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Variability in lung cancer response to ALK inhibitors cannot be explained by the diversity of ALK fusion variants
Biochimie 154 (2018) 19e24
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Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
Variability in lung cancer response to ALK inhibitors cannot be explained by the diversity of ALK fusion variants Natalia V. Mitiushkina a, *, Vladislav I. Tiurin a, Aglaya G. Iyevleva a, b, Maxim M. Kholmatov a, b, Elena A. Filippova c, Fedor V. Moiseyenko a, d, e, Nikita E. Levchenko a, Ivan S. Sardaryan f, Svetlana V. Odintsova f, Alexandra M. Lozhkina c, Nikita M. Volkov d, Nina A. Karaseva g, Vladimir M. Moiseyenko d, Sergey V. Orlov c, h, Evgeny N. Imyanitov a, b, c, d, e, h, i a
N.N. Petrov Institute of Oncology, St.-Petersburg, Pesochny, Leningradskaya 68, 197758, Russia St.-Petersburg Pediatric Medical University, St.-Petersburg, Litovskaya 2, 194100, Russia c I.P. Pavlov St.-Petersburg State Medical University, St.-Petersburg, Lev Tolstoy Street 6-8, 197022, Russia d City Cancer Center, St.-Petersburg, Pesochny, Leningradskaya 68A, 197758, Russia e I.I. Mechnikov North-Western Medical University, St.-Petersburg, Kirochnaya Street 41, 191015, Russia f LLC Bioeq, St.-Petersburg, Krasnogvardeisky Pereulok 23, 197342, Russia g City Cancer Dispensary, St.-Petersburg, Veteranov Prospect 56, 198255, Russia h Institute of Medical Primatology, Sochi, Veseloye, Mira Street 177, 354376, Russia i St.-Petersburg State University, St.-Petersburg, Universitetskaya Naberezhnaya 7/9, 199034, Russia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2018 Accepted 27 July 2018 Available online 30 July 2018
Multiple laboratory evidences indicate that distinct variants of ALK translocations differ in their biochemical properties and responsiveness to ALK tyrosine kinase inhibitors (TKIs). These data are supported by some clinical studies, which showed improved responses to crizotinib in non-small cell lung cancer (NSCLC) patients carrying particular variants of ALK translocation. We retrospectively considered 64 Russian patients with ALK-rearranged NSCLC, who were treated by crizotinib (n ¼ 23), ceritinib (n ¼ 39) or alectinib (n ¼ 2). ALK fusion variants were genotyped by PCR. Median progressionfree survival (PFS) approached to 18 and 21 months in subjects with “short” (v.3a/b, v.5a/b) vs. “long” (TAPE-domain containing) fusion variants (p ¼ 0.783), respectively; similar data were obtained while comparing EML4/ALK variant 1 vs. other ALK translocations (19 and 21 months, respectively; p ¼ 0.604). Objective response rates were also strikingly similar in the above groups (“short”: 88%, “long”: 77%, p ¼ 0.479; variant 1: 76%, other translocations: 81%, p ¼ 0.753). Furthermore, ALK variants did not influence the disease outcomes when patients treated by crizotinib and ceritinib were analyzed separately. Overall, PFS on ALK TKI did not depend on whether the drug was administered upfront or after chemotherapy. Ceritinib produced significantly longer PFS than crizotinib (p ¼ 0.022). In conclusion, this study revealed that distinct ALK translocation variants render similar clinical responsiveness to ALK inhibitors. © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: ALK fusion variants Non-small cell lung cancer Tyrosine kinase inhibitors Treatment outcome
1. Introduction Activating ALK rearrangements in non-small cell lung cancer (NSCLC) were discovered in the year 2007 through a systematic
* Corresponding author. E-mail address: [email protected] (N.V. Mitiushkina).
search for novel oncogenes implicated in NSCLC pathogenesis [1,2]. A corresponding tyrosine kinase inhibitor, PF-02341066, later named crizotinib, was by then already under clinical evaluation; it was originally developed for the inhibition of oncogenic tyrosine kinase MET, however biochemical studies identified its concurrent anti-ALK activity [3e6]. The extension of the ongoing phase I crizotinib trial to the patients with ALK rearrangements demonstrated excellent activity of this drug towards ALK-driven tumors.
https://doi.org/10.1016/j.biochi.2018.07.018 0300-9084/© 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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Subsequent large clinical studies showed that administration of crizotinib results in either objective disease response or prolonged disease stabilization in virtually all patients with ALK-rearranged NSCLC, thus warranting the approval of this TKI [7,8]. In contrast to crizotinib, second- and third-generation ALK inhibitors (ceritinib, alectinib, lorlatinib, brigatinib) were intentionally developed to target this kinase. These drugs are more potent with regard to interfering with ALK kinase activity and capable to target crizotinib-resistant ALK mutants [9]. The detection of ALK rearrangements in the tumor tissue is an absolute prerequisite for administration of these drugs. Given that about two dozen ALK fusion variants were identified soon after the discovery of ALK translocations, use of variant-specific PCR for ALK analysis was not considered feasible at the time of initial TKI trials. As result, almost all ALK TKI studies relied on the fluorescence in situ hybridization (FISH) break-apart assay; this test is capable to establish the mere fact of the presence of ALK rearrangement, however it cannot discriminate between distinct ALK fusion variants [10,11]. There are mounting evidences that ALK rearrangements may differ in their sensitivity to ALK inhibitors. Richards et al. [12,13] analyzed the most common NSCLC-specific fusion, EML4-ALK, and revealed that the presence of a tandem atypical beta-propeller in EML protein (TAPE) domain influences both the stability of the chimeric protein product and the response of cells to Hsp90 inhibitors. In fact, so-called short fusion variants (3a/b and 5a/b) lack TAPE domain and tend to be less responsive to the action of crizotinib than the longer (TAPE-containing) versions of the EML4ALK (variants 1, 2 etc.) [12e15]. These data are highly consistent with in vitro studies of Woo et al. [16] and Noh et al. [17], who also observed decreased sensitivity of short EML4-ALK proteins to ALK inhibitors. The role of the presence of the TAPE domain is less obvious in the earlier report of Heuckmann et al. [18]: