Impact of Tumor Shrinkage Pattern with Biweekly Triplet Gemcitabine+Cisplatin+S-1 Regimen for Biliary Tract Cancers: Implications for Neoadjuvant Therapy from the Data of KHBO1401 (KHBO1401-1A Study)

Abstract

Introduction

The multicenter randomized phase III KHBO1401 study (gemcitabine+cisplatin+S-1 [GCS] vs. GC in biliary tract cancers [BTC]) demonstrated that GCS not only prolonged patient survival but also achieved a high response rate and that it should be good for neoadjuvant therapy. Therefore, to explore the possibilities of neoadjuvant therapy, we investigated the tumor shrinkage pattern.

Methods

Among the total of 246 patients enrolled in the KHBO1401, the tumor shrinkage pattern and survival were investigated in patients with measurable BTC (n = 183, 74%; GCS, n = 91; GC, n = 92).

Results

The tumor shrinkage pattern could be divided into 4 categories based on the response at 100 days after enrollment: categories A (<–30% in size), B (−30–0%), C (0% to +20%), and D (>+20%). The GCS arm included more category A and B cases (61 [67%] vs. 33 [36%], p < 0.0001). Each category predicted the best response and overall survival (p < 0.0001). Category A showed sustained tumor response compared with category B; in GCS, the time to maximum tumor response was 165 ± 76 days in category A and 139 ± 78 in category B. Categories C and D did not achieve tumor shrinkage. The maximum tumor shrinkage size in category A was −53% in the GCS arm and −65% in the GC arm (p = 0.0892). Twenty percent of patients in the GCS showed tumor regrowth 154 ± 143 days later.

Conclusion

GCS provided faster and greater tumor shrinkage with better survival in comparison to GC, although 20% of patients showed regrowth after 6 cycles.

Introduction

Even with the increasing occurrence of biliary tract cancer (BTC) [1], treatment strategies are very limited [2]. Surgery is the only radical treatment and the only evidence-based systemic chemotherapy is the combination of gemcitabine and cisplatin [3–6]. There is limited evidence regarding adjuvant therapy [7, 8] and there is no evidence-based neo-adjuvant therapy. Recently, our group showed that biweekly gemcitabine, cisplatin, and S-1 (GCS) therapy prolonged patient survival in comparison to conventional gemcitabine and cisplatin (GC) in the KHBO1401 study [9]. This protocol was associated with a higher response rate (RR) (GCS, 42%; GC, 15%; p < 0.001) and a good conversion rate (GCS, 2.5%) in comparison to the previous systemic chemotherapy for BTC. Biweekly GCS not only prolonged patient survival in unresectable and metastatic BTC but also had the possibility of being applied as neoadjuvant chemotherapy. In addition to this, we proved that several chemotherapies were not appropriate after severe BTC surgery. At present, feasibility of adjuvant chemotherapy is limited after BTC surgery (KHBO1003, 1004, 1202 [10–12]); thus, it is necessary to investigate the possible application of GCS as neoadjuvant chemotherapy.

Several authors currently challenge to explorer-appropriate neo-adjuvant regimens. Gemcitabine-based treatment with or without radiotherapy has been the possible regimens explored for neoadjuvant therapy for resectable BTC [13–15]. Gemcitabine-combined radiotherapy achieved good local control [13]; however, there was an institutional bias with regard to radiotherapy. In contrast, gemcitabine-based chemotherapy had a limited tumor control rate [14, 16]. Recently, two gemcitabine-based triplet regimens, which have good RR and tumor control rate, have been developed [9, 17]. One is a gemcitabine, cisplatin, and nab-paclitaxel therapy [17]; the other is gemcitabine, cisplatin, and S-1 therapy [9]. We focused on GCS therapy, which we developed [9, 18]; however, there was no information regarding optimal treatment cycles for neoadjuvant therapy. Thus, we investigated patterns of the tumor shrinkage, to explore cycles that are appropriate for neoadjuvant therapy.

