Clinical Studies on Ultrafractionated Chemoradiation: A Systematic Review

Aim The efficacy of low-dose fractionated radiotherapy (LDFRT) and chemotherapy (CHT) combination has large preclinical but little clinical evidence. Therefore, the aim of this review was to collect and analyze the clinical results of LDRT plus concurrent CHT in patients with advanced cancers. Methods A systematic literature search was conducted on PubMed using the PRISMA methodology. Only studies based on the combination of LDFRT (< 1 Gy/fraction) and CHT were included. Endpoints of the analysis were tumor response, toxicity, and overall survival, with particular focus on any differences between LDFRT-CHT and CHT alone. Results Twelve studies (307 patients) fulfilled the selection criteria and were included in this review. Two studies were retrospective, one was a prospective pilot trial, six were phase II studies, two were phase I trials, and one was a phase I/II open label study. No randomized controlled trials were found. Seven out of eight studies comparing clinical response showed higher rates after LDFRT-CHT compared to CHT alone. Three out of four studies comparing survival reported improved results after combined treatment. Three studies compared toxicity of CHT and LDFRT plus CHT, and all of them reported similar adverse events rates. In most cases, toxicity was manageable with only three likely LDFRT-unrelated fatal events (1%), all recorded in the same series on LDFRT plus temozolomide in glioblastoma multiforme patients. Conclusion None of the analyzed studies provided level I evidence on the clinical impact of LDFRT plus CHT. However, it should be noted that, apart from two small series of breast cancers, all studies reported improved therapeutic outcomes and similar tolerability compared to CHT alone. Systematic Review Registration www.crd.york.ac.uk/prospero/, identifier CRD42020206639.


INTRODUCTION
Conventionally fractionated curative radiotherapy (RT) is delivered in 1.8-2.0 Gy daily fractions. Conversely, low-dose fractionated RT (LDFRT) is defined as the use of very small dose per fraction (< 1.0 Gy). In some experimental models, LDFRT resulted more effective than predicted by the linear quadratic model in terms of improved cell kill (1,2). In particular, in vitro experiments showed this phenomenon in several cell lines (3)(4)(5). Interestingly, the higher efficacy of LDFRT was confirmed in human cells by several laboratories using different assay techniques, conditions of cell growth, handling, and irradiation (1). On the contrary, a relative tumor cell radiation resistance was recorded when higher doses per fraction were used (6). The low-dose hyper-radiation sensitivity (HRS) phenomenon has been interpreted on the basis of a threshold effect in radiationinduced damage repair. In fact, DNA-repair mechanisms are triggered only above certain dose levels, while lower doses are ineffective in arresting irradiated cells in the G2 cell-cycle phase (7,8).
The peculiar efficacy of LDFRT has been interpreted also on the basis of immunological mechanisms. For example, Klug and colleagues (9) reported that local LDFRT produces efficient recruitment of tumor-specific T cells in human pancreatic carcinomas with T-cell-mediated tumor rejection and prolonged survival in otherwise immune refractory spontaneous and xenotransplant mouse tumor models. The authors used one single fraction with doses ranging between 0.5 and 6.0 Gy. They observed that the number of intratumoral T lymphocytes was higher after irradiation with the lowest dose (0.5 Gy) (9). Based on this preclinical evidence, LDFRT was tested also in a clinical study (10).
Concurrent chemoradiation is a standard treatment option in several tumors since CHT is able to act as a radiosensitizer. Interestingly, when delivered as LDFRT, also RT may act as a chemosensitizer. This peculiar synergistic effect of LDFRT and CHT was demonstrated by several preclinical studies, in different cell lines, and using different drugs such as cisplatin, carboplatin, docetaxel, and paclitaxel (11)(12)(13)(14)(15). It is worth noting that LDFRTinduced toxicity is significantly lower compared to conventional fractionation or hypofractionation. This higher tolerability allows LDFRT to be associated with "full-dose" CHT, with a clear benefit in terms not only of local response but also of systemic tumor control (16).
Considering these aspects, interest in the combination of LDFRT with CHT in the clinical management of cancer patients grew. LDFRT was proposed as a new systemic agent labeled with an "r" (e.g., gemcitabine plus LDFRT: rG) (17). Although some preliminary studies suggested the effectiveness of this combination (16,17), randomized trials, meta-analyses, and systematic reviews on this topic are lacking. Therefore, the aim of this review was to collect and analyze the results of LDFRT plus CHT, currently available in literature, in terms of tumor response, clinical outcomes, and treatment tolerability.

METHODS AND MATERIALS
Our systematic review protocol was registered (registration number: CRD42020206639) within the International Prospective Register of Systematic Reviews (PROSPERO, www. crd.york.ac.uk/prospero/) on 31 August 2020.

Inclusion Criteria
Human studies of any design, without limitations in terms of the number of enrolled patients, and based on LDFRT plus CHT combination, were included. Studies based on LDFRT without concurrent CHT were excluded. No restriction about total delivered dose, biological effective dose (BED), and RT technique was imposed.

