Arlene Oei

place within seconds after DNA is damaged and will be finished within a few hours. Therefore, our in vitro data on HR proficient cells suggest that the fast repair is less affected by mild hyperthermia alone. However, as re-oxygenation cannot be studied in in vitro experiments, the issue of the dominant mechanism in a clinical setting remains undecided. Furthermore, this result only concerns tumor cells and not normal tissue toxicity. The optimal time interval and sequence also covers the best balance between targeting the tumor tissue as much as possible, with tolerable damage to the normal tissue. Finally, direct cell death due to radiotherapy and hyperthermia does not give insight in tumor relapse. The in vitro experiments did show more cell death after a shorter time interval, but then again, this has not yet been tested in respect to the normal tissue. Research studying the effects on normal tissue toxicity is ongoing.

The importance of limiting the time interval between radiotherapy and hyperthermia was not only demonstrated in in vitro results. In patients, the optimal window between radiotherapy and hyperthermia to gain a beneficial effect is even shorter. This difference between tumor cells in vitro and patients is that, unlike the situation in human beings, cultured tumor cells are treated under optimal conditions, i.e. the temperature distribution is homogeneous and oxygen levels are perfectly regulated. The clinical lesson to be learned is that shortening the time interval between radiotherapy and hyperthermia is essential in achieving a higher overall survival. A limitation of the patient study was the relatively small number of patients (n=58). Moreover, among all patients with inoperable cervical cancer, these patients even had more dismal prognostic factors, which coincides with their contraindication against receiving chemotherapy. This group included a large number of elderly and physically unfit patients. If hyperthermia becomes a standard treatment for a larger group of patients, the impact of sequence and time interval must be re-evaluated prospectively in a randomized trial which includes patients with various ages and with different physical status. The impact should be compared for patients with different ages and various physical conditions as the sequence and time interval between radiotherapy and hyperthermia might be even more important in younger and physically fitter patients, effects may be larger and perhaps last longer, as the response of blood flow to hyperthermia may be better in younger patients.

HPV+ tumors, a special target for hyperthermia?
Remarkably, cervical carcinoma has a favorable clinical outcomes after combinational therapies including hyperthermia (Datta et al, 2016). In this thesis (Chapter 4), we explored one possible mechanism explaining the success of hyperthermia in this particular tumor type. Whereas hyperthermia interferes with the complex formation of HPV-E6 and p53 in HPV+ cervical carcinoma cell lines, thereby rescuing p53 to become active and induce tumor cells death, a new logical question rises whether hyperthermia has this effect on all HPV+ tumor types, such as anal cancer and tumors of the head and neck. These questions are definitely high ranking on our list of topics to be investigated in future projects. According to our results, hyperthermia is rather unique in interrupting the tumor promoting complex formation of E6 and p53; neither radiotherapy nor chemotherapy, or the combination of both is able to do so. Therefore, it may be important to support clinical application of hyperthermia in all patients with HPV+ tumors and not only for a selected group of patients. Whereas cervical carcinoma has a significantly better outcome when combined with hyperthermia, head and neck tumors which are caused by HPV have already been found to respond very well to radiotherapy alone. It would therefore be extremely interesting to investigate if hyperthermia could further increase clinical outcomes for this specific tumor type. Elucidating whether hyperthermia has similar effects in all HPV+ tumor types would be essential, in order to prevent over-treating patients. As there are also multiple investigations testing the effectiveness of hyperthermia in both HPV+ and HPV- head and neck tumors, also in clinical trials, the current clinical outcomes of treated head and neck tumors need to be improved. The effect on E6 is certainly not the only effect of heat. In HPV- tumors hyperthermia induces p53 dependent apoptosis as well. This raises the question whether hyperthermia affects MDM2, which is an important p53 regulator. This issue remains to be investigated.