these authors observed some differences between variants 2 and 3a, however, in contrast to the above data, variants 1 and 3 b showed similar response to crizotinib. The analysis of clinical impact of distinct ALK translocations is highly complicated. As mentioned above, the majority of NSCLC patients receiving ALK inhibitors in Europe and USA are diagnosed by FISH or immunohistochemistry (IHC) [10], therefore the type of their rearrangement remains unknown. Furthermore, the distribution of ALK rearrangements is not even: variants 1 and 3 are responsible for approximately 30e40% of ALK fusions each, and variant 2 accounts for another 10% of ALK rearrangements [19,20]. Rare EML4-ALK variants and translocations involving other than EML4 fusion partners account for no more than 10e30% of ALK alterations. Clinical studies evaluating the predictive role of ALK translocation subtype were performed mainly in Eastern Asian patients, given that guidelines for ALK testing in these countries are not restricted to FISH analysis and allow PCR testing [21]. Importantly, some data sets demonstrated that the type of ALK rearrangement may significantly influence treatment outcomes [16,17,22e24]. The analysis of predictive role of distinct ALK fusion variants is of high medical importance. If some categories of ALK rearrangements are characterized by low responsiveness to ALK inhibitors, one could conclude that FISH- or IHC-based ALK analysis is insufficient to guide the treatment. Here we report treatment outcomes for 64 ALK-rearranged NSCLCs treated with TKI therapy. 2. Materials and methods This study retrospectively considered ALK-rearranged NSCLC patients, who received ALK-specific therapy within years 2012e2017 in one of the cancer hospitals of St.-Petersburg and had
data on the treatment outcome. ALK translocation genotypes were determined by RT-PCR in tumor biopsies or surgically resected specimens obtained before the start of systemic treatment; this testing was performed in the Laboratory of Molecular Oncology, N.N. Petrov Institute of Oncology (St.-Petersburg, Russia) as a part of the routine diagnostic procedure [25,26]. In addition to PCR analysis, each patient had a confirmation of the presence of ALK fusions being performed either by FISH or IHC. All patients belonged to Caucasian race. 53 patients were treated in I.P. Pavlov Medical University, St.-Petersburg, five subjects received TKI in St.Petersburg City Cancer Hospital and six cases were managed in St.-Petersburg Oncological Dispensary. The study considered responses to ALK inhibitors only in TKI-naïve patients. 37 subjects received their first ALK TKI as an upfront treatment, 20 patients were exposed to TKI starting from second-line therapy, and 7 patients were administered TKI at the third line (Table 1). 23 patients received crizotinib, 39 were treated with ceritinib and 2 were treated with alectinib. The study was conducted in accordance with
Table 1 Patient characteristics. Characteristic
Number of patients (%)
Median age at diagnosis, years (range) Sex, females Smoking status Ever-smokers Never-smokers Unknown Histology Adenocarcinoma Adenosquamous Squamous Poorly differentiated Giant-cell carcinoma Brain metastases Present before TKI treatment Emerged during TKI treatment Absent Unknown First TKI treatment Crizotinib Ceritinib Alectinib Treatment line for the first TKI exposure 1 2 3 Response to the first TKI treatment CR PR SD PD Progression at last assessment Median PFS (95% CI), months Overall number of sequential TKIs 1 2 3 ALK variant EML4ex13/ALKex20 (v.1) EML4ex6/ALKex20 (v.3) EML4ex18/ALKex20 EML4ex20/ALKex20 (v.2) EML4ex2/ALKex20 (v.5) EML4ex13/ins69ALKex20 (v.6) EML4ex19/ALKex20 EML4ex21/ALKex20 KIF5bex17/ALKex20 Variant undetermined Total
52.5 (25e78) 39 (60.9%) 11 (19.3%) 46 (80.7%) 7 60 (93.8%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 6 (9.4%) 26 (40.6%) 31 (48.4%) 1 (1.6%) 23 (35.9%) 39 (60.9%) 2 (3.1%) 37 (57.8%) 20 (31.3%) 7 (10.9%) 7 (10.9%) 43 (67.2%) 13 (20.3%) 1 (1.6%) 35 (54.7%) 19 (15e35) 48 (75%) 15 (23.4%) 1 (1.6%) 33 (51.6%) 16 (25%) 4 (6.3%) 3 (4.7%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 3 (4.7%) 64
Abbreviations: TKI, tyrosine kinase inhibitor; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; PFS, progression-free survival.
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the Helsinki Declaration and followed the rules of the local ethics committee. All statistical tests were performed using R software (http://www.r-project.org/) version 3.4.2. Survival analysis was done with the R package Survival [27]. 3. Results ALK translocation genotype was successfully determined by variant-specific PCR in 61 out of 64 analyzed patients. Three patients had rare variants of rearrangements, which were recognized by unbalanced expression of 5’/30 -ends of ALK transcript [26] and FISH, but could not be classified by available variant-specific PCR tests. EML4-ALK variant 1 was detected in 33/64 (52%) cases; EML4ALK variant 3 was the second most common, being present in 16/64 (25%) NSCLC samples. Other ALK fusion variants were rare (Table 1). While performing comparisons of treatment outcomes, we considered two approaches. First, we classified EML4-ALK rearrangements into short and long forms, as supported by functional data [12]; we identified 17 patients with short EML4-ALK fusions, 16 of whom carried variant 3 translocation (Table 1). Secondly, we considered variant 1 against all others, as suggested by Yoshida et al. [22]. Data on the tumor response by RECIST are presented in Table 2. Frequencies of objective response were strikingly similar among various patient categories. Only 14 (22%) out of 64 patients were non-responders; lack of substantial tumor shrinkage could not be explained by the presence of particular ALK fusion variant. PFS was also similar across patient subgroups (Fig. 1). This trend did not change when crizotinib or ceritinib were considered separately.
Fig. 1. Kaplan-Meier plots of progression-free survival (PFS) according to ALK fusion type. ALK variants are grouped in two different ways: “short” EML4-ALK variants (v.3a/ b and v.5a/b, containing 6 or less EML4 exons) vs “long” EML4-ALK variants (v.1 and other variants) (a) or EML4-ALK variant 1 vs all other ALK fusion species (b). p-values are calculated using the log-rank test.
