Immune-Checkpoint Inhibitors for Metastatic Colorectal Cancer: A Systematic Review of Clinical Outcomes

Simple Summary In 2018, colorectal cancer (CRC) was declared the fourth most deadly cancer worldwide. Unfortunately, a quarter of all patients are diagnosed at late stages, when curative surgery is not possible, rendering systemic therapy and/or best supportive care as the only options. To our knowledge, this is the first systematic review assessing response and survival rates in patients with mCRC treated with immune-checkpoint inhibitors (ICIs). Our study established that ICIs show potentially superior response rates in mCRC patients, though only in those with high microsatellite instability (MSI). Nivolumab + regorafenib was reported to provide encouraging response in low-MSI (MSI-L) patients; however, additional studies using cohort randomization are required. Further studies are required, particularly regarding the mechanism of resistance to ICIs in MSI-L patients. Abstract Background. Colorectal cancer (CRC) is the fourth most deadly cancer worldwide. Unfortunately, a quarter of the patients are diagnosed at late stages, when surgical options are limited. Targeted therapies, particularly immune-checkpoint inhibitors (ICIs), are the latest addition and have been studied herein regarding their efficacy outcomes. Methods. Clinical studies were identified through the PubMed, Scopus and Cochrane databases. Any trial that evaluated ICIs in patients with metastatic CRC (mCRC) and reported the objective response rate was deemed eligible. Data analysis was performed by employing the random-effects model in STATA v.17. Results. A total of 461 articles were identified; 13 clinical trials were included, encompassing a total cohort of 1209 patients. Our study determined that a single PD-1/PD-L1 checkpoint blockade provides durable clinical response in mCRC patients with high microsatellite instability (MSI-H). The combinatorial therapy of CTLA-4 + PD-1 inhibitors also showed high response rates in pre-treated MSI-H patients. The single-arm REGONIVO trial reported durable clinical response in patients with microsatellite stable (MSS) status. Conclusions. Our study surmises that PD-1/PD-L1 inhibitors as well as combination therapy with CTLA-4 and PD-1 inhibitors show encouraging response rates in mCRC patients, albeit exclusively in patients with cancer that are of MSI-H status. A single study suggests that nivolumab + regorafenib can reach a durable response rate in MSS patients; however, further studies in larger randomized settings are required.