For neoadjuvant therapy, the following factors are important: a high tumor control rate, rapid effect, survival benefit, and less regrowth. In this study, we analyzed the spider plots for each patient and were able to divide each curve into 4 categories according to tumor shrinkage at 100 days: <-30%, −30–0%, 0 to +20%, and >+20%. To perform this analysis, we divided stable disease (SD), according to the Response Evaluation Criteria in Solid Tumours (RECIST [19]) (−30% to +20%) into two categories because the margin status in neoadjuvant therapy is very important for BTC surgery. Rather than early tumor shrinkage (ETS), we evaluated the time to the maximum effect and time to exceeding the initial size because we wanted to know the maximum allowable duration as neoadjuvant chemotherapy. In this study, we showed that the tumor shrinkage pattern (maximum response and time to maximum effect) was dependent on the type of chemotherapy and that it affected patient survival.

Methods

KHBO1401 Study

We conducted sub-analyses for tumor shrinkage and survival in order to explore an appropriate neoadjuvant regimen using the KHBO1401 study database. We showed a schematic illustration of the KHBO1401 study [9, 18]. The eligibility criteria were as follows: unresectable (locally advanced or metastatic) and recurrent BTC, histologically confirmed adenocarcinoma or adenosquamous carcinoma, no prior chemotherapy, age ≥20 years, and an adequate organ function. We included patients of >75 years of age in this study because we previously showed the safety and efficacy of chemotherapy in patients in this age group [20]. The treatment protocol is summarized in online supplementary sTable 1 (for all online suppl. material, see https://doi.org/10.1159/000533669). Conventional GC therapy was performed as follows: gemcitabine/cisplatin was infused at a dose of 1,000/25 mg/m² over 30/60 min on day 1 and day 8, every 3 weeks [6]. Biweekly GCS [9, 18] was performed as follows: gemcitabine/cisplatin was infused at a dose of 1,000/25 mg/m² over 30/60 min on day 1, every 2 weeks. S-1 was administered orally, twice a day, for seven consecutive days. The dose of S-1 was calculated according to the body surface area (BSA) as follows: BSA <1.25 m², 80 mg/day; BSA ≥1.25 m²–1.5 m², 100 mg/day; and BSA ≥1.5 m², 120 mg/day. Randomization was stratified as follows: unresectable/recurrent according to the results from our previous study [10]: gall bladder cancer/other cancers, and performance status (PS) 0-1/PS 2. Follow-up included a monthly check of tumor markers (carcinoembryonic antigen [CEA] and carbohydrate antigen 19-9 [CA19-9]) and imaging tests every 12 weeks (computed tomography or magnetic resonance imaging). A total of 246 patients were enrolled between July 2014 and February 2016, and the study revealed the superiority of biweekly GCS to conventional GC (hazard ratio [HR] = 0.791; 90% confidence interval [CI] = 0.628–0.996, one-sided p = 0.046) [9].

Statistical Analysis

To investigate tumor shrinkage patterns, we evaluated the efficacy of each protocol in the patients with measurable BTC (Fig. 1). The measurable BTC were estimated by RECIST. The response was evaluated in a total of 183 patients (74%, GC arm, n = 92; GCS arm, n = 91). The best response, RR, and disease control rate of each treatment arm are summarized in online supplementary sTable 2. The best responses of 4 cases were revised by the imaging central review committee from the original study in November 2018 [9]. The tumor shrinkage pattern (best response, timing, response at 100 days (14 weeks, approx. 6 cycles in GCS, and 4 cycles in GC, etc.)) and survival were investigated. The timing of evaluation was set to half of the treatment period (24 weeks) with consideration of the time from assignment to initial treatment.

Fig. 1.

Patients’ flow diagram GC, conventional gemcitabine + cisplatin; GCS, biweekly gemcitabine + cisplatin + S-1; OS, overall survival; PFS, progression-free survival; NE, not evaluated.

The data were expressed as the median (range), mean ± SD, or number (%). Differences between arms were tested by Student’s t-test or χ² test. p values of <0.05 were considered to indicate statistical significance. Overall survival (OS) rates from the day of treatment assignment were estimated using the Kaplan-Meier method and were compared using a log-rank test. Patients who were alive at the time of the last follow-up examination were censored. HRs and CIs were determined by Cox proportional hazards regression. The gemcitabine-related relative dose intensity (RDI) was calculated as the actual administered dose divided by the scheduled full dose of gemcitabine. All statistical analyses were conducted using the StatView software program (version 5.0, SAS Institute, Cary, NC, USA).