Indeed, ceritinib produced significantly better response rates and PFS as compared to crizotinib, however ALK fusion variants did not contribute to this difference (Table 2, Figs. S1eS3). Noticeably, PFS on TKI did not depend on whether the patients received ALK inhibitor while being treatment-naïve or if the TKI was administered after chemotherapy (Fig. 2). Overall survival (OS) was not influenced by the type of ALK translocation (Fig. 3). This lack of differences in OS was unlikely to be attributed to known confounding factors: for example, the analyzed groups of patients were relatively well balanced with regard to the presence of brain metastases or number of ALK TKIs received (Table 2; Figs. S4eS5).
Table 2 Patient characteristics according to ALK fusion variant. EML4-ALK short variantsa Median age at diagnosis, 54 (34e78) years (range) Sex, females 10 (58.8%) Smoking status Ever-smokers 6 (37.5%) Never-smokers 10 (62.5%) Histology, adenocarcinoma 16 (94.1%) Brain metastases Present before TKI 1 (5.9%) treatment Emerged during TKI 5 (29.4%) treatment Absent 11 (64.7%) First TKI Crizotinib 4 (23.5%) Ceritinib 12 (70.6%) Alectinib 1 (5.9%) Treatment line 1 12 (70.6%) 2-3 5 (29.4%) Response to the first TKI treatment CR 2 (11.8%) PR 13 (76.5%) SD 2 (11.8%) PD 0 ORR 15 (88.2%) ORR, crizotinib-treated 3 (75%) patients ORR, ceritinib-treated 11 (91.7%) patients Overall number of sequential TKIs 1 14 (82.4%) 2e3 3 (17.6%) Total N ¼ 17
EML4-ALK long variants
p-value short vs long variants
51 (25e76)
0.257 (Kruskal-Wallis rank 51 (25e76) sum test) 1 (c2-test) 22 (66.7%)
26 (60.5%) 4 (10.8%) 33 (89.2%) 42 (97.7%)
0.05 (Fisher's exact test)
5 (11.9%)
EML4ex13/ALKex20 (variant 1)
Non-variant 1 ALK p-value fusions variant 1 vs other variants 53 (34e78) 17 (54.8%)
0.476 (Kruskal-Wallis rank sum test) 0.443 (c2-test)
8 (28.6%) 20 (71.4%) 28 (90.3%)
0.103 (Fisher's exact test)
0.49 (Fisher's exact test)
3 (10.3%) 26 (89.7%) 32 (97%)
0.49 (Fisher's exact test)
4 (12.1%)
2 (6.7%)
0.281 (Fisher's exact test)
18 (42.9%)
16 (48.5%)
10 (33.3%)
19 (45.2%)
13 (39.4%)
18 (60%)
0.347 (Fisher's exact test)
18 (41.9%) 24 (55.8%) 1 (2.3%)
0.434 (Fisher's exact test)
12 (36.4%) 20 (60.6%) 1 (3%)
11 (35.5%) 19 (61.3%) 1 (3.2%)
1 (Fisher's exact test)
22 (51.2%) 21 (48.8%)
0.281 (Fisher's exact test)
15 (45.5%) 18 (54.5%)
22 (71%) 9 (29%)
0.07 (Fisher's exact test)
4 (9.3%) 29 (67.4%) 9 (20.9%) 1 (2.3%) 33 (76.7%) 11 (61.1%)
0.753 (Fisher's exact test)
3 (9.7%) 22 (71%) 6 (19.4%) 0 25 (80.6%) 7 (63.6%)
1 (Fisher's exact test)
0.479 (Fisher's exact test) 1 (Fisher's exact test)
4 (12.1%) 21 (63.6%) 7 (21.2%) 1 (3%) 25 (75.8%) 7 (58.3%)
0.753 (Fisher's exact test) 1 (Fisher's exact test)
21 (87.5%)
1 (Fisher's exact test)
17 (85%)
17 (89.5%)
1 (Fisher's exact test)
31 (72.1%) 12 (27.9%) N ¼ 43
0.52 (Fisher's exact test)
25 (75.8%) 8 (24.2%) N ¼ 33
23 (74.2%) 8 (25.8%) N ¼ 31
1 (Fisher's exact test)
Abbreviations: TKI, tyrosine kinase inhibitor; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; ORR, overall response rate. a EML4-ALK variants 3a/b and 5a/b, which contain shorter portion of EML4 gene and are more structurally stable than other (“long”) EML4-ALK variants [13].
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Fig. 2. Kaplan-Meier plots of progression-free survival (PFS) for patients receiving ALK TKI upfront (treatment line 1) vs. subjects treated by an ALK inhibitor after chemotherapy (treatment lines 2 or 3). p-values are calculated using the log-rank test.
Fig. 3. Kaplan-Meier plots of overall survival (OS) according to ALK fusion type. (a) Survival curves for “short” EML4-ALK variants vs “long” EML4-ALK variants. (b) Survival curves for EML4-ALK variant 1 vs all other ALK fusions. p-values are calculated using the log-rank test.
