Predictive biomarkers of immune response and immunotherapy

Aleš Ryška

The Fingerland Department of Pathology, Charles University Medical Faculty Hospital, Hradec Králové, Czech Republic

There are not many areas of oncology with such fast development in the last decade as diagnostics, classification and treatment of non-small cell lung cancer.

Some while ago, the basic differentiation between small cell- and non-small cell carcinoma was fully sufficient as in the group of NSCLC there were no differences in therapeutic approach between e.g. adenocarcinoma and squamous cell carcinoma. Thus, any additional subclassification of NSCLC was beyond the therapeutic needs. Only discovery of novel treatment options, which do work best in certain subgroups of NSCLC required distinction of heterogeneous NSCLC category into adenocarcinoma, squamous cell carcinoma, large cell carcinoma etc. However, even this precise morphological typing is not sufficient anymore.

The emerging biologic treatment targeting various molecular signaling pathways is efficient only in patients with neoplasms bearing certain molecular changes, most often one of the so called driver mutations – typically EGFR activating mutation or EML4/ALK gene rearrangement. Thus the classical morphologic diagnosis must be – at least in certain tumor types – accompanied (supplemented) by result(s) of molecular test(s). According to our current knowledge, individual driver mutations are usually mutually exclusive, therefore EGFR positive NSCLC virtually never shows e.g. ALK rearrangement. Therefore, in addition to morphological classification, tumors can be classified also on the basis of their molecular

characteristics. This approach helps the physician in decision making what particular drug(s) should be considered for treatment.

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Based on these new data, the diagnostic guidelines have been updated in many countries (including Czech Republic) and molecular testing is nowadays a diagnostic procedure implemented as routine step in complex diagnostics of NSCLS.

Unfortunately, majority of clinical studies focused on use of targeted therapy, such as TKI, show promising results regarding prolongation of progression free survival, but only very few studies were successful in demonstration of prolongation of overall survival. Thus, majority of patients on targeted treatment do benefit of it for certain period, however, sooner or later does the neoplasm progress and the treatment fails. The mechanisms of development of resistance to treatment are still not fully understood. For example, in case of TKI treatment, secondary resistance mutations such as T790M do develop and are responsible for overgrowth of neoplastic clone resistant to original therapy. Despite the fact that new generations of TKI are on the way and these can more or less effectively target even recurring neoplasms bearing these resistance mutations, the fact that even these drug inevitably will fail and the cancer will progress is highly probable. The reason for development of resistance is quite clear. Malignant neoplasms are extremely plastic

“organisms” and as such, they are able to adapt in accordance to the changes of the environment. Thus, any targeted treatment represents a selective pressure on neoplastic population and cells with signaling alternative bypassing the blocked molecular pathway can survive, grow and sooner or later replace the original tumor mass.

Are there any chances how to overcome or eliminate this principal limitation of our therapeutic efforts? To be able to answer this question, one has to return back to our understanding of tumor biology. The most important point which we ignore in the current targeted approach is the fact that malignant tumor is not just a cluster of neoplastic cells. These cells are able to grow, multiply and survive only thanks to the neoplastic stroma, which represents an integral part of the tumorous lesion and as such has strong influence on the biological behavior of the disease. Stroma does not only serve as a “skeleton” of the neoplasm, it modifies its properties, supplies via network of capillary vessels oxygen and various nutrients, stromal fibroblasts produce signaling and regulatory molecules such as cytokines and growth factors, which are distributed to the neoplastic cells both via the microvessels as well as by diffusion through the extracellular matrix. Another essential component of the tumor stroma are various types of inflammatory elements – granulocytes, histiocytes and lymphoid cells. Each component plays a crucial role and only successful

orchestration of all factors together gives the neoplasm full invasive potential.

The current strategies in systemic therapy are mostly targeted on the neoplastic population itself. The only exception is blockage of microvessels by the use of antiangiogenic therapy. However, there remains significant – so far not fully harnessed – potential in the influencing of other components of tumor stroma. The currently most promising are of novel approaches is influence of anti-tumor immune response by immune checkpoint inhibitors.

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For the tumor to become clinically significant, neoplastic population must escape from the immune surveillance of the organism. This is usually achieved by active blockage of the immunity using both suppression of effector populations of immune cells (such as NK lymphocytes) and stimulation of regulatory T-cells (Tregs) with inhibiting effect on anti-tumor immunity. The idea of unblocking of immune response and thus helping the organism to fight the cancer by own means have proven to be effective in the clinical trials with new therapeutic antibodies targeted against different molecules regulating the immune response, such as anti-CTLA4 ipilimumab or anti-PD1 nivolumab or pembrolizumab. Other molecules are emerging and the results from additional clinical studies should be available quite soon.

Unfortunately, these quite promising molecules prove to be efficient only in minority of patients. At this moment, there are no evidence based clinical or molecular markers which could be used to predict the effect of the treatment in individual patients. Therefore, there is an enormous need for identification of such markers to make the treatment more efficient.

While there are no putative predictive markers available so far for the ipilimumab therapy, there has been done a lot in the research of predictive markers for anti-PD1 treatment (pembrolizumab and nivolumab). At least in certain tumor types, one of the ligands for PD1 receptor, namely PD-L1 molecule is evaluated as a potential predictor of anti-PD1 treatment efficacy.

This ligand is expressed in many neoplasms and as such can be detected by immunohistochemistry in the samples of tumor tissue. Unfortunately, only little is known about the pattern of expression of the molecule – both temporal and spatial heterogeneity exists and it is not established, what is the optimal way for evaluation of expression regarding the distribution of the molecule (center of the tumor vs. its periphery), significance of expression in different cell types (positivity in neoplastic cells vs. expression by the lymphoid infiltrate) or threshold of positivity. In addition, only very little is known about the dynamics of the expression during the course of the disease, how much is the expression influenced by the coincident treatment by other medicaments modifying the immune reaction (such as cytotoxic chemotherapy or use of corticosteroids). Yet another variable not solved so far is the fact that different clinical studies used different methods for detection of the PD-L1 expression in the tissue and so far, there exists no study comparing the pattern and intensity of expression of PD-L1 detected by various primary antibodies and detection kits.

Another puzzling issue is the fact that whereas PD-L1 is strongly predictive of treatment effect in certain neoplasms (such as lung non-squamous carcinoma), it has not been predictive in other neoplasms (such as melanoma or squamous cell lung carcinoma). Therefore, although it is not fully clear if the expression of PD-L1 can be used in the latter mentioned tumors, it is highly probable that this marker will be used as a predictor required for starting the treatment in the lung

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adenocarcinoma. Therefore, a network of histopathological laboratories, which are currently testing predictive markers for other drugs (such as EGFR mutations for the use of TKI or EML4/ALK rearrangement for the use of anti-ALK therapy) will need to introduce standardized immunohistochemical protocols for these tumors.

To guarantee constant optimal performance of the testing, all laboratories have to use appropriate internal controls as well as participate in the external quality assurance program.

In conclusion, the testing of predictive markers enabling us to select those patients who will with highest probability benefit from the immunotherapy has to be established. However, there are several key questions which have to be answered before the predictive value of these markers can be considered as fully evidence based.

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