Outcome Measures
We reported the main findings of the analyzed papers with particular focus on clinical tumor response, overall survival, and treatment-related toxicity. Moreover, any differences between LDFRT-CHT and CHT alone were recorded and reported.

Bibliographic Search
We conducted a search based on PubMed from the earliest date to 20 May 2020. In our review, we considered only studies published in the English language. We used various combinations of the subsequent terms in PubMed such as low-dose, radiotherapy, ultrafractionation, hyper-radiation-sensitivity, chemosensitization, concurrent, and chemotherapy. Finally, the following two search strategies were used in PubMed: i) low-dose[All Fields] AND ("radiotherapy"[Subheading] OR "radiotherapy"[All Fields] OR "radiotherapy"[MeSH Terms]) AND concurrent[All Fields] AND ("drug therapy"[Subheading] OR ("drug"[All Fields] AND "therapy"[All Fields]) OR "drug therapy"[All Fields] OR "chemotherapy"[All Fields] OR "drug therapy"[MeSH Terms] OR ("drug"[All Fields] AND "therapy"[All Fields]) OR "chemotherapy"[All Fields]); and ii) "hyper radiation sensitivity" OR (("ultrafractionation" OR "ultrafractionated") AND ("radiotherapy" OR "irradiation" OR "radiation")) OR ("chemosensitization" AND ("radiotherapy" OR "irradiation" OR "radiation")). We found 396 studies with the first strategy and 253 with the second one. We removed duplicates, and we made the first selection based on titles and abstracts. Moreover, a further search through the references of the selected studies was performed. After reading the full-text articles, six studies were excluded: three used the term "ultrafractionation" or "low-dose RT," but the delivered dose/fraction was ≥ 1 Gy; two studies did not use LDFRT plus CHT combination, and one study reported duplicated patients. Finally, 12 articles fulfilled our criteria (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27).

Study Selection and Quality Assessment
We used the PRISMA guidelines as a guide to select the items to be included within the review (28,29). Title, abstract, and keywords of the identified articles were independently analyzed by two researchers (ES, AZ), and disagreements were solved by the senior author (AM). Potentially eligible studies were retrieved, and full-text evaluation was performed based on the inclusion and exclusion criteria by two different authors (ES, AZ) with disagreements resolved by consensus-based discussion. Subsequently, the following data were collected independently by two authors (ES, MB) from each article, with disagreements resolved by the senior author (AM): authors' name and year of publication, study design, accrual period, patients and setting, treatment (LDFRT and CHT), and main outcomes. Papers were evaluated based on the ROBINS-I Risk of Bias tool (30). Two reviewers (ES, AZ) assessed the quality of the included studies, and discrepancies were resolved on agreement.

Study Design and Risk of Bias
Two studies were retrospective (22,26), one was a prospective pilot trial (18), six were phase II studies (16,19,21,23,25,27), two were phase I trials (20,24), and one was a phase I/II open label study (17). No randomized controlled trials were found. All were considered to own moderate to serious risk of bias according to the ROBINS-I tool (30). Appendix 1 shows the risk of bias rating per study based on the ROBINS-I tool.

Treated Tumors
The characteristics and stage of primary tumors in the analyzed papers are shown in Table 1.

Patients and Treatment
Patients' median age ranged from 21 to 84 years (median 57.6) (16-18, 20, 22-26). Median follow-up ranged from 6.5 to 48 months (median: 22.5 months). The RT total dose ranged from 1.6 to 67.5 Gy. CHT was based on different schedules depending on tumor features. RT details and CHT schedules are shown in Table 1.

Treatment Results
Toxicity results are shown in Table 2. In most studies, the treatment was reasonably tolerated, despite obvious differences due to the different used CHT regimens (16)(17)(18)(19)(20)(21)(23)(24)(25)(26)(27). In the phase II trial conducted by Beauchesne et al. (19) on LDFRT plus temozolomide in glioblastoma multiforme (GBM), three cases of fatal adverse events were reported: one due to hematological toxicity and two due to pulmonary infections. Moreover, Regine and colleagues (17), in their trial on gemcitabine plus LDFRT in pancreatic and small bowel cancers, reported one grade 3 infection out of six patients treated with 0.6 Gy/fraction and one grade 3 infection and one grade 3 diarrhea out of four patients treated with 0.7 Gy/fraction. Table 3 reports details on tumor response and outcome. The results are very inhomogeneous as expected considering the different treated tumors and clinical settings.