4. Discussion The predictive role of distinct ALK variants was previously investigated in several Asian studies. Yoshida et al. [22] evaluated 35 patients treated by crizotinib and revealed that NSCLC with variant 1 EML4-ALK translocation had longer progression-free survival as compared to others. Lei et al. [28] compared 22 patients with EML4-ALK variant 1, 18 subjects with variants 3a/b and 21 NSCLC with other ALK fusions and found no differences in treatment outcome between these three groups. Cha et al. [29] analyzed 52 patients with ALK rearrangements treated by cytotoxic and/or targeted therapy. Among 35 subjects exposed to pemetrexed, PFS was longer in EML4-ALK cases as compared to patients with non-EML4-ALK rearrangements; this difference was attributed mainly to improved PFS in patients with EML4-ALK variant 1. ALK fusion variants did not influence PFS for ALK inhibitors (n ¼ 32) or platinum-based chemotherapy (n ¼ 40). Also, there was no difference in RECIST response rates according to ALK fusion variant for the patients receiving ALK inhibitors (n ¼ 37), pemetrexed (n ¼ 40) or platinum-based therapy (n ¼ 40). Woo et al. [16] considered 51 patients with EML4-ALK rearrangements, who were treated with crizotinib, alectinib or ceritinib (n ¼ 51). The 2-year PFS was higher for NSCLC EML4-ALK variants 1/2/others as compared to patients with 3a/b fusions. Sufficient number of cases were available only for crizotinib (n ¼ 44), and the difference remained statistically significant after the exclusion of alectinib- and ceritinib-treated patients. Noh et al. [17] revealed that tumor samples carrying short variants of EML4-ALK showed higher ALK expression by IHC. In
addition, short EML4-ALK fusions were associated with more advanced stage of the disease and presence of metastatic spread. Median PFS in 46 crizotinib-treated patients tended to be worse for NSCLC with EML4-ALK short variants as compared to subjects with long EML4-ALK fusions. Li et al. [24] investigated 60 NSCLC patients (variant 1: n ¼ 14; variant 2: n ¼ 9; variant 3a/b: n ¼ 20; rare EML4ALK translocations: n ¼ 11; non-EML4-ALK fusions: n ¼ 6); they observed improved PFS in patients with variant 2 rearrangements. A few non-Asian studies have been published very recently. McLeer-Florin et al. [30] considered 6 patients carrying EML4-ALK 1/2/others variants versus 8 NSCLC cases with EML4-ALK 3a/b translocations and found numerically better PFS in the former group. Lin et al. [31] compared treatment outcomes in patients with variant 1 and variant 3 ALK rearrangements. The analysis of tumor responsiveness to crizotinib was undertaken for 99 patients (variant 1: n ¼ 51; variant 3: n ¼ 48) and demonstrated no significant differences; interestingly, a third-generation ALK inhibitor lorlatinib produced significantly better PFS in subjects with variant 3 when given after the failure of crizotinib and at least 1 secondgeneration TKI. Christopolous et al. [23] considered 67 ALKrearranged NSCLCs with variant 1 (n ¼ 26), variant 2 (n ¼ 7) or variant 3 (n ¼ 34) translocation. EML4-ALK variant 3 was associated with higher metastatic burden at diagnosis, inferior PFS both on TKI and on chemotherapy, poor response to cerebral radiotherapy and inferior overall survival. Here we presented a relatively large data set evaluating clinical differences between various ALK variants in non-Asian patients. In contrast to EGFR mutations, distribution of ALK translocations appears to be relatively similar in Orientals and Caucasians, therefore the contribution of racial factor in the observed relationships may not be of importance. Current investigation did not confirm favorable impact of EML4-ALK variant 1 or long EML4-ALK forms on PFS or other treatment outcomes. However, several limitations of this study have to be acknowledged. First of all, it utilized a group of patients treated by two distinct drugs, crizotinib and ceritinib. This is the first study, where most of the patients were treated with the second-generation ALK-inhibitor, ceritinib. Neither crizotinib (n ¼ 23) nor ceritinib (n ¼ 39) treatment outcomes were affected by the type of ALK rearrangement. However, the number of patients in the crizotinib group was small; therefore, if only crizotinib but not ceritinib efficacy is modulated by the type of ALK rearrangement, this study lacked the power to detect the differences. Similarly to other studies [16,17,22e24,28,29,31], this report considered both treatment-naïve and chemotherapy-pretreated patients. However, even if TKI was administered after the failure of chemotherapy, the PFS on TKI treatment was virtually identical to the one in patients receiving TKI upfront (Fig. 2). It is also of notice, that the spectrum of distinct ALK translocations is a subject of interstudy variations: for example, the share of EML4-ALK variant 1 in the present study was somewhat higher than in several previous reports [16,17,23,24], while the frequency of variant 3 appeared to be lower. These deviations are unlikely to be caused by methodological reasons, given that the currently utilized ALK genotyping methods (variant-specific PCR or next generation sequencing) are highly reliable. Clinical and biological factors affecting the distribution of ALK variants deserve further investigation. We believe that this study is sufficient to exclude major contribution of ALK fusion variants in the treatment outcomes for ALK inhibitors. Nevertheless, it is important to consider further analysis utilizing larger and more carefully selected NSCLC subgroups.
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Conflicts of interest Natalia V. Mitiushkina, Vladislav I. Tiurin, Aglaya G. Iyevleva, Maxim M. Kholmatov, Elena A. Filippova, Fedor V. Moiseyenko, Nikita E. Levchenko, Ivan S. Sardaryan, Svetlana V. Odintsova, Alexandra M. Lozhkina, Nikita M. Volkov, Nina A. Karaseva, Vladimir M. Moiseyenko, Sergey V. Orlov and Evgeny N. Imyanitov declare that they have no conflict of interest.
[3]
Author contribution
[4]
Natalia V. Mitiushkina e conception and design of the study, interpretation of data, drafting the article and the final approval of the manuscript; Vladislav I. Tiurin e acquisition of data on ALK fusion types in selected samples, manuscript preparation and approval; Aglaya G. Iyevleva e analysis of clinical data and interpretation of results, manuscript preparation and approval; Maxim M. Kholmatov e statistical analysis, revision and approval of the manuscript; Elena A. Filippova e acquisition of clinical data, revision and approval of the manuscript; Fedor V. Moiseyenko e acquisition of clinical data, revision and approval of the manuscript; Nikita E. Levchenko e acquisition of clinical data, revision and approval of the manuscript; Ivan S. Sardaryan e acquisition of clinical data, revision and approval of the manuscript; Svetlana V. Odintsova e acquisition of clinical data, revision and approval of the manuscript; Alexandra M. Lozhkina e acquisition of clinical data, revision and approval of the manuscript; Nikita M. Volkov e acquisition of clinical data, revision and approval of the manuscript; Nina A. Karaseva e acquisition of clinical data, revision and approval of the manuscript; Vladimir M. Moiseyenko e acquisition of clinical data, revision and approval of the manuscript; Sergey V. Orlov e acquisition and interpretation of clinical data, manuscript preparation and approval; Evgeny N. Imyanitov - conception and design of the study, drafting the article and the final approval of the manuscript. Declaration of interest
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
None. [13]
Acknowledgements This study has been supported by the Russian Scientific Fund (grant 16-15-10396).
[14]
Appendix A. Supplementary data
[15]
Supplementary data related to this article can be found at https://doi.org/10.1016/j.biochi.2018.07.018.