Results

Response in Each Patient according to the Treatment Cycles

We summarized the response curves for size in Figure 2a (GC arm) and 2B (GCS arm). “0” indicates the initial size, and an increase in size indicates a positive value and a decrease in size indicates a negative value in these spider plots. According to the response at 100 days after assignment, we could divide each curve into four categories: category A, >30% reduction (<–30% in y-axis); category B, 0–30% reduction (−30% to 0 in the y-axis); category C, 0–20% progression (0 to +20% in the y-axis); and category D, >20% progression (>+20% in the y-axis). The GCS arm included more category A patients (Table 1). Some category A patients in the GCS arm (but not the GC arm) experienced tumor regrowth (Fig. 2b; Table 1). In the GC arm, tumor shrinkage was sustained in the category A and B patients (Fig. 2a; Table 1). In contrast, various tumor marker (CEA and CA19-9) responses were observed, and these could not be divided into any categories (online suppl. Fig. 1a–d).

Fig. 2.

Dynamics of response (Spider plot) to GC or GCS in the analysis set. The individual lines represent the percentage variation of the size at different time points. a, b Change of tumor size (%) for GC (a) or GCS (b). The X-axis indicates the days after assignment. The Y-axis indicates the variation of size versus baseline ((variation of size – baseline size)/(baseline size), %). We categorized each case as A to D according to size change rate at day 100: category A, reduction >–30%; category B, reduction –30–0%; category C, 0–20%; category D, progression >20%. GC, conventional gemcitabine + cisplatin; GCS, biweekly gemcitabine + cisplatin + S-1.

Table 1.

Time to maximum effect and over initial size in each category

	n	Time to maximum effect, days	Change of tumor size, %	Time to exceeding initial size, days
Category A
GCS	40	164.6±76.4	−52.6±22.4	n = 8 (20.0%), 153.5±142.8
GC	13	225.3±190.5	−64.6±20.7	n = 0 (0%), N/A
p value		0.2826	0.0892	0.0801, N/A
Category B
GCS	21	138.5±78.4	−17.5±11.0	n = 9 (42.9%), 143.6±98.9
GC	20	153.9±81.9	−18.7±10.5	n = 7 (33.3%), 157.9±63.8
p value		0.5613	0.7285	0.6062, 0.7433

Data are expressed as the mean ± SD.

GC, conventional gemcitabine + cisplatin; GCS, biweekly gemcitabine + cisplatin + S-1. Bold typeface indicates p < 0.1.

Baseline Patient Characteristics in Each Category

We summarized the patients’ baseline characteristics in each category in Table 2. There were no significant differences among the categories in each arm with regard to age, sex, unresectable or recurrent, primary lesion, and initial tumor size. It seemed that category A included more recurrent tumor cases and cases with a smaller initial tumor size; however, these differences did not show statistical significance.

Table 2.

Baseline patient characteristics in each category

Treatment – category	GC-A	GC-B	GC-C	GC-D	GCS-A	GCS-B	GCS-C	GCS-D	p
N	13	20	36	23	40	21	19	11
Age	68.3±12.6	66.5±9.9	67.2±8.4	64.7±9.4	69.2±7.9	65.3±10.4	64.9±10.0	67.0±5.7	N.S.
Gender, n (%)
Male	6 (46)	11 (55)	20 (56)	12 (52)	22 (55)	12 (57)	9 (47)	4 (36)	0.9584
Female	7 (54)	9 (45)	16 (44)	11 (48)	18 (45)	9 (43)	10 (53)	7 (64)
Diagnosis, n (%)
Unresectable	9 (69)	16 (80)	30 (84)	19 (83)	25 (63)	18 (86)	16 (84)	9 (82)	0.3124
Locally advanced	2 (15)	7 (35)	10 (28)	4 (17)	3 (8)	5 (24)	4 (21)	1 (9)	0.3806
Metastatic	7 (54)	9 (45)	20 (56)	15 (65)	22 (55)	13 (62)	12 (63)	8 (73)
Recurrent	4 (31)	4 (20)	6 (16)	4 (17)	15 (37)	3 (14)	3 (16)	2 (18)
Primary tumor, n (%)
Gall bladder	6 (46)	4 (20)	13 (36)	10 (43)	13 (33)	6 (29)	11 (58)	4 (36)	0.4124
Extrahepatic bile duct	3 (23)	5 (25)	8 (22)	4 (17)	13 (33)	4 (19)	4 (21)	5 (46)
Intrahepatic bile duct	4 (31)	10 (50)	15 (42)	8 (35)	13 (33)	10 (47)	3 (16)	2 (18)
Ampullary	0	1 (5)	0	1 (5)	1 (1)	1 (5)	1 (5)	0
Initial tumor size, mm	46.5±28.4	61.2±36.7	58.8±34.0	65.0±42.9	54.2±32.7	65.2±39.0	55.8±41.2	56.5±37.2	N.S.

