Does salmon calcitonin cause cancer? A review and meta-analysis

As noted above, calcitonin elicits its biologic effect by binding to CTr, which is a member of a subfamily of the seven trans-membrane domain G-protein-coupled receptor superfamily that includes glucagon-like peptide 1, glucagon, pituitary adenylate cyclase (AC)-activating polypeptide, corticotropin-releasing factor, gastric inhibitory polypeptide receptors, and PTH/PTH-related peptide [9]. It has long been known that some malignant cells express CTr [10]. Binding to CTr activates AC which results in an increase of intracellular cyclic adenosine monophosphate (cAMP). Increases in cAMP directly activate two classes of effector proteins: protein kinase A (PKA) and the guanine nucleotide exchange factor EPAC. With respect to cancer-relevant phenomena, cAMP/PKA is known to regulate cell proliferation, apoptosis, and motility; in most cells, cAMP serves to inhibit cell growth [11]. The growth-suppressive activities of cAMP/PKA are mediated by a number of signaling pathways, and this effect of cAMP, mediated via sCT, has been shown, e.g., in a line of malignant breast cancer cells [12]. The best understood link between PKA and suppression of proliferation occurs by repressing ERK. In addition, it should be noted that one of the best studied prostate cancer cell lines, DU-145, has been reported to show decreased ERK activity and decreased c-Jun expression in response to calcitonin [13]. Since ERK activity is closely correlated to cell cycle progression, it is doubtful that calcitonin would promote proliferative behavior in this particular prostate cancer cell line.

The Shah group has reported that (a) exogenously added hCT induces DNA synthesis in prostate cancer LNCaP and PC-3M cells [14], (b) hCT, acting through Gs/cAMP and Ca2+ pathways, induces production of a CD44 splice variant that is associated with increased invasiveness [15‐17], (c) calcitonin stimulates angiogenesis [18], and (d) calcitonin can promote in vivo metastases of PC3 prostate cancer cells [19]. These data point to an oncogenic effect for calcitonin, via activation of AC and increases in cAMP, in these prostate cancer cell lines. These results, however, conflict with other published studies of the biology of calcitonin. For example, Macchia et al. reported that elevated cAMP inhibits the growth of LNCaP cells and the Ritchie group demonstrated that hCT decreased proliferation of both PC-3 and DU-145 cells, consistent with the role of elevated cAMP suppressing cell growth [20, 21]. These results are further supported by Segawa, who reported that DU-145 cells treated with calcitonin resulted in decreased ERK activity and decreased c-Jun [22]. Since ERK activity is closely correlated with suppression of cell proliferation, these results support the findings of Ritchie with respect to the results for PC-3 and DU-145 cell lines. Nakamura reported that gastrin-releasing peptide and calcitonin gene-related peptide (CGRP) increased tumor cell invasion of DU-145 cells whereas calcitonin did not [23]. GRP and CGRP also increased the tumor invasiveness of PC-3 cells while calcitonin did not. The effect of CGRP is of interest since the exposure required for calcitonin to achieve a positive effect on LNCaP cells was 10 ng/ml, approximately 1000 times greater than the Cmax achieved with nasal sCT [21]. These highly elevated levels of calcitonin have the potential to cross react with CGRP receptors and might explain the some of the contradictory findings of Shah et al., calling into question his postulate linking calcitonin to prostate cancer.

More recently, Tsagaraki et al. conducted an experiment in which MG63 osteosarcoma cells exposed to calcitonin demonstrated an NF-κB-dependent induction of fibronectin, an extracellular matrix component, and matrix metalloproteinase-9 (MMP-9), a combination that is often associated with oncogenic processes [24]. However, in response to calcitonin, these same cells were also found to express elevated levels of tissue inhibitor of metalloprotease (TIMP)-1 and TIMP-2, which oppose the action of MMP-9 and MMP-2, respectively. Given these complexities, and the fact that these studies were carried out in vitro in a single cell line, their clinical relevance is not clear.

In summary, there are no data which demonstrate that calcitonin can induce carcinogenic transformation. The data with respect to calcitonin’s ability to promote tumor aggressiveness are inconsistent and contradictory.