Hyperthermia to target CSC?
Treatment failure after local radiotherapy and systemic chemotherapy in women with cervical cancer is caused by distant metastases, regional failure and local recurrence. The initial response to treatment is usually favorable, but recurrences still occur, probably caused by a small percentage of cells that become therapy-resistant. These therapy-resistant cells, typically localized in the hypoxic area of the tumor, often show similar characteristics to normal tissue stem cells, and are therefore referred to as cancer stem cells (CSCs). These CSCs have the capacity to re-populate and re-establish an entirely new tumor. In Chapter 10, we argued that hyperthermia targets exactly the areas in which radioresistant cells are located, and may therefore strengthen the forces in the battle against CSCs. Indeed, in tumors located in previously irradiated areas, low doses of radiation combined with hyperthermia proved to be just as effective as high doses of radiation. This protocol is chosen to prevent too much normal tissue toxicity caused by ionizing radiation. Hyperthermia can then be added to prevent cells from becoming radioresistant. It would therefore be interesting to investigate whether hyperthermia can sensitize CSCs to chemotherapy and radiotherapy. In vitro and in vivo experiments are needed to confirm this hypothesis. This could shed light on why hyperthermia can increase the local disease free survival in clinical trials raises the question whether hyperthermia should be applied already in primary tumors to avoid cells from getting therapy-resistant (by reaching all areas of the tumor with the combinational anti-cancer treatment), or whether hyperthermia is more effective when specifically applied to treat when radioresistant cells are known to be present and posing a problem for conventional therapies. The outcome of this debate depends on whether hyperthermia has the ability not only to reduce hypoxia in primary tumors, but more importantly, to make usually well oxygenated recurrences and metastases more vulnerable to conventional therapy by targeting the CSCs. This approach can not only increase tumor response and time to recurrence, but can also increase cancer cure rates, if hyperthermia actually can kill all remaining tumor cells.

Multimodality treatments to improve the effectiveness of hyperthermia
As mentioned before, hyperthermia is not effective as a single modality treatment of cancer, but is very effective after combination with radiotherapy or chemotherapy. If tumor recurrence is located in previously irradiated areas, it is preferred to avoid radiotherapy. Combinational treatments with hyperthermia, cisplatin and PARP-inhibitors have been investigated in in vitro and in vivo experiments. Once radiotherapy is contraindicated for treatment, hyperthermia and cisplatin is an established combination for retreatment of these therapy resistant tumors. The rationale to combine hyperthermia with PARP-inhibitors, originates from the induction of synthetic lethality by treating BRCA-deficient cancer cells with PARP-inhibitors. Since PARP plays a crucial role in the repair of single strand breaks, treatment with a PARP-inhibitor will convert single strand breaks into double strand breaks. These will normally be repaired by HR, in which BRCA-proteins are essential. Therefore, patients who have BRCA1/2-deficient tumors are, theoretically, likely to benefit from PARP-inhibitor without too much normal tissue toxicity, since only the tumor cells lack functional BRCA. In Chapter 7, rats treated with cisplatin, hyperthermia and PARP-inhibitor showed a tumor growth control, without observing any side-effects. However, in four different studies (all using Olaparib), a slower tumor progression was found, but patient survival was not improved. Worse, serious side-effects such as nausea, fatigue, vomiting and anaemia were observed (Ledermann et al, 2012; Wiggans et al, 2015). In a fifth study, also treating epithelial ovarian cancer, Veliparib (another PARP-inhibitor) was used, demonstrating fewer severe side-effects, but the patient numbers were too small to draw any firm conclusions (Wiggans et al, 2015). Either lower doses of PARP-inhibitors should be applied to reduce side-effects, or the clinical effects of a newer generation PARP-inhibitors with lower toxicities should be investigated. Since hyperthermia is a local therapy that can temporarily downregulate BRCA2, we argue that hyperthermia creates a time window to treat all tumor types, including BRCA-proficient tumor types. The trimodality of cisplatin, hyperthermia and a PARP-inhibitor shows a higher tumor cell death than only thermochemotherapy (Chapter 7).

Cisplatin is a very effective and widely used chemotherapeutic agent. However, as cisplatin is used as a systemic therapy, it does not distinguish between tumor cells and fast replicating non-tumor cells as cisplatin targets all fast replicating cells. The – sometimes irreversible - side-effects can be serious, as it can result into hearing impairment, sensory loss and kidney failure. The side-effects can already be reduced by so-called hyper-hydration of the patient before cisplatin is administered. However, hyper-hydration increases the clearance of the drug from the body, and may thereby reduce the effectiveness of cisplatin. As shown in Chapter 8, the addition of a PARP-inhibitor to hyperthermia and reduced cisplatin levels, is equally effective as standard cisplatin dose with hyperthermia. This finding can be clinically interesting to maintain tumor control at reduced cisplatin, thereby reducing severe and irreversible toxicity. Moreover, it would also be interesting to evaluate if addition of PARP-inhibitor can compensate for hyperthermia treatment at a lower temperature, or a high temperature for a shorter treatment time. Another approach to boost the effectiveness of hyperthermia is achieved by interfering with counteracting chaperone proteins (for example Heat Shock Proteins, HSPs) that protect cells from hyperthermia stress. Several in vitro and in vivo studies demonstrate targeting of HSPs combined with other anti-cancer treatments, can improve treatment outcome (Ciocca et al, 2010). Also, promising results are shown in clinical trials (Calderwood, 2010). As the effects of hyperthermia were temporary, a simultaneous application of HSP inhibitor would be required for further boosting effectiveness of HT. In vivo studies are the first step towards clinical application, and when results are positive this may eventually encourage to start clinical trials to validate these novel multi-modality strategies. A particularly important question is evaluation of the toxicity profile of the proposed multi-modality schedules. For instance, stress factors like hyperthermia, chemotherapy and radiotherapy can induce HSPs. As HSP90 regulates protein folding, prevents misfolding and assists in the function of various proteins, also in healthy cells, inhibition of this protein may result in some toxicity.