Introduction
Colorectal cancer (CRC) is the fourth most common cause of cancer-related mortality, accounting for 10% of all annually diagnosed malignancies worldwide [1]. It is the second most common cancer in women and the third most common in men [2], with the yearly incidence to surpass 2.5 million by 2035 [3]. Despite the availability of screening programs, 25% of all new CRC cases are still diagnosed at advanced stages [4,5] (Figure 1). For decades, chemotherapy has been the primary treatment for metastatic CRC [6], particularly fluorouracil (5-FU/F), oxaliplatin (OX), irinotecan (IRI) and capecitabine (CAP), alone or in combination [7]. Unfortunately, high risk of systemic toxicity, poor response rates and low efficacy have driven the search for better treatment options with higher tumor-specific selectivity, especially among patients with late-stage disease progression [7].  [8]. TNM staging is based on the size of the tumor, growth into lymph nodes and distant metastases to organs and/or tissues. Relative survival rate is an epidemiological characteristic comparing people with the specific histological type and stage of cancer to the overall population in a specific area/region of our country. 1-, 3-and 5-year relative survival rates reduce drastically as CRC progresses from stage 0 to 4. Targeted therapies provide an alternative for patients with metastatic CRC. Targeted therapies function by blocking specific molecules involved in cancer growth and dissemination [9]. In CRC, several potential targets have been studied over the past 20 years (Figure 2), including epidermal growth factor receptor (EGFR) inhibition [10] as well as suppression of the Ras-Raf-MEK-ERK pathway, which is responsible for cell growth, proliferation and survival [11,12]. Vascular endothelial growth factor-A (VEGF-A), which promotes tumor angiogenesis [13][14][15], is another critical target. The first anti-VEGF-A mAb, bevacizumab, was approved in 2004 [16]. Since then, the Food and Drug Administration (FDA) has granted approval to aflibercept (inhibitor of VEGF-A), ramucirumab (fully humanized mAb against VEGFR-2) and regorafenib (inhibitor of VEGFR-2) for the treatment of CRC [16]. However, treatment resistance is inevitable and novel treatment options are needed.  [8]. TNM staging is based on the size of the tumor, growth into lymph nodes and distant metastases to organs and/or tissues. Relative survival rate is an epidemiological characteristic comparing people with the specific histological type and stage of cancer to the overall population in a specific area/region of our country. 1-, 3-and 5-year relative survival rates reduce drastically as CRC progresses from stage 0 to 4. Targeted therapies provide an alternative for patients with metastatic CRC. Targeted therapies function by blocking specific molecules involved in cancer growth and dissemination [9]. In CRC, several potential targets have been studied over the past 20 years (Figure 2), including epidermal growth factor receptor (EGFR) inhibition [10] as well as suppression of the Ras-Raf-MEK-ERK pathway, which is responsible for cell growth, proliferation and survival [11,12]. Vascular endothelial growth factor-A (VEGF-A), which promotes tumor angiogenesis [13][14][15], is another critical target. The first anti-VEGF-A mAb, bevacizumab, was approved in 2004 [16]. Since then, the Food and Drug Administration (FDA) has granted approval to aflibercept (inhibitor of VEGF-A), ramucirumab (fully humanized mAb against VEGFR-2) and regorafenib (inhibitor of VEGFR-2) for the treatment of CRC [16]. However, treatment resistance is inevitable and novel treatment options are needed. (1) Anti-EGFR (epidermal growth factor receptor) monoclonal antibodies (mAbs) cetuximab and panitumumab; (2) encorafenib, an inhibitor of the Raf protein as part of the MAPK/ERK signaling pathway; (3) VEGF-A (vascular endothelial growth factor-A) inhibitors aflibercept and bevacizumab; (4) VEGF receptor inhibitors regorafenib and ramucirumab; (5) anti-PD-1 (programmed cell death-1) mAbs nivolumab and pembrolizumab and anti-PD-L1 (programmed cell death ligand 1) mAbs atezolizumab, durvalumab and avelumab; (6) anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) mAb ipilimumab. Current targeted therapies inhibit three major processes crucial for cancer growth: unrestricted proliferation, neoangiogenesis and suppression of T cell immune responses.
A recent therapeutic addition to the CRC treatment regimen are immune-checkpoint inhibitors (ICIs) [17]. ICIs are mAbs targeting inhibitors of T cell receptor (TCR) activation, particularly programmed cell death-1 (PD-1), programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) expressed in T cells and antigen-presenting cells [17]. They have become widely used in many cancers, including melanoma, RCC, NSCLC and HCC [18]. In mCRC, this treatment modality has already shown an encouraging clinical response [19] in patients with high microsatellite instability (MSI-H) in mismatch repair (MMR) genes such as MutL homolog 1 (MLH1), MutS homolog 2, 6 (MSH2, MSH6), PMS2 and tumor-associated calcium signal transducer 1/epithelial cell adhesion molecule (TACSTD1/EPCAM) [20]. However, MSI-H CRC accounts for 5% of the total cases, with the remaining 95% of CRC cancers classified as microsatellite stable (MSS) or low microsatellite instability (MSI-L) [20], where the efficacy of ICIs is yet to be defined. This study aims to comprehensively summarize existing knowledge regarding response and survival rates and outline major barriers to ICI treatment in patients with mCRC.

Eligibility Criteria and Data Collection
For the purpose of this study, we utilized the following eligibility criteria: (1) patients with mCRC; (2) PD-1, PD-L1, CTLA-4 inhibitors used for treatment either as a single agent or in combination with chemotherapeutic/biological agents; (3) ORR was reported; (4) published in English. Systematic reviews and meta-analysis, case reports, studies with no ORR data and studies not published in the English language were excluded. A recent therapeutic addition to the CRC treatment regimen are immune-checkpoint inhibitors (ICIs) [17]. ICIs are mAbs targeting inhibitors of T cell receptor (TCR) activation, particularly programmed cell death-1 (PD-1), programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) expressed in T cells and antigenpresenting cells [17]. They have become widely used in many cancers, including melanoma, RCC, NSCLC and HCC [18]. In mCRC, this treatment modality has already shown an encouraging clinical response [19] in patients with high microsatellite instability (MSI-H) in mismatch repair (MMR) genes such as MutL homolog 1 (MLH1), MutS homolog 2, 6 (MSH2, MSH6), PMS2 and tumor-associated calcium signal transducer 1/epithelial cell adhesion molecule (TACSTD1/EPCAM) [20]. However, MSI-H CRC accounts for 5% of the total cases, with the remaining 95% of CRC cancers classified as microsatellite stable (MSS) or low microsatellite instability (MSI-L) [20], where the efficacy of ICIs is yet to be defined. This study aims to comprehensively summarize existing knowledge regarding response and survival rates and outline major barriers to ICI treatment in patients with mCRC.

