The depot somatostatin analogs (SSA) octreotide LAR and lanreotide autogel are currently the mainstay of the pharmacological treatment of acromegaly.1,2 Early studies reporting efficacy rates as high as 60% suffered from a significant selection bias.3,4 More recent multicentric trials without such a selection bias have established more realistic efficacy rates that range from 25% to 30%; thus, a considerable number of patients do not respond to SSA and remain with clinically and biochemically active disease.5,6 Predicting the pharmacological response to SSA has been an elusive goal. The value of the subcutaneous octreotide test in identifying those patients with acromegaly who will eventually respond to long-term SSA therapy has been a matter of controversy; some studies claim that this test is highly predictive of the pharmacological response,7–9 while others conclude that it should only be used to establish tolerance.10–12 Similarly, 111In-Octreotide scanning cannot discern responsive from resistant patients, due to the background uptake by the normal pituitary gland.13 More recently, hypointense lesions on T2-weighted magnetic resonance images (MRI) have been associated with a favorable response to SSA; yet, the positive predictive value of such a finding is only 62% and thus cannot be used by itself to decide whether or not to treat a patient with SSA.14

These SSA bind preferentially to the subtype 2 of somatostatin receptors (SSTR2) and to a lesser extent to subtype 5 (SSTR5).15,16 In fact, they were designed to bind these receptor subtypes precisely because they are the most abundantly expressed in the vast majority of GH-secreting adenomas.15,16 SSTR2 expression by the tumor at both the mRNA (by RT/PCR)17,18 and protein (by IHC)19–23 levels has been positively associated with the response to SSA. However, a significant number of patients with tumors expressing high levels of SSTR2 are resistant to these pharmacological agents.24 Compared to SSTR2, SSTR5 is expressed by a greater proportion of these tumors; yet its significance in terms of predicting response to SSA is less well understood.18–24 Although in vitro SSTR5 appears to be fundamental for SSA-induced GH inhibition,25 a low SSTR2/SSTR5 mRNA ratio was found to be associated with a poor pharmacological response to octreotide LAR.18 At the protein level, while SSTR2 immunostaining is found in SSA-responsive patients, SSTR5 is found in SSA-resistant patients as well.20 Despite these observations suggesting a negative effect of SSTR5 expression in determining pharmacological response to SSA, subsequent IHC studies have focused mainly on SSTR2.21,22

In the present study we evaluated the expression of SSTR 2 and 5 by both RT/PCR and IHC in a large group of patients with acromegaly, with the aim of finding patterns associated with the pharmacological response to SSA.

Table 1 depicts the biochemical and imaging characteristics of the patients. Although GH and IGF-1 levels were similar in patients who were treated preoperative with octreotide and in those who did not, subjects who received the SSA before surgery had smaller and less invasive tumors (Table 1). Of the 36 patients who received octreotide LAR treatment, 10 (27%) achieved full biochemical control targets (GH <2.5ng/mL and IGF-1<1.2×ULN), 7 (19%) responded partially (>50% decrease in GH and >30% decrease in IGF-1, compared to baseline levels) and 19 (52.7%) were non-responders. GH at baseline was significantly lower among octreotide responsive patients (complete and partial responders, analyzed together) compared to those who did not respond to the SSA (p=0.008). Although IGF-1 levels also tended to be lower, the difference did not reach statistical significance (p=0.08). As a group, patients responding to octreotide had smaller adenomas than those in whom the SSA was not effective (median tumor volume [IQR]: responders 1172mm3 [79–21760], non-responders 6070mm3 [2590–9210], p=0.004).

Immunohistochemistry

A total of 37 GH-secreting tumors were included in a TMA for immunohistochemical analysis; 17 (46%) were octreotide naïve, whereas 20 (54%) had been treated preoperatively with octreotide LAR (5 complete responders, 6 partial responders and 9 non-responders). Low to moderate intensity SSTR2 and SSTR5 immunostaining was found in all tumors studied taking into account the cytoplasmic algorithm. The mean SSTR5 immunostaining score was significantly higher than that of SSTR2 when the whole group was analyzed together (SSTR5 70.1±12.3 vs. SSTR2 54.6±14.9, p<0.001), and this remained so when we analyzed octreotide naïve (SSTR5 69.1±15.8 vs. SSTR2 56.3±15.2, p=0.02), octreotide responsive (SSTR5 70.18±10.1 vs. SSTR2 56.6±16.2, p=0.03) and octreotide non-responsive (SSTR5 69.1±8.6 vs. SSTR2 48.8±12.9, p=0.001) patients separately (Fig. 1, supplementary Table 1).