Future perspectives for hyperthermia in cervical carcinoma
The Dutch Deep Hyperthermia Trial (van der Zee et al, 2000) demonstrated significant increased tumor control and survival benefit if hyperthermia was added to radiotherapy in cervical carcinoma. In this trial, radiotherapy was compared to radiotherapy combined with hyperthermia in patients with advanced bladder, cervical and rectal tumors. In cervical cancer patients, the complete response rate was significantly higher after radiotherapy combined with hyperthermia (83%) compared to radiotherapy alone (57%). Similar results were reported in other randomized trials evaluating the effect of combining. However, the current standard treatment of patients with locally advanced cervical cancer is not radiotherapy alone, but cisplatin based chemoradiotherapy. In the RADCHOC trial (Lutgens et al, 2016), hyperthermia combined with radiotherapy was compared to chemoradiotherapy, and did not show a significant difference between the two approaches. It should be noted that many patients in the RADCHOC trial received radiotherapy in the nearest radiotherapy facility, but had to travel sometimes several hours to receive hyperthermia in a distant facility. In our retrospective patient series, described in Chapter 6, we discovered that a long time interval between radiotherapy and hyperthermia is detrimental for treatment outcome, and that the time interval between the two modalities should preferable be as short as possible, mandating delivery of both in the same facility. Then, the outcome of thermoradiation may eventually be much better as was suggested by the RADCHOC study.

Taking it to the next level, standard chemoradiotherapy versus hyperthermia plus chemoradiotherapy has also been investigated in a multicentre randomised clinical trial in Japan in locally advanced cervical carcinoma (Harima et al, 2016). A trend was seen demonstrating a higher five-year overall survival of 77.8% in the arm treated with hyperthermia and chemoradiotherapy vs. 64.8% for chemoradiotherapy alone. Complete response rates were significantly higher when hyperthermia was added to chemoradiotherapy (88% vs. 77.6%). Moreover, the triple modality was well tolerated, no additional toxicities were observed. A comparable multicentre trial randomizing between the same two arms, initiated from Duke University Medical Center, the Academic Medical Centre Amsterdam, and other international partners (Westermann et al, 2012) did show a non significant trend towards a benefit of adding hyperthermia to chemoradiation regarding local control (70% to 78%) but no benefit regarding overall survival. The latter may be due to a considerable number of patients that had para-aortic lymph node metastases beyond the heated region of the primary tumor. This warrants the development of hyperthermia techniques capable of safely heating even larger volumes, or to target the para-aortic lymph nodes separately. Considering the cytotoxicity to the normal tissue due to cisplatin-based treatment, the rationale to prefer hyperthermia over cisplatin or to give trimodality treatment with a significantly reduced cisplatin dose seems logical. Further clinical studies to confirm these data are warranted. Multiple clinical trials are under investigation. For example, a clinical trial is ongoing at the University of Texas Health Science Center, in which the effectiveness of triple-modality treatment for inoperable or metastastic pancreatic cancer is investigated (www.clinicaltrials.gov). Also, clinical trials in which combinational treatments of hyperthermia with PARP-inhibitor are explored.

In conclusion, hyperthermia is a local treatment, which has proven its effectiveness in multiple clinical trials (Datta et al, 2016) and still many new improvements are explored. Not only did several studies demonstrate favorable clinical outcomes, in none of the patients severe toxicities were reported associated with hyperthermia. A good treatment not only targets the tumor cells, it must also spare the normal tissue as much as possible to reduce toxicity, both to prolong life and maintain a good quality of life for patients after treatment. After elucidating the mechanisms responsible for the effectiveness of hyperthermia, application in clinical practice of hyperthermia may be improved, which hopefully is a step towards routine clinical practice for all patients who may benefit from hyperthermia as I feel hyperthermia is potentially beneficial for a much larger group of patients than the relatively small number of patients who are presently treated with hyperthermia.

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