DISCUSSION
To the best of our knowledge, this is the first review of clinical studies on combined LDFRT plus CHT. Five studies compared clinical response rates after LDFRT-CHT with literature data on CHT in similar patients, reporting higher ORR rates (16,21,23,26,27). Similarly, four studies compared OS after LDFRT-CHT and reported improved outcome compared to CHT alone (17,19,20,22). Finally, four studies compared toxicity after LDFRT plus CHT versus CHT alone reporting similar adverse event rates (16,21,24,25). Interestingly, clinical findings regarding LDFRT-CHT were published in 12 studies between 2004 and 2017, and no further studies were published thereafter. The lack of prospective studies, moreover with no control groups, could explain the disinterest in this combined modality therapy.    (21) reported 42% ORR and 17 months median OS in stage III-IV non-small cell lung cancer treated with LDFRT plus concurrent pemetrexed. These results were better compared to 9.1% ORR and 8.3 months median OS recorded in a similar patient population treated with pemetrexed alone (41). Valentini et al. (26) reported higher response rates in patients with lung (ORR: 41.6%) and head and neck cancer (ORR: 57%) treated with LDFRT-CHT compared to literature data on lung (ORR: 5-10%) (42,43) and head and neck tumors (ORR: 10-35%) (44-47) treated with CHT alone (similar regimens). Das et al. (27) reported 100% ORR and 100% 2year OS in locally advanced carcinoma of the uterine cervix treated with LDFRT plus induction CHT followed by radical chemoradiation. These figures were higher compared to the ones registered in a similar patient population treated with the same CHT induction regimen followed by standard chemoradiation (48). Only two studies did not show improved results after LDFRT plus CHT compared to CHT alone. In fact, Nardone et al. (24,25) treated stage IIA/B-IIIA breast cancer patients with LDFRT plus CHT and reported similar response rates compared to CHT alone. However, it should be noted that the sample size of these studies was particularly small, with only 10 (24) and 21 patients (25) enrolled, respectively.   (17). The first series included GBM patients treated with LDFRT plus temozolomide. Three cases of fatal adverse events were recorded: one after severe hematological toxicity and two due to pulmonary infections (19). It should be noted that these complications are not uncommon in patients treated with temozolomide alone. In particular, pneumonitis can occur when prophylactic treatment against pneumocystis carinii infections is not prescribed. In the second study, based on LDFRT plus gemcitabine in pancreatic and small bowel cancers, two grade 3 infections and one grade 3 diarrhea were reported (17). The irradiation of the entire upper abdomen could almost partially explain these adverse events.
A comparison within the same study between LDFRT-CHT and CHT was reported only by Morganti et al. As previously described, after CHT based on the FOLFIRI-bevacizumab regimen, the ORR rate was 83.4% in metastatic lesions undergoing LDFRT and 33.3% in non-irradiated lesions (p: 0.02) (23). This review has several limitations including lack of randomized trials, heterogeneity of the study design with inclusion of two retrospective studies (22,26), small sample size with a median number of 23 patients per study (range:  and four studies with less than 20 patients, and heterogeneity in terms of tumor and treatment characteristics. More specifically, the outcome results reported in two phase I (24) and phase I/II (17) trials, each enrolling only 10 patients, must be interpreted with caution due to the very small sample size. The usefulness of a literature review with these limitations could be debatable. However, due to lack of evidence from large prospective trials, we considered it useful to review the available data. Furthermore, it should be emphasized the uniformity between the analyzed series in terms of results, since all studies reported better outcomes after LDFRT-CHT compared to CHT alone, apart from two small studies on breast cancer (24,25).
Based on the low level of evidence of the selected studies, the use of LDFRT-CHT in current clinical practice does not seem justified. However, especially in advanced cancers resistant to systemic therapies, enrollment of patients in prospective studies would be useful. Further studies in this field could have the following design or aims: (i) randomized comparison between LDFRT-CHT versus CHT alone; (ii) definition of the optimal dose and fractionation in LDFRT-CHT; (iii) definition of the optimal CHT regimens in this setting; and (iv) evaluation of LDFRT plus immunotherapy combination, given some evidence on the immune-enhancement effect of LDFRT (51).

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.  Efficacy results compared to CHT alone (median OS of metastatic patients around 6 months in locally advanced disease with gemcitabine alone versus 9.1 months with LDFRT + CHT) Balducci et al., 2014 (18) LDFRT + CHT showed a very low toxicity profile when compared to the same group of patients treated with different approaches (36) Beauchesne et al., 2015 (19) Median OS of 16 months higher than OS rates reported in EORTC/NCIC trial (conventional RT + CHT versus conventional RT alone) Das et al., 2015 (27) ORR (100% with 40% CR and 60% PR, based on MRI findings) and 3y-OS (80%) with LDFRT + CHT followed by CHT + RT versus RR (70%) and 3y-OS (68%) with CHT + RT (the latter treatment scheme done with more CHT cycles). Lower toxicity grade with LDFRT+CHT followed by CHT + RT compared to treatment scheme using CHT+RT (the latter done with higher cycles of CHT) Morganti et al., 2016 (23) 2y PFS: 63.9 and 31.2%, ORR: 83.3% and 33.3% in irradiated and not irradiated lesions, respectively Mattoli et al., 2017 (22) Median OS higher than CHT alone RT, radiotherapy; CHT, chemotherapy; SCCHN, advanced squamous cell carcinoma of the head and neck; HN, head and neck; ORR, response rate; CR, complete response; PR, partial response; PD, progression disease; PFS, progression free survival; RECIST, response evaluation criteria in solid tumors; BID, bis in die (twice daily); NRC, neoadjuvant radiochemotherapy; NAC, conventional neoadjuvant chemotherapy; PO, per oral; PMRR, pathological major response rate; TRG, tumor regression grade; DLT, dose-limiting toxicity.