GC, conventional gemcitabine + cisplatin; GCS, biweekly gemcitabine + cisplatin + S-1.

N.S. indicates p > 0.1 among categories.

Maximum Effect in Each Category

The best response and the RR of each category are summarized in Tables 3, 4. The maximum effect was related to category in both the GC and GCS arms, and SD was divided into categories B and C (Table 3). The tumor response (RR) was remarkable in category A and was even noted in category C of GCS (Table 4).

Table 3.

Maximum effect in each category

	n	Category A	Category B	Category C	Category D	p value
GC	92	13	20	36	23	<0.0001
CR	1	1	0	0	0
PR	14	12	2	0	0
SD	47	0	15	27	5
PD	30	0	3	9	18
GCS	91	40	21	19	11	<0.0001
CR	3	2	0	1	0
PR	36	33	2	1	0
SD	35	5	19	9	2
PD	17	0	0	8	9

Best response was evaluated according to RECIST version 1.1 [19].

GC, conventional gemcitabine + cisplatin; GCS, biweekly gemcitabine + cisplatin + S-1; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; RR, response rate; DCR, disease control rate.

Bold typeface indicates p < 0.1.

Table 4.

Maximum RR in each category

RR	Total	Category A	Category B	Category C	Category D
GC: CR + PR/total, GC-RR%	15/92, 16%	13/13, 100%	2/20, 10%	0/36, 0%	0/23, 0%
GCS: CR + PR/total, GCS-RR%	39/91, 43%	34/40, 85%	2/21, 10%	3/19, 16%	0/11, 0%
p value	<0.0001	0.1381	0.9590	0.0142	N/A

Best response was evaluated according to RECIST version 1.1 [17].

N/A, not available.

GC, conventional gemcitabine + cisplatin; GCS, biweekly gemcitabine + cisplatin + S-1.

Bold typeface indicates p < 0.1.

Time to Maximum Effect and Exceeding the Initial Size in Each Category

Table 1 summarizes the time to maximum effect and the time to exceeding the initial size in categories A and B of each arm. In category A, the time to maximum effect was longer in the GC arm than in the GCS arm (not statistically significant), while the change in the tumor size tended to be greater in the GC arm than in the GCS arm (p = 0.0892). In the comparison of categories A and B, interestingly, the time to maximum effect was longer in category A. Size exceeding the initial size was noted in category A and B in the GCS arm and category B in the GC arm, and was not noted in category A in the GC arm. The time to exceeding initial size was approximately 150 days (5 months, nearly equal to 10 cycles of GCS) in each category. We compared the baseline characteristics between regrowth and no-regrowth group of category A in the GCS arm as shown in online supplementary sTable 3. There were no significant differences between the group regarding baseline characteristics.

Overall Survival in Each Category

Figure 3 and online supplementary Figure 2 summarizes the OS of each treatment arm in each category. There was no difference between the GCS and GC arms in any of the categories (online suppl. sFig. 2a, b, c, d), and OS depended on the category rather than treatment arm (Fig. 3a, b, c). Treatment cycles supported this (Fig. 4a) with the same RDI in each category (Fig. 4b). There were no difference of rate of neutropenia and thrombocytopenia in each category (Fig. 4c); on the contrary, in only category D of both of GC and GCS arm, there was a high rate of biliary tract infections (35–45% in category D and 8–16% in categories A to C). This may cause worse OS in category D (Fig. 3).

Fig. 3.

Survival analyses of each treatment arm (a: total, b: GC, c: GCS) among categories GC, conventional gemcitabine + cisplatin; GCS, biweekly gemcitabine + cisplatin + S-1.

Fig. 4.