Study Endpoints
The primary endpoint is to systematically determine the ORR (according to Response Evaluation Criteria in Solid Tumors version 1.1 [RECIST v1.1]) in patients with mCRC treated with ICI-based therapies. In addition, we summarized median progression-free survival (PFS) with 95% confidence interval (CI), if reported across selected studies.

Literature Search and Study Endpoints
This systematic review is reported in accordance with PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) reporting guidelines and is registered at the PROSPERO database (CRD42020197617) [33].
Three authors (D.S., L.A. and G.A.) independently conducted a comprehensive literature review through the electronic databases Scopus, MEDLINE, Web of Science and Cochrane Library from 30 September 2004 to 30 April 2021 using keywords (immunecheckpoint inhibitors, metastatic colorectal cancer, colon cancer, rectal cancer, targeted therapy, monoclonal antibodies, pembrolizumab, nivolumab, ipilimumab, atezolizumab) linked by operators "AND" and "OR." Abstracts presented at annual meetings of the American Society of Clinical Oncology (ASCO) and the European Society for Medical Oncology (ESMO) were also examined. Abstracts were considered eligible if the study was published for the first time and matched eligibility criteria. Finally, we searched through bibliographies of selected articles as well as clinical trial registries (www.clinicaltrials.gov (accessed from 1 May 2021 to 25 May 2021) using the aforementioned keywords.
The records found through primary search were initially screened by title and abstract. The full text of potentially eligible studies was reviewed, and if eligible the study was included in the analysis. Selected studies were reviewed by all authors and all discrepancies were solved by consensus. If one study was reported multiple times, the study with the most comprehensive and up to date data was included.

Data Analysis
Risk of bias in randomized studies was assessed using a revised Cochrane tool to assess risk of bias in randomized trials [34,35] whereas single-arm studies were assessed by the Newcastle-Ottawa tool for Cohort Studies [36]. PFS is defined as time (in months) from randomization to recurrence [37]. Study heterogeneity was calculated with I 2 . I 2 values less than 25%, 50% and 75% were considered to be of low, moderate and high study heterogeneity, respectively. The random-effects model (DerSimonian and Laird procedure) was applied to investigate the best therapeutic outcome among the selected studies. Results were deemed statistically significant if p-level <0.05. Data synthesis was performed using Stata version 17 (Stata Corp. LLC) software.

Results
A total of 461 articles were identified using the defined keywords, of which 13 clinical trials were included in the qualitative and quantitative analyses based on the outlined eligibility criteria with a total cohort of 1209 patients. The PRISMA flow chart is shown in Figure 3. also examined. Abstracts were considered eligible if the study was published for the first time and matched eligibility criteria. Finally, we searched through bibliographies of selected articles as well as clinical trial registries (www.clinicaltrials.gov (accessed from May 1, 2021 to May 25, 2021) using the aforementioned keywords.
The records found through primary search were initially screened by title and abstract. The full text of potentially eligible studies was reviewed, and if eligible the study was included in the analysis. Selected studies were reviewed by all authors and all discrepancies were solved by consensus. If one study was reported multiple times, the study with the most comprehensive and up to date data was included.

Data Analysis
Risk of bias in randomized studies was assessed using a revised Cochrane tool to assess risk of bias in randomized trials [34,35] whereas single-arm studies were assessed by the Newcastle-Ottawa tool for Cohort Studies [36]. PFS is defined as time (in months) from randomization to recurrence [37]. Study heterogeneity was calculated with I 2 . I 2 values less than 25%, 50% and 75% were considered to be of low, moderate and high study heterogeneity, respectively. The random-effects model (DerSimonian and Laird procedure) was applied to investigate the best therapeutic outcome among the selected studies. Results were deemed statistically significant if p-level <0.05. Data synthesis was performed using Stata version 17 (Stata Corp. LLC) software.