Agents for use as human pharmaceuticals are required to undergo rigorous in vitro and in vivo testing for general toxicity, genotoxicity, and carcinogenicity prior to licensure. The latter is typically performed in multiple species. In such studies, sCT was not mutagenic in multiple bacterial strains, nor was it clastogenic in chromosome aberration tests of Chinese hamster ovary V79 cells [5]. Neither 1-year chronic toxicity studies in Sprague-Dawley rats nor formal carcinogenicity studies of sCT in Sprague-Dawley rats and CD-1 mice dosed for up to 2 years at doses 39 to 390 times the maximum recommended subcutaneous nor intranasal doses yielded evidence of carcinogenicity. The sole issue arising in nonclinical testing was the finding of decreased latency period in nonmalignant pituitary adenomas in rodents, which occur with a background frequency of 34–94 % [7]. However, there was no evidence of malignant transformation of these tumors, nor is it believed to be relevant to human use. The prescribing information for current calcitonin products states “In female Sprague Dawley rats, the incidence of pituitary adenomas after two years was high in all treatment groups (between 80 % and 92 % including the control groups) such that a treatment-related effect could not be distinguished from natural background incidence” [5].

Consistent with these data, Sondergaard et al. created transgenic mice over-expressing sCT. These mice were not noted to have an increased risk of cancer, although the publication did not state that toxicologic examination was performed [25].

In summary, in vivo animal data did not identify a risk of malignancy following exposure to sCT.

If calcitonin does not itself cause cancer, a possible alternative hypothesis to support a putative relationship between calcitonin and increased cancer risk is to suggest that calcitonin elicits a nonspecific tumor-promoting effect. An experiment of nature is medullary thyroid cancer (MTC), an unusual but not rare tumor of the parafollicular (C) cells of the thyroid. It may occur sporadically or as a component of several multiple endocrine neoplasia (MEN) constellations. Individuals with MTC may elaborate extraordinarily high levels of hCT, and serum hCT is used as both a diagnostic and prognostic factor in the approach to treatment [26]. If the hypothesis that calcitonin is a nonspecific oncogen is correct, it could be expected that individuals with MTC would have a high likelihood of second primary cancers, as many tumor types are associated with bidirectional risk of a second primary malignancy. The US Surveillance, Epidemiology and End Results (SEER) program collects data on all persons diagnosed with cancer in specific geographic regions. Using nine SEER registries, Ronckers et al. examined the likelihood of a second primary malignancy after the occurrence of thyroid cancer(s) [27]. The authors were able to identify 884 cases of MTC which were followed for a median of 8 years. These individuals did not show evidence of an increased risk of secondary malignancies, as opposed to individuals with papillary and follicular thyroid carcinoma, who did show evidence of increased risk. These data do not support an association between hCT and increased risk of malignancy.

In November 2010, the sponsor of an investigational oral calcitonin product being studied in two phase 3 trials (each 2-year duration) of OA informed regulatory authorities of an imbalance in prostate cancer in male study participants [6, 7]. At the time of the notification, one of the trials was ongoing and the other complete. Following an extensive investigation, including retrospective screening of study participants and measurement of serum prostate-specific antigen (PSA) levels and completion of the trials, final data did not ultimately reveal an increased risk of prostate cancer (5.4 % [20/365] of male calcitonin recipients vs. 4.0 % [16/405] of placebo recipients) [7]. In subjects diagnosed with prostate cancer, Gleason scores did not differ significantly for subjects who had received sCT compared to placebo subjects (6.9 vs. 6.4, p = 0.27). Most of these individuals were found to have had high PSA levels at baseline which would have been discovered on routine screening. PSA levels were not accelerated in sCT recipients compared to placebo.

Following the initial signal of a potential increase in prostate cancer incidence, a literature search was conducted, and the sponsor, Novartis Pharmaceuticals, conducted a meta-analysis of 17 historical studies of nasal spray calcitonin [6, 28]. The methods and results from this analysis have been extensively reported in publically available regulatory documents [6, 7, 29]. All but two of these trials were limited to women. They included daily doses ranging from 100 to 400 IU per day (the licensed dose of Miacalcin^® nasal spray is 200 IU/day). The results of this analysis suggested a small increased risk of developing any cancer in calcitonin recipients compared to placebo (odds ratio [OR] 1.61 [95 % confidence interval (CI), 1.11–2.34]). The authors then excluded four trials in which no cases of cancer occurred “according to the Peto method” [28]. In this analysis, the OR was 1.9 (CI 1.27–2.84). The most frequently reported cancer was basal cell carcinoma, but many different types of cancer were reported in calcitonin and placebo recipients. Analysis by dose of calcitonin did not suggest any dose response, and the duration of exposure was similar in calcitonin-treated cases to placebo-treated cases (CI 21.8 vs. 22.4 months). The increased risk observed in this analysis was almost entirely attributable to one large study, known as the PROOF trial [29, 30].