Number of treatment cycles (a), gemcitabine-related relative dose intensity (b), and rate of major adverse events (maximum grade 3-4) (c) in each category The X-axis indicates the treatment arm (GC or GCS) and category (A, B, C, or D). GEM-RDI, gemcitabine-related relative dose intensity; GC, conventional gemcitabine + cisplatin; GCS, biweekly gemcitabine + cisplatin + S-1.

Discussion

In this sub-analysis of KHBO1401, we showed a higher RR in the GCS arm, where the responses were related to the type of chemotherapy rather than the RDI, that the response at 100 days predicted maximum tumor shrinkage and patient survival in each treatment arm, that earlier tumor shrinkage was achieved in the GCS arm, and that 20% of patients experienced tumor regrowth after 10 cycles of GCS.

Sub-analyses for the tumor shrinkage pattern were performed in several large-scale prospective chemotherapy studies. The main purpose of these analyses was to explore prognostic markers that could be measured at an early stage [21, 22]. For example, early tumor shrinkage (ETS) is well-known prognostic marker that is used to decide whether to continue first-line chemotherapy or to move to second-line chemotherapy [22]. From the KHBO1401 data, ETS also reflected the maximum tumor response and patient survival (data not shown). In the other analyses, regarding responses, the depth of response (DpR) was often analyzed [22]. In our analysis, DpR also predicted patient survival. However, there have been few sub-analyses of factors associated with tumor shrinkage in BTC [23] and it was difficult to compare the present study to previous studies. We considered that there were two possible reasons for the small number of sub-analyses: the first reason is that few prospective studies of chemotherapy have been conducted and the second is that it is difficult to evaluate the tumor size. From another viewpoint, in this study, we evaluated tumor shrinkage at 100 days (not early), to learn the shrinkage pattern (which has implications for neoadjuvant therapy), time to maximum response, duration of response, time to regrowth, and effect on survival. From our data, the tumor shrinkage pattern at 100 days not only reflected the maximum response and patient survival but also differed according to the type of chemotherapy. One of the most important points, regarding implications for neoadjuvant therapy, in approximately 67% of tumors, the size was maintained until 10 cycles of GCS. To achieve longer patient survival, an earlier response or an effective response is necessary. Based on our analysis of the KHB1401 data, we clarified that biweekly GCS was more effective for this than conventional GC. These features are also useful for the development of neoadjuvant therapy.

The main purpose of the present study was to determine the appropriate duration of biweekly GCS for neoadjuvant chemotherapy. Our group has also developed adjuvant chemotherapy; however, the dose intensity was limited after several types of surgery, for example, major hepatectomy [10–12, 24]. S-1 has potential application for adjuvant chemotherapy after major hepatectomy, but gemcitabine is not feasible after major hepatectomy despite the normal concentration of gemcitabine metabolites [24, 25]. Additionally, the authors presented several preoperative imaging markers that predict patient survival after surgery [26, 27]. Based on these lines of evidence, we also tried to develop neoadjuvant chemotherapy for BTC patients with FDG accumulation in the lymph nodes in the KHBO1201 study (UMIN000009831, NCT01821248). In the KHBO1201 study, we employed 3 to 6 cycles of GCS for neoadjuvant chemotherapy in these patients. These data from the KHB1401 study support that 3 or 6 cycles of GCS is reasonable and appropriate for neoadjuvant therapy; however, whether 3 cycles or 6 cycles is best for neoadjuvant chemotherapy remained a question. Furthermore, there was another problem as to whether GCS could maintain the tumor size after neoadjuvant chemotherapy and before surgery. Previously, we administered full-dose gemcitabine in combination with radiotherapy; the RR was 70%, the R0 resection rate was 96%, and the survival benefit was retrospectively revealed by an inverse probability of treatment weighting analysis [13]. However, systemic chemotherapy would be desirable for universal application; thus, we explored a possible regimen. In comparison to chemoradiotherapy, chemotherapy without radiotherapy was associated with limitations in terms of the response and the efficacy in maintaining the tumor size until surgery. Our study based on the KHBO1401 data revealed that the size was maintained until 10 cycles if tumor shrinkage was achieved at 100 days. Indeed, as after neoadjuvant chemotherapy and before surgery there were no data regarding the maintenance of tumor size, we need to address these questions and conduct a randomized study to investigate the optimal number of cycles of GCS in the future.