Results
A total of 461 articles were identified using the defined keywords, of which 13 clinical trials were included in the qualitative and quantitative analyses based on the outlined eligibility criteria with a total cohort of 1209 patients. The PRISMA flow chart is shown in Figure 3.    [19]. PEMBRO-treated ORR reached 43.8% (95% CI: 35.8 to 52), with 11.1% (n = 17) and 32.7% (n = 50) of the examined patients with complete (CR) and partial responses (PR), respectively. In total, 67% (n = 102) and 30% (n = 42) had rightand left-side colon malignancy, respectively, and 75% (n = 115) did not receive any prior therapy. The study concluded that PFS is consistently longer in patients treated with PEMBRO and resulted in the recent update of therapeutic guidelines for mCRC patients with MSI-H/dMMR [38].

Checkpoint Blockade in Colorectal Cancer Patients with High Microsatellite Instability
A phase 2 single-arm trial, KEYNOTE-164 (NCT02460198), determined the efficacy of PEMBRO 200 mg Q3W in mCRC patients with MSI-H status previously treated with ≥2 (cohort A, n = 61) and ≥1 (cohort B, n = 63) lines of systemic therapies [22]. The median PFS reached 2.3 months (95% CI: 2.1 to 8.1) and 4.1 months (95% CI: 2.1 to 18.9) in cohorts A and B, respectively [22]. The ORR in both cohorts was 33%, including 2 CRs and 18 PRs in cohort A and 5 CRs and 16 PRs in cohort B [22]. The data regarding tumor location were not reported. Finally, the authors concluded that PEMBRO provides durable clinical response in MSI-H/dMMR patients progressed on ≥2 and ≥1 lines of previous systemic treatment [22].
With regard to nivolumab (NIVO) monotherapy, the CheckMate142 (NCT02060188) phase 2 single-arm study established an ORR of 32% (95% CI: 22 to 44) with the median PFS of 14.3 months (95% CI: 4.3 to NR) [23]. Whereas 99% (n = 73) of the patients had at least one line of prior chemotherapy, 100% of the patients were MSI-H/dMMR [23]. The authors concluded that NIVO 3 mg/kg Q2W reached a durable clinical response and survival rates and could be considered in further clinical trials; however, the primary tumor location was not reported [23].

Single PD-L1 Inhibitor
A phase 1b trial (NCT01633970) investigated atezolizumab (ATEZO) + various chemotherapeutic/biological (bevacizumab) regimens in patients with advanced solid tumors [24] and recorded an ORR in mCRC patients of 30% (95% CI: 6.7 to 65.3), with the median PFS not reached by the time of data cutoff [24]. ATEZO 10 mg/kg Q3W was used in combination with bevacizumab 15 mg/kg Q3W among n = 10 enrolled patients, of whom n = 7 and n = 3 had ≥2 and 1 line(s) of previous chemotherapy, respectively [24]. Therapeutic efficacy was encouraging, and follow-up is ongoing, with the final results not yet reported.
A phase 2 single-arm study (NCT01693562) investigating durvalumab (DURVA) in patients with advanced solid tumors demonstrated an ORR of 22% (95% CI: 10 to 39) and a median PFS of 6 months (95% CI: 3 to 20) in n = 36 mCRC patients [25]. DURVA 10 mg/kg Q2W showed promising antitumor activity in pre-treated mCRC patients, with the final results of this trial anticipated by Fall 2021. Finally, the examined studies showed that PD-L1 inhibitors can also provide a durable clinical response in mCRC patients with MSI-H status.
To summarize, the aforementioned trials validated ICIs as a therapeutic alternative for mCRC patients with MSI-H, particularly PEMBRO. NIVO as monotherapy or in combination with IPI has shown encouraging response rates and has also been approved by the FDA for mCRC patients progressed on standard chemotherapy. Finally, PD-L1 inhibitors have also shown encouraging response and survival rates; however, larger randomized trials are required (Figure 4).
To summarize, the aforementioned trials validated ICIs as a therapeutic alternative for mCRC patients with MSI-H, particularly PEMBRO. NIVO as monotherapy or in combination with IPI has shown encouraging response rates and has also been approved by the FDA for mCRC patients progressed on standard chemotherapy. Finally, PD-L1 inhibitors have also shown encouraging response and survival rates; however, larger randomized trials are required (Figure 4).