[16]
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Research paper
Variability in lung cancer response to ALK inhibitors cannot be explained by the diversity of ALK fusion variants Natalia V. Mitiushkina a, *, Vladislav I. Tiurin a, Aglaya G. Iyevleva a, b, Maxim M. Kholmatov a, b, Elena A. Filippova c, Fedor V. Moiseyenko a, d, e, Nikita E. Levchenko a, Ivan S. Sardaryan f, Svetlana V. Odintsova f, Alexandra M. Lozhkina c, Nikita M. Volkov d, Nina A. Karaseva g, Vladimir M. Moiseyenko d, Sergey V. Orlov c, h, Evgeny N. Imyanitov a, b, c, d, e, h, i a
N.N. Petrov Institute of Oncology, St.-Petersburg, Pesochny, Leningradskaya 68, 197758, Russia St.-Petersburg Pediatric Medical University, St.-Petersburg, Litovskaya 2, 194100, Russia c I.P. Pavlov St.-Petersburg State Medical University, St.-Petersburg, Lev Tolstoy Street 6-8, 197022, Russia d City Cancer Center, St.-Petersburg, Pesochny, Leningradskaya 68A, 197758, Russia e I.I. Mechnikov North-Western Medical University, St.-Petersburg, Kirochnaya Street 41, 191015, Russia f LLC Bioeq, St.-Petersburg, Krasnogvardeisky Pereulok 23, 197342, Russia g City Cancer Dispensary, St.-Petersburg, Veteranov Prospect 56, 198255, Russia h Institute of Medical Primatology, Sochi, Veseloye, Mira Street 177, 354376, Russia i St.-Petersburg State University, St.-Petersburg, Universitetskaya Naberezhnaya 7/9, 199034, Russia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2018 Accepted 27 July 2018 Available online 30 July 2018
Multiple laboratory evidences indicate that distinct variants of ALK translocations differ in their biochemical properties and responsiveness to ALK tyrosine kinase inhibitors (TKIs). These data are supported by some clinical studies, which showed improved responses to crizotinib in non-small cell lung cancer (NSCLC) patients carrying particular variants of ALK translocation. We retrospectively considered 64 Russian patients with ALK-rearranged NSCLC, who were treated by crizotinib (n ¼ 23), ceritinib (n ¼ 39) or alectinib (n ¼ 2). ALK fusion variants were genotyped by PCR. Median progressionfree survival (PFS) approached to 18 and 21 months in subjects with “short” (v.3a/b, v.5a/b) vs. “long” (TAPE-domain containing) fusion variants (p ¼ 0.783), respectively; similar data were obtained while comparing EML4/ALK variant 1 vs. other ALK translocations (19 and 21 months, respectively; p ¼ 0.604). Objective response rates were also strikingly similar in the above groups (“short”: 88%, “long”: 77%, p ¼ 0.479; variant 1: 76%, other translocations: 81%, p ¼ 0.753). Furthermore, ALK variants did not influence the disease outcomes when patients treated by crizotinib and ceritinib were analyzed separately. Overall, PFS on ALK TKI did not depend on whether the drug was administered upfront or after chemotherapy. Ceritinib produced significantly longer PFS than crizotinib (p ¼ 0.022). In conclusion, this study revealed that distinct ALK translocation variants render similar clinical responsiveness to ALK inhibitors. © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: ALK fusion variants Non-small cell lung cancer Tyrosine kinase inhibitors Treatment outcome
1. Introduction Activating ALK rearrangements in non-small cell lung cancer (NSCLC) were discovered in the year 2007 through a systematic
* Corresponding author. E-mail address: [email protected] (N.V. Mitiushkina).
search for novel oncogenes implicated in NSCLC pathogenesis [1,2]. A corresponding tyrosine kinase inhibitor, PF-02341066, later named crizotinib, was by then already under clinical evaluation; it was originally developed for the inhibition of oncogenic tyrosine kinase MET, however biochemical studies identified its concurrent anti-ALK activity [3e6]. The extension of the ongoing phase I crizotinib trial to the patients with ALK rearrangements demonstrated excellent activity of this drug towards ALK-driven tumors.
https://doi.org/10.1016/j.biochi.2018.07.018 0300-9084/© 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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Subsequent large clinical studies showed that administration of crizotinib results in either objective disease response or prolonged disease stabilization in virtually all patients with ALK-rearranged NSCLC, thus warranting the approval of this TKI [7,8]. In contrast to crizotinib, second- and third-generation ALK inhibitors (ceritinib, alectinib, lorlatinib, brigatinib) were intentionally developed to target this kinase. These drugs are more potent with regard to interfering with ALK kinase activity and capable to target crizotinib-resistant ALK mutants [9]. The detection of ALK rearrangements in the tumor tissue is an absolute prerequisite for administration of these drugs. Given that about two dozen ALK fusion variants were identified soon after the discovery of ALK translocations, use of variant-specific PCR for ALK analysis was not considered feasible at the time of initial TKI trials. As result, almost all ALK TKI studies relied on the fluorescence in situ hybridization (FISH) break-apart assay; this test is capable to establish the mere fact of the presence of ALK rearrangement, however it cannot discriminate between distinct ALK fusion variants [10,11]. There are mounting evidences that ALK rearrangements may differ in their sensitivity to ALK inhibitors. Richards et al. [12,13] analyzed the most common NSCLC-specific fusion, EML4-ALK, and revealed that the presence of a tandem atypical beta-propeller in EML protein (TAPE) domain influences both the stability of the chimeric protein product and the response of cells to Hsp90 inhibitors. In fact, so-called short fusion variants (3a/b and 5a/b) lack TAPE domain and tend to be less responsive to the action of crizotinib than the longer (TAPE-containing) versions of the EML4ALK (variants 1, 2 etc.) [12e15]. These data are highly consistent with in vitro studies of Woo et al. [16] and Noh et al. [17], who also observed decreased sensitivity of short EML4-ALK proteins to ALK inhibitors. The role of the presence of the TAPE domain is less obvious in the earlier report of Heuckmann et al. [18]: these authors observed some differences between variants 2 and 3a, however, in contrast to the above data, variants 