In 2013, the US FDA performed its own meta-analysis, incorporating data from Novartis’ investigational oral calcitonin into the above-described nasal spray analysis (and one additional historical nasal spray study) [7]. These data were subsequently included in the prescribing information for all calcitonin products in the USA [5]. In this analysis, which was expressed in terms of risk difference (RD) rather than OR, the RD for the development of any cancer in sCT-treated patients exceeded that of placebo-treated patients by 1 % (95 % CI, 0.3, 1.6). When basal cell cancer was excluded, the risk was not significant as the RD was 0.5 % (95 % CI, −0.1, 1.2), despite which the prescribing information states that “imbalances in risk were still observed” [5].

An alternative formulation of recombinant sCT for oral administration is in late clinical development for postmenopausal osteoporosis [31]. Based upon publically available data and data from Tarsa Therapeutics’ phase 2 and 3 studies of its investigational oral sCT, one of the authors (GW) performed an additional meta-analysis to further explore a possible association between sCT and cancer. The analysis included data from 22 randomized, controlled clinical trials with calcitonin-salmon (nasal spray or oral investigational oral formulations). Essentially, this analysis incorporated available data from the original 17 trial meta-analyses of Heep [28] (including the studies with zero events), the three large oral phase 3 trials of an investigational oral of calcitonin formulation studies sponsored by Novartis (including the two phase 3 osteoarthritis studies and a third study in postmenopausal women with osteoporosis), and the two trials of a different oral formulation of sCT sponsored by Tarsa Therapeutics, Inc (Fig. 1, study 201 and study 801). These two trials, each approximately 1 year in duration, have been described elsewhere; neither trial showed an increased incidence of cancer in calcitonin recipients, nor was there an excess risk when data from the two trials were pooled [31‐33]. The analysis employed methods that were intended to replicate the data in Table 9 of the FDA’s background document for the joint advisory committee meeting [7]. Therefore, this analysis considered RD estimates for each trial and a meta-analysis using a Mantel-Haenszel-type method to estimate the pooled RD for all strata, assuming a fixed effects model.

First, the data displayed in Table 9 of the FDA’s briefing book were reproduced utilizing RevMan software version 5.2 (http://​tech.​cochrane.​org/​RevMan). This software employs the same fixed effect method and uses a forest plot to display the frequency data and RD estimates with 95 % CIs (provided both in numeric and graphic formats). In addition, the weight of the contribution of each study to the overall, meta-analytic RD result is provided. The standard tests for association as well as the Cochran Q-test and I ² measure for heterogeneity were calculated. The data derived by the US FDA were replicated exactly, except for slight differences in the 95 % CI values for zero-event studies: the overall RD was identical (please note that there is an apparent addition error in the denominator for the placebo group in Table 9 of the FDA’s background document, which should total 4706 and not 4687). Secondly, the adverse events consistent with cancer were incorporated from the two Tarsa-sponsored studies. The resultant meta-analysis and forest plot are displayed in Fig. 1. A total of 11,489 unique individuals, including males, were included in this analysis. The trials ranged in duration from 6 months to 5 years; the two Tarsa-sponsored studies were 48 weeks and 1 year in duration [31, 32].

The forest plot displays the frequency data and RD estimates with 95 % CIs (provided in both numeric and graphic formats) for each of the studies. The Cochrane Q-test (p = 0.44) and the I ² measure indicate that heterogeneity is not an issue. As a result, weights based on the fixed effects model were used to combine the study estimates, yielding the meta-analytic RD of 0.90 % (95 % CI, 0.21 to 1.58 %) and the test of association indicates that this RD is significant (p = 0.01). As noted previously, the data are heavily influenced by a single large 5-year trial (CT320 [PROOF]), resulting in a slight increased risk of any malignancy [30].

Data derived from spontaneous reports of adverse events to regulatory authorities have well-known limitations including absence of proof of causality, the presence of co-morbidities, and other limitations such as knowledge of duration of use and total exposure. Nonetheless, sCT has been available and widely used for many decades, such that even a faint signal might be apparent. In anticipation of the 2013 advisory committee, the US FDA performed a review of spontaneously reported adverse events of malignancy in calcitonin recipients and a data mining exercise to look at a possible association [7]. Usage data included reports from the US Adverse Event Reporting System (AERS) and multiple proprietary drug utilization databases. The agency concluded that its findings “did not identify any potential signal for prostate cancer or other malignancies in the postmarketing data,” nor in the data mining exercise [7].