cancer patients can be stimulated with low doses of cytokines for better immune responses against virus infected tumour cells in cervical cancer [63]. Thus, LAK-based immunotherapy needs clinical trials to study their efficacy in cervical cancers.

NK cells are lymphocytes with the ability to target and kill tumour cells and virus-infected cells without the need for any antigenic-specific recognition mechanisms. They are negative for CD3 and positive for CD16 or CD56. They represent 5–20 % of peripheral blood lymphocytes [64]. Studies on NK cells in cervical cancer have been going on from the 1980s. Increased NK cell activity in peripheral blood correlates with reduced cancer risk [65]. Breast cancer cells inoculated in NOD/SCID mice, which possessed NK cell activity, showed development of only a small tumour at the size of inoculation without organ metastasis, while injection of breast cancer cells in NOD/SCID/ccnull (NOG) mice lacking T cell, B cell, and NK cell activity resulted in formation of a relatively large tumour and spontaneous organ metastasis [66]. NK cells have been used as therapeutic agents in several clinical studies for various malignancies. In a phase I trial on ex vivo expanded NK cells in 11 patients with metastatic colorectal cancer and one patient with non-small cell lung cancer, there were no adverse effects in any of the patients and safety was confirmed [67]. Autologous NK cell therapy in nine patients with recurrent malignant glioma resulted in three partial responses (PR), two mixed or minor responses (MR), four no change (NC) and seven progressive disease (PD) in a total of 16 courses of treatment [68]. In a HER-2-positive breast cancer patient with lung metastasis, who was refractory to treatment with various agents including anti-HER-2 therapy, trastuzumab, and lapatinib, re-induction of trastuzumab in addition to NK cell therapy resulted in decrease in level of tumour markers and after combining taxane and capecitabine, lung metastases reduced and the progression-free survival time was 10 months [69]. With reference to cervical cancer, Seltzer et al.’s study examined the cytotoxic ability of NK cells in cervical cancer patients and identified that there was a decrease in cytotoxic ability of peripheral blood (PB) derived NK cells in CIN III and with advanced cervical carcinoma patients. However, after treatment with interferon, there was enhancement of cytotoxic activity of the NK cells in those patients except those with advanced cervical carcinoma [70]. NK cells exert their cytotoxic function through granule-dependent cytotoxicity and the apoptosis pathway in the target cells. Tumour cells have evolved immune evasion mechanisms and HPV in cervical cancer also have strategies for immune cell evasion. NK cell activity is governed by a balance of inhibitory and activating receptors. Studies have shown a deregulation of the receptors in HPV infection. Particularly, down-regulation of NKp30 and NKp46 receptors have been reported to correspond to lower cytotoxic activity of NK cells in cervical cancer patients [71]. A study by Pillai et al. indicated a beneficial effect of in vitro of interleukin 2 (IL-2) and interferon in stimulating the spontaneous cell mediated cytotoxicity of NK cells in cervical cancer especially in early stages [72]. It has also been demonstrated that in patients with cervical cancer undergoing chemotherapy using cisplatin and bleomycin, the quantity of NK cells in the peripheral blood were higher in patients in whom there was good clinical response to chemotherapy. Also, the stimulation with IL-12 increased the cytolytic activity only in those patients who showed good response and thus assessment of NK cells can be considered as an assay to assess the response of the tumour to therapy [73]. Infusion of NK cells and CD3(+) CD16(+) CD56(+) CIK cells in five patients with advanced solid tumors showed that these CIK cells had lytic activity on the cervical cancer cells and the median survival was 4.5 months from the first infusion of the CIK cells [74]. In a Stage IV-A cervical cancer patient with residual lymphadenopathy after radiation therapy, infusion of in vitro expanded NK cell and activated T lymphocyte-based autologous immune enhancement therapy (AIET) resulted in complete resolution of residual lymph nodes with no evidence of local lesion after six infusions [75]. As cancer cells have been identified to develop strategies to escape immune surveillance and NK cells from cancer patients have diminished cytotoxicity compared to healthy individuals, allogenic NK cell therapies have been postulated and are being studied. In a phase II study on allogenic NK cells in recurrent ovarian and breast cancer, infusion of allogenic NK cells resulted in PR in four ovarian cancer patients, stable disease (SD) in eight ovarian cancer and four breast cancer patients and progressive disease (PD) in one ovarian cancer and two breast cancer patients [76]. Allogenic NK cells have been applied in renal cell carcinoma too with positive results [77]. Other strategies that have been postulated to overcome immune evasion by cancer cells is using allogeneic NK cell lines and genetic modification of NK cells to express cytokines, Fc receptors and/or chimeric tumor-antigen receptors [78]. While speaking of NK cell-based immunotherapy, a point that needs to be emphasized is the ability of NK cells to target and lyse cancer stem cells [79, 80]. Cancer stem cells are a population of cells in the tumour that are responsible for initiation of cancer and also play a role in cancer resistance [81] by being usually resistant to conventional therapies like chemotherapy [82]. Cancer stem cells have been identified in cervical cancer and targeting the cancer stem cells in cervical cancer has been proposed as a possible approach to obtain a favorable prognosis in patients with relapsed and metastatic cervical cancer [81]. Thus, NK cell therapy may be a potential therapeutic strategy for cervical cancer due to its ability to deal with cancer stem cells.

CTLs are components of the adaptive immune system which have the capacity to kill target cells using a combination of granule (perforin/granzyme)- and receptor (Fas/tumour necrosis factor) mediated mechanisms [83]. The difference between NK cells and CTLs is that CTLs are antigen specific and they recognize the antigens using a clonally unique T cell receptor (TCR). Target cells are presented to T cells by the antigen presenting cells (APCs) (e.g., DCs). APCs process the antigens and present them to T cells via carriers such as MHC molecules. CTLs are potential anti-tumour therapeutic agents for two reasons. One is the widespread expression of MHC class I molecules that makes it possible to use CTLs against a diverse variety of tumours and the second reason is that the target recognition by CTLs is very sensitive as even a single peptide – MHC class I complex has the ability to stimulate highly active effector CTLs. CTLs have also effector mechanisms like production of interferon γ which has many direct and indirect anti-tumour properties [83]. CTLs have been applied for immunotherapy in several solid tumours. In seven patients with recurrent ovarian cancer, intraperitoneal infusion of tumour specific CTLs resulted in decrease of CA-125 tumour marker and the median survival was 11.5 months [84]. In metastatic melanoma patients with progressive disease, infusion of tumor-reactive T cells resulted in objective clinical response in 5 out of 10 patients [85]. For cervical cancer, cord blood derived CD3+ CTLs induced apoptotic cell death and tumor remission in NOD/SCID mice with human cervical tumors [86]. Cancer stem cell lines derived from cervical cancer have shown susceptibility to lysis by CTLs [87] and thus CTL-based immunotherapy can be a valuable therapeutic strategy in cervical cancer.