1 and 3 b showed similar response to crizotinib. The analysis of clinical impact of distinct ALK translocations is highly complicated. As mentioned above, the majority of NSCLC patients receiving ALK inhibitors in Europe and USA are diagnosed by FISH or immunohistochemistry (IHC) [10], therefore the type of their rearrangement remains unknown. Furthermore, the distribution of ALK rearrangements is not even: variants 1 and 3 are responsible for approximately 30e40% of ALK fusions each, and variant 2 accounts for another 10% of ALK rearrangements [19,20]. Rare EML4-ALK variants and translocations involving other than EML4 fusion partners account for no more than 10e30% of ALK alterations. Clinical studies evaluating the predictive role of ALK translocation subtype were performed mainly in Eastern Asian patients, given that guidelines for ALK testing in these countries are not restricted to FISH analysis and allow PCR testing [21]. Importantly, some data sets demonstrated that the type of ALK rearrangement may significantly influence treatment outcomes [16,17,22e24]. The analysis of predictive role of distinct ALK fusion variants is of high medical importance. If some categories of ALK rearrangements are characterized by low responsiveness to ALK inhibitors, one could conclude that FISH- or IHC-based ALK analysis is insufficient to guide the treatment. Here we report treatment outcomes for 64 ALK-rearranged NSCLCs treated with TKI therapy. 2. Materials and methods This study retrospectively considered ALK-rearranged NSCLC patients, who received ALK-specific therapy within years 2012e2017 in one of the cancer hospitals of St.-Petersburg and had
data on the treatment outcome. ALK translocation genotypes were determined by RT-PCR in tumor biopsies or surgically resected specimens obtained before the start of systemic treatment; this testing was performed in the Laboratory of Molecular Oncology, N.N. Petrov Institute of Oncology (St.-Petersburg, Russia) as a part of the routine diagnostic procedure [25,26]. In addition to PCR analysis, each patient had a confirmation of the presence of ALK fusions being performed either by FISH or IHC. All patients belonged to Caucasian race. 53 patients were treated in I.P. Pavlov Medical University, St.-Petersburg, five subjects received TKI in St.Petersburg City Cancer Hospital and six cases were managed in St.-Petersburg Oncological Dispensary. The study considered responses to ALK inhibitors only in TKI-naïve patients. 37 subjects received their first ALK TKI as an upfront treatment, 20 patients were exposed to TKI starting from second-line therapy, and 7 patients were administered TKI at the third line (Table 1). 23 patients received crizotinib, 39 were treated with ceritinib and 2 were treated with alectinib. The study was conducted in accordance with
Table 1 Patient characteristics. Characteristic
Number of patients (%)
Median age at diagnosis, years (range) Sex, females Smoking status Ever-smokers Never-smokers Unknown Histology Adenocarcinoma Adenosquamous Squamous Poorly differentiated Giant-cell carcinoma Brain metastases Present before TKI treatment Emerged during TKI treatment Absent Unknown First TKI treatment Crizotinib Ceritinib Alectinib Treatment line for the first TKI exposure 1 2 3 Response to the first TKI treatment CR PR SD PD Progression at last assessment Median PFS (95% CI), months Overall number of sequential TKIs 1 2 3 ALK variant EML4ex13/ALKex20 (v.1) EML4ex6/ALKex20 (v.3) EML4ex18/ALKex20 EML4ex20/ALKex20 (v.2) EML4ex2/ALKex20 (v.5) EML4ex13/ins69ALKex20 (v.6) EML4ex19/ALKex20 EML4ex21/ALKex20 KIF5bex17/ALKex20 Variant undetermined Total
52.5 (25e78) 39 (60.9%) 11 (19.3%) 46 (80.7%) 7 60 (93.8%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 6 (9.4%) 26 (40.6%) 31 (48.4%) 1 (1.6%) 23 (35.9%) 39 (60.9%) 2 (3.1%) 37 (57.8%) 20 (31.3%) 7 (10.9%) 7 (10.9%) 43 (67.2%) 13 (20.3%) 1 (1.6%) 35 (54.7%) 19 (15e35) 48 (75%) 15 (23.4%) 1 (1.6%) 33 (51.6%) 16 (25%) 4 (6.3%) 3 (4.7%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 3 (4.7%) 64
Abbreviations: TKI, tyrosine kinase inhibitor; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; PFS, progression-free survival.
N.V. Mitiushkina et al. / Biochimie 154 (2018) 19e24
21
the Helsinki Declaration and followed the rules of the local ethics committee. All statistical tests were performed using R software (http://www.r-project.org/) version 3.4.2. Survival analysis was done with the R package Survival [27]. 3. Results ALK translocation genotype was successfully determined by variant-specific PCR in 61 out of 64 analyzed patients. Three patients had rare variants of rearrangements, which were recognized by unbalanced expression of 5’/30 -ends of ALK transcript [26] and FISH, but could not be classified by available variant-specific PCR tests. EML4-ALK variant 1 was detected in 33/64 (52%) cases; EML4ALK variant 3 was the second most common, being present in 16/64 (25%) NSCLC samples. Other ALK fusion variants were rare (Table 1). While performing comparisons of treatment outcomes, we considered two approaches. First, we classified EML4-ALK rearrangements into short and long forms, as supported by functional data [12]; we identified 17 patients with short EML4-ALK fusions, 16 of whom carried variant 3 translocation (Table 1). Secondly, we considered variant 1 against all others, as suggested by Yoshida et al. [22]. Data on the tumor response by RECIST are presented in Table 2. Frequencies of objective response were strikingly similar among various patient categories. Only 14 (22%) out of 64 patients were non-responders; lack of substantial tumor shrinkage could not be explained by the presence of particular ALK fusion variant. PFS was also similar across patient subgroups (Fig. 1). This trend did not change when crizotinib or ceritinib were considered separately.
Fig. 1. Kaplan-Meier plots of progression-free survival (PFS) according to ALK fusion type. ALK variants are grouped in two different ways: “short” EML4-ALK variants (v.3a/ b and v.5a/b, containing 6 or less EML4 exons) vs “long” EML4-ALK variants (v.1 and other variants) (a) or EML4-ALK variant 1 vs all other ALK fusion species (b). p-values are calculated using the log-rank test.