The published data of the RR in patients who received other systemic chemotherapy regimens were as follows: GC (gemcitabine+cisplatin), 28% [16]; GS (gemcitabine+S-1), 36% (JCOG0805) [28]; and GCS (KHBO1401), 42%. Additionally, a new triplet regimen consisting of gemcitabine, cisplatin, and nab-paclitaxel therapy was examined by MD Anderson and the Mayo Clinic group (GCnP) [17], and the RR was 32%. The TCR was 75% in GC, 82% in GCnP, and 80% in GCS. Our analyses of the KHBO1401 data showed that the tumor response would contribute to patient survival; the GCnP regimen is also expected to improve patient survival. Regarding neoadjuvant therapy, similarly to GCS, the appropriateness of 2–4 months of GCnP before surgery should be examined, and several groups have conducted trials of this regimen (NCT03579771 in the USA and UMIN000029490 in Japan). As abovementioned RR and TCR, several triplet regimens would be promising for the neo-adjuvant therapy of resectable BTC. Recently, the study to compare overall survival after surgery first and that after neo-adjuvant GCS with surgery had started (JCOG1920; jRCTs031200388).

Limitation: our analysis is limited to measurable lesions by RECIST (10 mm or more in size). As small lesions of BTC (less than 10 mm in size) are considered nonmeasurable lesions, our interpretation for shrinkage speed and time to regrowth could not be applied.

In conclusion, biweekly GCS provided faster and greater tumor shrinkage with better survival in the comparison of GC, although 20% of patients showed regrowth after 6 cycles. Biweekly GCS can be expected to be applied as a neoadjuvant regimen.

Acknowledgments

We would also like to especially acknowledge the members of the Data and Safety Monitoring Committee and the members of the Audit Committee. We are also grateful to the members of the KHBO Data Center for their support in data management (Masami Kashibo and Hiromi Oura). The study content was presented in part at the American Society of Clinical Oncology 2019 Annual Meeting, Chicago, USA, June 3rd, 2019.

This manuscript, including the concept, analyses, statistics, interpretation, and conclusion, was also reviewed by other members of KHBO group than the authors; Dr. Satoru Seo at the Division of Hepato-Biliary-Pancreatic Surgery and Transplantation, Department of Surgery, Graduate School of Medicine, Kyoto University; Dr. Kunihito Gotoh at the Department of Surgery, National Hospital Organization Osaka National Hospital; and Dr. Yoshitaro Shindo at the Department of Gastroenterological, Breast and Endocrine Surgery, Graduate School of Medicine, Yamaguchi University. This manuscript was proofread by a professional editor who is a native speaker of English at Japan Medical Communication (http://www.japan-mc.co.jp//).

Statement of Ethics

The study protocol was reviewed and approved by the Institutional Review Board of Kyoto University, and written informed consent was obtained from each patient for KHBO1401 (approval number: C863-2). The study was performed in accordance with the Declaration of Helsinki.

Conflict of Interest Statement

M.K. has received personal fees from Chugai. M.T. has received personal fees from Ono. K.Y. has received personal fees from Ohara, Nihon-Shinyaku, Sysmex, Chugai Pharma, Eli-Lilly, AstraZeneca, Otsuka, Novartis, Eisai, Nihon-Kayaku, Boehringer Ingelheim, Taiho, and Pfizer. E.H. has received personal fees from Eli-Lilly and Taiho. T.I. has received personal fees from Incyte, Chugai, Yakult Honsha, Taiho Pharmaceutical, Ono Pharm, Servier, Daiichi Sankyo, Nihon Zouki, Eli-Lilly, Otsuka, Novartis, AstraZeneca, and Abbott. The other authors declare that they have no competing interest.

Funding Sources

This investigation was funded by Taiho Pharmaceutical under a research contract.

Author Contributions

S.K., E.H., H.N., and T.I.: conception and design. S.K., H.W., D.S., H.B., M.K., H.K., T.T., M.U., M.T., M.S., K.Y., E.H., H.N., and T.I.: interpretation of data. S.K., H.W.,. D.S., H.B., M.K., H.K., T.T., M.U., M.T., and M.S.: acquisition of data. S.K., K.Y., and T.I.; analysis of data.

Funding Statement

This investigation was funded by Taiho Pharmaceutical under a research contract.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.