**6. Classic chemotherapy and rationale for combination with immunotherapy**

Introduction of immunotherapy into the classic chemotherapy regimens is undoubtedly a challenge. The use of chemotherapy aims complete direct cancer cell eradication, which frequently is not achieved. Post chemotherapy exposure to a tumor cell death may be induced, leading to cancer antigen release. These antigens could be subsequently processed by the APCs and the cytotoxic CTLs. Besides a direct cytotoxic effect, such immune modulating effects have been proven for gemcitabine [78,79]; induction of immunogenic cancer cell death or immune sensitization for T-lymphocytes killing of the cancer cell has also been described for platinum compounds [29,80,81]. The effector cells of the immune system seem to remain unaffected [82], thus suggesting a possible rationale for searching of increased synergistic antitumor activity by combination of chemotherapy agents and immunotherapy. A large number of clinical trials are already running in multitude of solid tumor localizations. An example of a combination with chemotherapeutic agent is the emtansine/trastuzumab complex that is used in the treatment of HER-2 positive metastatic breast cancer.

**4.5. Predictive and prognostic biomarkers for immunotherapy**

90 Immunopathology and Immunomodulation

Research is ongoing in order to identify potential biomarkers for cancer immunotherapy. In order to optimize this process, we shall recently be in great demand of predictive/prognostic factors, justifying the selection of patients, who would be the best candidates for such novel, expensive, and potentially toxic treatments. PD-L1-positive cancers are associated with poorer prognoses than PD-1 negative. A correlation of PD-L1 expression and response rate was demonstrated in patients with the highest levels of PD-L1 expression and PD-L1-positive TILs [72]. The potential role of PD-L1 as well as TILs as a biomarkers remain to be elucidated.

The presence or absence of TILs also remains to be clarified. There are data that the immune system plays an important role in the process of recurrence of solid tumors. There is a multicenter study over 603 patients with colorectal cancer that showed the importance of the adaptive immune response and the presence/absence of T-lymphocytes in the resected tumor was a factor that correlated more accurately with clinical outcomes than the current parameters considered as gold standards for prognosis, histopathologically determined tumor stage (T) and nodal status (N), yielding a place for TILs as a potential prognostic marker in colorectal cancer [73] and potentially in other localizations of malignant tumors. It has also been proven for patients with large early-stage cervical cancer [74], muscle-invasive urothelial bladder carcinoma [75], and breast cancer [76]. All these findings suggest that assessment and consid‐ eration of the local intratumoral immune response in the primary tumor may have prognostic

Adverse events (AE) are graded using NCI Common Terminology Criteria for Adverse Events Version 4.0. Their management is important as the population of treated patients frequently consists of patients with disseminated disease or patients who have been previously treated with multiple treatment lines. Most frequent drug-related AEs with potential immune-related mechanism are hepatitis, pneumonitis, infusion reactions, colitis, arthralgia, and rash, necessitating sometimes the use of corticosteroids [77]. Fatigue, decreased appetite, nausea, dyspnea, diarrhea or constipation, vomiting, pyrexia, vitiligo, and headache are also described

Introduction of immunotherapy into the classic chemotherapy regimens is undoubtedly a challenge. The use of chemotherapy aims complete direct cancer cell eradication, which frequently is not achieved. Post chemotherapy exposure to a tumor cell death may be induced, leading to cancer antigen release. These antigens could be subsequently processed by the APCs

value and should be evaluated in the process of treatment decision taking.

**6. Classic chemotherapy and rationale for combination with**

**5. Adverse effects of immunotherapy**

as immune-related AEs.

**immunotherapy**

Synergistic combinations with immunotherapy are also possible with radiotherapy [83], targeted agents [84], antiangiogenic drugs, or combining two immunotherapeutic agents with complementary mechanism of action [85]. There are multiple phase I–III trials, recruiting patients with solid tumors (MEL, NSCLC, RCC, CRC, etc.) where combinations of two immune checkpoint inhibitors are used in combination, e.g., anti-CTLA-4 MAB (ipilimumab) with PD-1 or PD-L1 inhibitors. CTLA-4 inhibitors stimulate the T cell activation in lymphatic tissues and increase the frequency of tumor-specific T cells, while the inhibition of the PD-1/PD-L1 axis modulates the T cell effector phase in order to overcome T cell anergy present in the tumor microenvironment [86].