Indeed, ceritinib produced significantly better response rates and PFS as compared to crizotinib, however ALK fusion variants did not contribute to this difference (Table 2, Figs. S1eS3). Noticeably, PFS on TKI did not depend on whether the patients received ALK inhibitor while being treatment-naïve or if the TKI was administered after chemotherapy (Fig. 2). Overall survival (OS) was not influenced by the type of ALK translocation (Fig. 3). This lack of differences in OS was unlikely to be attributed to known confounding factors: for example, the analyzed groups of patients were relatively well balanced with regard to the presence of brain metastases or number of ALK TKIs received (Table 2; Figs. S4eS5).
Table 2 Patient characteristics according to ALK fusion variant. EML4-ALK short variantsa Median age at diagnosis, 54 (34e78) years (range) Sex, females 10 (58.8%) Smoking status Ever-smokers 6 (37.5%) Never-smokers 10 (62.5%) Histology, adenocarcinoma 16 (94.1%) Brain metastases Present before TKI 1 (5.9%) treatment Emerged during TKI 5 (29.4%) treatment Absent 11 (64.7%) First TKI Crizotinib 4 (23.5%) Ceritinib 12 (70.6%) Alectinib 1 (5.9%) Treatment line 1 12 (70.6%) 2-3 5 (29.4%) Response to the first TKI treatment CR 2 (11.8%) PR 13 (76.5%) SD 2 (11.8%) PD 0 ORR 15 (88.2%) ORR, crizotinib-treated 3 (75%) patients ORR, ceritinib-treated 11 (91.7%) patients Overall number of sequential TKIs 1 14 (82.4%) 2e3 3 (17.6%) Total N ¼ 17
EML4-ALK long variants
p-value short vs long variants
51 (25e76)
0.257 (Kruskal-Wallis rank 51 (25e76) sum test) 1 (c2-test) 22 (66.7%)
26 (60.5%) 4 (10.8%) 33 (89.2%) 42 (97.7%)
0.05 (Fisher's exact test)
5 (11.9%)
EML4ex13/ALKex20 (variant 1)
Non-variant 1 ALK p-value fusions variant 1 vs other variants 53 (34e78) 17 (54.8%)
0.476 (Kruskal-Wallis rank sum test) 0.443 (c2-test)
8 (28.6%) 20 (71.4%) 28 (90.3%)
0.103 (Fisher's exact test)
0.49 (Fisher's exact test)
3 (10.3%) 26 (89.7%) 32 (97%)
0.49 (Fisher's exact test)
4 (12.1%)
2 (6.7%)
0.281 (Fisher's exact test)
18 (42.9%)
16 (48.5%)
10 (33.3%)
19 (45.2%)
13 (39.4%)
18 (60%)
0.347 (Fisher's exact test)
18 (41.9%) 24 (55.8%) 1 (2.3%)
0.434 (Fisher's exact test)
12 (36.4%) 20 (60.6%) 1 (3%)
11 (35.5%) 19 (61.3%) 1 (3.2%)
1 (Fisher's exact test)
22 (51.2%) 21 (48.8%)
0.281 (Fisher's exact test)
15 (45.5%) 18 (54.5%)
22 (71%) 9 (29%)
0.07 (Fisher's exact test)
4 (9.3%) 29 (67.4%) 9 (20.9%) 1 (2.3%) 33 (76.7%) 11 (61.1%)
0.753 (Fisher's exact test)
3 (9.7%) 22 (71%) 6 (19.4%) 0 25 (80.6%) 7 (63.6%)
1 (Fisher's exact test)
0.479 (Fisher's exact test) 1 (Fisher's exact test)
4 (12.1%) 21 (63.6%) 7 (21.2%) 1 (3%) 25 (75.8%) 7 (58.3%)
0.753 (Fisher's exact test) 1 (Fisher's exact test)
21 (87.5%)
1 (Fisher's exact test)
17 (85%)
17 (89.5%)
1 (Fisher's exact test)
31 (72.1%) 12 (27.9%) N ¼ 43
0.52 (Fisher's exact test)
25 (75.8%) 8 (24.2%) N ¼ 33
23 (74.2%) 8 (25.8%) N ¼ 31
1 (Fisher's exact test)
Abbreviations: TKI, tyrosine kinase inhibitor; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; ORR, overall response rate. a EML4-ALK variants 3a/b and 5a/b, which contain shorter portion of EML4 gene and are more structurally stable than other (“long”) EML4-ALK variants [13].
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Fig. 2. Kaplan-Meier plots of progression-free survival (PFS) for patients receiving ALK TKI upfront (treatment line 1) vs. subjects treated by an ALK inhibitor after chemotherapy (treatment lines 2 or 3). p-values are calculated using the log-rank test.
Fig. 3. Kaplan-Meier plots of overall survival (OS) according to ALK fusion type. (a) Survival curves for “short” EML4-ALK variants vs “long” EML4-ALK variants. (b) Survival curves for EML4-ALK variant 1 vs all other ALK fusions. p-values are calculated using the log-rank test.