Immune-Checkpoint Inhibitors + Chemotherapeutic or Biological Agents
A phase 1b single-arm REGONIVO study (NCT03406871) investigated NIVO 3 mg/kg Q2W in combination with the multikinase inhibitor regorafenib 80-160 mg in patients with advanced gastric cancer or CRC [27]. It demonstrated an ORR of 36% (95% CI: 18 to 57.5) with a median PFS of 7.9 months (95% CI: 2.0 to NR) in the mCRC cohort (n = 25) [27]. Of note, 96% (n = 24) of the participants had MSS status with 20% (n = 5) and 80% (n = 20) Cancers 2021, 13, 4345 9 of 14 had 2 and ≥3 lines of prior chemotherapy, respectively [27]. In 80% (n = 20) of the patients, the primary tumor was located in the left side or the rectum [27]. Overall, Fukuoka et al. reported encouraging antitumor activity in MSS mCRC patients, which are currently deemed ineligible for checkpoint blockade therapies. Further studies should be conducted to determine the outcomes of ICIs and kinase inhibitors in MSS patients.
A phase 3 randomized controlled trial, IMblaze 370 (NCT02788279), examined ATEZO 840 mg Q2W + cobimetinib 60 mg (cohort A) or ATEZO 1200 mg Q3W (cohort B) compared to regorafenib 160 mg in mCRC patients with MSS status [30]. The primary endpoint was not observed with no significant difference in overall survival compared to the control group treated with regorafenib [30]. A total of 93% (n = 170) and 92% (n = 83) of the patients in cohorts A and B, respectively, had MSS status [30]. Of note, the earlier trial (NCT01988896, phase 1b) also established a low clinical efficacy in MSS patients treated with ATEZO + cobimetinib [28]. These data have underscored the challenge of exploring ATEZO-based regimens in mCRC patients with MSS status.
A phase 2 randomized, placebo-controlled trial BACCI (NCT02873195) compared the outcomes between capecitabine + bevacizumab + placebo (arm A, n = 46) and capecitabine + bevacizumab + ATEZO (arm B, n = 82) in mCRC patients [26]. A total of 85.7% (n = 70) of patients had MSS status [26]. No significant differences between study and control groups were established [26]. Nonetheless, study investigators have decided to conduct a phase 3 trial to further examine this regimen in mCRC patients with MSS status.

Combination of CTLA-4 + PD-L1 Inhibitors
A phase 2 randomized trial (NCT02870920) investigating DURVA + tremelimumab in mCRC patients with MSS status compared to the best supportive care (BSC) demonstrated no CR in the study group. In addition, only one patient (total cohort included n = 119) achieved PR with the median PFS of 1.8 months [32]. No significant differences in PFS between the studied regimens were observed; however, stable disease (SD) was recorded in 22.7% (n = 27) and 6.6% (n = 4) of the patients treated with DURVA-based regimen and BSC, respectively [32]. As the patients were of MSS status and had received at least one line of prior chemotherapy, the authors concluded that DURVA + tremelimumab may be of potential benefit for this patient population due to the lack of other treatment options [32].
To summarize, ICIs did not show encouraging clinical efficacy in mCRC patients with MSS status. The mechanisms underlying this resistance are currently unknown.

Study Limitations
Our study has limitations worth outlining. First, some of the selected studies did not define the number of therapeutic lines used prior to ICIs. Secondly, our analysis focused on determining efficacy clinical outcomes, disregarding safety outcomes in mCRC patients.