A theory hypothesizes that combining immunotherapy with targeted agents could be syner‐ gistic as targeted agents promote apoptosis in tumor cells, thus enhancing tumor antigen presentation without adversely effecting immune effector cells; they can also directly modulate the immune response and improve immune-cell function, essentially acting as immunesensitizing agents through different mechanisms [84]. The combination of immunotherapy with anti-angiogenic agents (e.g., *bevacizumab*, *sunitinib*, or *pazopanib*) is also supported by a strong biologic rationale as it has been shown that *bevacizumab* increases the maturation of DCs and antigen presentation process while *sunitinib* decreases the number of MDSCs and Tregs in the tumor microenvironment [87].

The rationale behind combination with radiotherapy is multidimensional, including radiationinduced tumor cell damage, leading to the spill of tumor-associated antigens, attracting the immune effector cells [80,88]. Radiotherapy also sensitizes the tumor cells, thus making them more susceptible to immune-mediated killing; it is partially due to the expression of MHC class I and death receptors [88]. There is a phase II trial in metastatic malignant melanoma (NCT01689974), which compares the use of ipilimumab as monotherapy or in combination with radiotherapy. Another important issue to be addressed in order to optimize the effect of this strategy is related to the timing of radiotherapy related to the administration of the immunotherapy [89].

The combination of agents always upfronts the question of antitumor effects and potential additive toxicity that is largely considered today. Currently, the most frequent combination remains the administration of immune adjuvants, e.g., IL-2 or GM-CSF with MAB or cancer vaccines, in order to stimulate the recruitment/activation of immune effector cells.

#### **7. Tumor response evaluation of immunotherapy**

An issue that has been recently recognized is the measurement of antitumor effect of immu‐ notherapy. The cytotoxicity of chemotherapeutic agents often produces a measurable change in the size of the target lesions within weeks of the initial administration. Response for solid tumors is most frequently assessed using WHO or RECIST criteria [90,91]. For cytotoxic agents, these guidelines assume that an early increase in tumor growth and/or appearance of new lesions signal progressive disease (PD) and the term "progression" became synonymous with drug failure. Cessation of the currently used chemotherapy is thus recommended once PD has been detected.

On the other hand, immunotherapeutic agents enhance antitumor immune responses [92] and achievement of stable disease (SD) may also be viewed as an indicator of meaningful thera‐ peutic effect. Beyond that, additional novel response patterns, observed with these agents, raise concerns about the interpretation and characterization of WHO or RECIST criteria. In studies with cytokines, cancer vaccines, and monoclonal antibodies, response classified as CR, PR, or SD has been shown to occur after an initial increase in tumor burden characterized as PD by WHO or RECIST criteria [93-96]. Therefore, conventional response criteria may not adequately assess the activity of immunotherapeutic agents because PD (by initial radio‐ graphic evaluation) does not necessarily reflect therapeutic failure. Thus, in order to system‐ atically characterize additional patterns of response in patients treated with immunotherapy, underlying WHO criteria were evolved into immune-related response criteria (irRC) [97]. The core novelty of the irRC is the incorporation of measurable new lesions into "total tumor burden" and comparison of this variable to baseline measurements (before and after WHO PD, but not after confirmed irPD). Clinical activity often appears to be delayed following immunotherapeutic treatment and a period of apparent progression (as defined by the existing response criteria) may occur, followed by response. Four types of distinct response patterns have been described (two conventional and two new, unique to immunotherapy): 1) imme‐ diate response; 2) durable stable disease; 3) response after tumor burden increase; and 4) response in the presence of new lesions. The apparent increase in tumor burden that sometimes precedes response in patients receiving immune therapy may reflect either continued tumor growth until a sufficient immune response develops or transient immune-cell infiltration into the tumor with or without edema [97].

The use of irRC for response evaluation with immunotherapeutic treatment is considered clinically meaningful as they appear to be related to favorable survival. However, they are still in early development and prospective trials need to evaluate their role and potential associa‐ tion with survival.

#### **8. Conclusion**

A lot of scientific evidence has been recently accumulated over the role of the immune system in the prevention, development and progression of solid tumors. All this knowledge is continuously enriched in order to implicate it into meaningful clinically relevant therapeutic strategies and use immunotherapy either alone or in combination with other systemic anticancer treatments. These new strategies will hopefully lead to improvement of the outcomes of patients with solid malignancies.