4. Discussion The predictive role of distinct ALK variants was previously investigated in several Asian studies. Yoshida et al. [22] evaluated 35 patients treated by crizotinib and revealed that NSCLC with variant 1 EML4-ALK translocation had longer progression-free survival as compared to others. Lei et al. [28] compared 22 patients with EML4-ALK variant 1, 18 subjects with variants 3a/b and 21 NSCLC with other ALK fusions and found no differences in treatment outcome between these three groups. Cha et al. [29] analyzed 52 patients with ALK rearrangements treated by cytotoxic and/or targeted therapy. Among 35 subjects exposed to pemetrexed, PFS was longer in EML4-ALK cases as compared to patients with non-EML4-ALK rearrangements; this difference was attributed mainly to improved PFS in patients with EML4-ALK variant 1. ALK fusion variants did not influence PFS for ALK inhibitors (n ¼ 32) or platinum-based chemotherapy (n ¼ 40). Also, there was no difference in RECIST response rates according to ALK fusion variant for the patients receiving ALK inhibitors (n ¼ 37), pemetrexed (n ¼ 40) or platinum-based therapy (n ¼ 40). Woo et al. [16] considered 51 patients with EML4-ALK rearrangements, who were treated with crizotinib, alectinib or ceritinib (n ¼ 51). The 2-year PFS was higher for NSCLC EML4-ALK variants 1/2/others as compared to patients with 3a/b fusions. Sufficient number of cases were available only for crizotinib (n ¼ 44), and the difference remained statistically significant after the exclusion of alectinib- and ceritinib-treated patients. Noh et al. [17] revealed that tumor samples carrying short variants of EML4-ALK showed higher ALK expression by IHC. In
addition, short EML4-ALK fusions were associated with more advanced stage of the disease and presence of metastatic spread. Median PFS in 46 crizotinib-treated patients tended to be worse for NSCLC with EML4-ALK short variants as compared to subjects with long EML4-ALK fusions. Li et al. [24] investigated 60 NSCLC patients (variant 1: n ¼ 14; variant 2: n ¼ 9; variant 3a/b: n ¼ 20; rare EML4ALK translocations: n ¼ 11; non-EML4-ALK fusions: n ¼ 6); they observed improved PFS in patients with variant 2 rearrangements. A few non-Asian studies have been published very recently. McLeer-Florin et al. [30] considered 6 patients carrying EML4-ALK 1/2/others variants versus 8 NSCLC cases with EML4-ALK 3a/b translocations and found numerically better PFS in the former group. Lin et al. [31] compared treatment outcomes in patients with variant 1 and variant 3 ALK rearrangements. The analysis of tumor responsiveness to crizotinib was undertaken for 99 patients (variant 1: n ¼ 51; variant 3: n ¼ 48) and demonstrated no significant differences; interestingly, a third-generation ALK inhibitor lorlatinib produced significantly better PFS in subjects with variant 3 when given after the failure of crizotinib and at least 1 secondgeneration TKI. Christopolous et al. [23] considered 67 ALKrearranged NSCLCs with variant 1 (n ¼ 26), variant 2 (n ¼ 7) or variant 3 (n ¼ 34) translocation. EML4-ALK variant 3 was associated with higher metastatic burden at diagnosis, inferior PFS both on TKI and on chemotherapy, poor response to cerebral radiotherapy and inferior overall survival. Here we presented a relatively large data set evaluating clinical differences between various ALK variants in non-Asian patients. In contrast to EGFR mutations, distribution of ALK translocations appears to be relatively similar in Orientals and Caucasians, therefore the contribution of racial factor in the observed relationships may not be of importance. Current investigation did not confirm favorable impact of EML4-ALK variant 1 or long EML4-ALK forms on PFS or other treatment outcomes. However, several limitations of this study have to be acknowledged. First of all, it utilized a group of patients treated by two distinct drugs, crizotinib and ceritinib. This is the first study, where most of the patients were treated with the second-generation ALK-inhibitor, ceritinib. Neither crizotinib (n ¼ 23) nor ceritinib (n ¼ 39) treatment outcomes were affected by the type of ALK rearrangement. However, the number of patients in the crizotinib group was small; therefore, if only crizotinib but not ceritinib efficacy is modulated by the type of ALK rearrangement, this study lacked the power to detect the differences. Similarly to other studies [16,17,22e24,28,29,31], this report considered both treatment-naïve and chemotherapy-pretreated patients. However, even if TKI was administered after the failure of chemotherapy, the PFS on TKI treatment was virtually identical to the one in patients receiving TKI upfront (Fig. 2). It is also of notice, that the spectrum of distinct ALK translocations is a subject of interstudy variations: for example, the share of EML4-ALK variant 1 in the present study was somewhat higher than in several previous reports [16,17,23,24], while the frequency of variant 3 appeared to be lower. These deviations are unlikely to be caused by methodological reasons, given that the currently utilized ALK genotyping methods (variant-specific PCR or next generation sequencing) are highly reliable. Clinical and biological factors affecting the distribution of ALK variants deserve further investigation. We believe that this study is sufficient to exclude major contribution of ALK fusion variants in the treatment outcomes for ALK inhibitors. Nevertheless, it is important to consider further analysis utilizing larger and more carefully selected NSCLC subgroups.
N.V. Mitiushkina et al. / Biochimie 154 (2018) 19e24
Conflicts of interest Natalia V. Mitiushkina, Vladislav I. Tiurin, Aglaya G. Iyevleva, Maxim M. Kholmatov, Elena A. Filippova, Fedor V. Moiseyenko, Nikita E. Levchenko, Ivan S. Sardaryan, Svetlana V. Odintsova, Alexandra M. Lozhkina, Nikita M. Volkov, Nina A. Karaseva, Vladimir M. Moiseyenko, Sergey V. Orlov and Evgeny N. Imyanitov declare that they have no conflict of interest.
[3]
Author contribution
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Natalia V. Mitiushkina e conception and design of the study, interpretation of data, drafting the article and the final approval of the manuscript; Vladislav I. Tiurin e acquisition of data on ALK fusion types in selected samples, manuscript preparation and approval; Aglaya G. Iyevleva e analysis of clinical data and interpretation of results, manuscript preparation and approval; Maxim M. Kholmatov e statistical analysis, revision and approval of the manuscript; Elena A. Filippova e acquisition of clinical data, revision and approval of the manuscript; Fedor V. Moiseyenko e acquisition of clinical data, revision and approval of the manuscript; Nikita E. Levchenko e acquisition of clinical data, revision and approval of the manuscript; Ivan S. Sardaryan e acquisition of clinical data, revision and approval of the manuscript; Svetlana V. Odintsova e acquisition of clinical data, revision and approval of the manuscript; Alexandra M. Lozhkina e acquisition of clinical data, revision and approval of the manuscript; Nikita M. Volkov e acquisition of clinical data, revision and approval of the manuscript; Nina A. Karaseva e acquisition of clinical data, revision and approval of the manuscript; Vladimir M. Moiseyenko e acquisition of clinical data, revision and approval of the manuscript; Sergey V. Orlov e acquisition and interpretation of clinical data, manuscript preparation and approval; Evgeny N. Imyanitov - conception and design of the study, drafting the article and the final approval of the manuscript. Declaration of interest
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None. [13]
Acknowledgements This study has been supported by the Russian Scientific Fund (grant 16-15-10396).
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at https://doi.org/10.1016/j.biochi.2018.07.018.
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