Discussion
Targeted therapy has opened a new chapter in the management of CRC, with ICIs providing a new hope for patients with advanced colonic malignancies [39]. In particular, the humanized anti-PD-1 mAb PEMBRO has recently become a new first-line therapeutic alternative for mCRC patients with MSI-H status [19]. The randomized KEYNOTE-177 trial reported significantly higher PFS and ORR among patients treated with PEMBRO compared to patients treated with SoC chemotherapy (mFOLFOX6 or FOLFIRI +/− bevacizumab or cetuximab) [19]. PEMBRO has also shown high efficacy in pre-treated (>1 and >2 lines of standard chemotherapy) mCRC patients with MSI-H status [22]. Another single PD-1 inhibitor, NIVO, has also shown durable clinical response in pre-treated mCRC patients with MSI-H status [23]. Similarly, PD-L1 inhibitors (ATEZO, Avelumab and DURVA) also demonstrated encouraging response rates in pre-treated MSI-H mCRC patients in early phases of clinical trials, with further studies ongoing [24,25,29]. These results emphasized the higher efficacy of single ICIs in mCRC patients with MSI-H status. In addition to these findings, the CheckMate142 trial reported that NIVO + IPI reached an impressive response rate in heavily pre-treated mCRC patients with MSI-H status (99% had ≥1 lines of chemotherapy) [21]. Further investigation of doublet ICI therapy is currently ongoing.
By contrast, PD-1/PD-L1 inhibitors showed no clinical benefit in patients with MSS status [26,28,30,32]. The factors impacting lower response rates of ICI therapy in CRC patients with MSS status remain unknown. An analysis conducted by Mlecnik et al. [40] revealed that patients with MSI-H status possess a higher rate of mutations in ACVR2A (activin A receptor type 2A), FBXW7 (F-box and WD repeat domain-containing 7) and CTNNB1 (catenin beta 1) genes, and fewer mutations in APC (adenomatous polyposis coli), KRAS and TP53 (tumor protein 53) genes compared to patients with MSS status [41]. The underlying mechanisms to explain these differences are unknown. Other studies reported that MSI-H patients possessed higher tumor infiltration of CD3+, CD8+ and CD45RO+ T cells compared to their MSI-L counterparts [42,43]. Unfortunately, clinical trials using ICIs in patients with mCRC do not commonly conduct genome sequencing or phenotyping of tumor-infiltrating lymphocytes (TILs); thus, it is difficult to establish the impact of tumor mutational burden (TMB) or TILs across MSS patients within the selected clinical trials. The encouraging outcomes of REGONIVO trial (NIVO + regorafenib) [27] could perhaps be due to known activity of regorafenib to modulate the tumor immune microenvironment via polarization of antigen-presenting cells, particularly macrophages [44]. Ou et al. established that regorafenib is capable of inducing the p38MAPK/Creb1/Klf4 signaling pathway responsible for the activation of tumor-associated macrophages [44], and subsequent production of pro-inflammatory cytokines (IL-10, IL-12 and IL-23) responsible for activation of cytotoxic T cells [45]. Following on these insights, it is of particular interest to examine combinatorial regimens of ICIs with kinase inhibitors. Lastly, the expression of other checkpoint molecules (ICOS, TIM-3, LAG-3, GITR and OX40) and their role in T cell inhibition or activation in the context of CRC may explain the resistance to currently used PD-1/PD-L1 inhibitors [20]. Although the efficacy of novel checkpoint inhibitors is yet to be fully defined, there is hope that in the near future, the majority of CRC patients may be benefited from ICI-based therapies.

Conclusion and Future Perspectives
Our analysis suggests that PD-1 and PD-L1 inhibitors may have significant potency in mCRC patients with MSI-H/dMMR status. FDA approval has already been granted to some regimens for use as therapeutic alternatives in aforementioned patient populations. Nonetheless, further evaluation in larger phase 3 clinical trials is necessary and is ongoing ( Table 2). Despite the promising efficacy of checkpoint blockade, it is commonly regarded as ineffective in patients with MSS status. Nonetheless, NIVO + regorafenib shows promise, though the trial was non-randomized and in a small cohort. Although clinical trials determining novel ICI-based combinatorial regimens in MSS patients are ongoing, it is also critical to conduct specifically designed translational studies to establish precise mechanisms of resistance in these patients. These studies may reveal previously unknown molecular patterns of CRC in patients with MSI-L and provide new strategies for overcoming therapeutic barriers. Understanding these roadblocks may, one day, result in successful and effective implementation of ICI therapy in a majority of patients with severely progressed or refractory colorectal cancer.   Data Availability Statement: All data can be obtained from the corresponding author on a reasonable request via e-mail (Golo.Ahlenstiel@health.nsw.gov.au).

Conflicts of Interest:
All authors declare that there are no competing interests that could have inappropriately influenced results of this study.