Figure 1.

SSTR2 and SSTR5 immunohistochemical score. The total group includes octreotide treated and naïve patients. The octreotide-responsive group consists of 5 complete responders and 6 partial responders. OCT NAÏVE: octreotide naïve group; OCT RESP: octreotide-responsive group; OCT NON-RESP: octreotide non-responsive group.

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Among the 17 patients who did not receive octreotide preoperatively, SSTR5 was the predominantly expressed receptor in 8 (47%); in the remaining 9 patients, no clear predominance of any of the two receptors could be found. Of the 11 subjects who responded to octreotide, 8 (72.7%) expressed SSTR5 as the predominant receptor subtype, whereas SSTR2 immunostaining was more intense in two (27.3%). SSTR5 was clearly predominant in 8 out of the 9 (88.8%) patients who did not respond to octreotide; in the remaining non-responsive patient immunostaining scores for SSTR2 and SSTR5 were similar. Thus, although SSTR5 predominance tended to be more apparent in non-responders than in responders, the difference did not reach statistical significance. Neither SSTR2 nor SSTR5 protein expression correlated with baseline and post-octreotide GH, baseline and post-octreotide IGF-1, or tumor volume (Table 2). The SSTR5/SSTR2 ratio tended to be higher among octreotide non-responders, but the difference did not reach statistical significance (responders 1.3±0.32, non-responders 1.47±0.31, p=0.2).

Table 2.

Correlations between SSTR2 and SSTR5 protein (IHC) and mRNA (PCR) expression with baseline and post-octreotide GH and IGF-1.

	Baseline GH	Post Oct GH	% GH reduction	Baseline IGF-1	Post Oct IGF-1	% IGF-1 reduction
SSTR2 (IHC)	r=−0.42p=0.06	r=−0.48p=0.07	r=0.10p=0.65	r=−0.18p=0.4	r=−0.24p=0.30	r=0.14p=0.53
SSTR5 (IHC)	r=−0.271p=0.24	r=−0.29p=0.20	r=−0.21p=0.36	r=−0.38p=0.09	r=−0.24p=0.30	r=0.25p=0.28

In the present study we evaluated a large number of patients with acromegaly, of whom two-thirds had received preoperative treatment with octreotide-LAR for 3–6 months while one-third had not. We found SSTR5 to be the more frequently and abundantly expressed somatostatin receptor subtype in all studied tumors, both by IHC and by RT/PCR, but we found no significant correlations between any of the two receptor subtypes and the response to octreotide LAR treatment. It has been suggested that preoperative SSA treatment may modify receptor expression.21–24 More specifically, SSTR2 protein expression but not mRNA is reduced in tumors from patients who received octreotide prior to surgery, perhaps reflecting the presence of ligand-induced internalization of the receptor but not transcription down-regulation.23 In our study, both octreotide naïve and octreotide-treated patients had the same expression pattern of SSTR2 and SSTR5 by IHC. However, the clear predominance of SSTR5 over SSTR2 mRNA expression was found only in the octreotide-treated subjects. Taken together, our results agree with the previously mentioned studies.

Although SSTR5 predominance has been consistently reported in earlier studies evaluating somatostatin receptor subtype expression in GH-secreting adenomas,18,20,24 recent investigations seem to have neglected the study of this receptor, focusing mostly on SSTR2.21–22 A positive correlation between SSTR2 expression and the response to SSA has been found at both mRNA and protein levels.17–18,21–23 A high sensitivity and negative predictive value between SSTR2 protein expression and biochemical response to SSA have been recently reported.21–22 Yet, SSTR2 expression specificity and positive predictive value are considerably low.15–16 The latter arises from a large number of SSA-resistant patients whose tumors express large amounts of SSTR2. Thus, although SSTR2 expression appears to be necessary in order to achieve a pharmacological response to SSA, in a large number of patients it is not sufficient.

The discrepancy between our results and the aforementioned studies may result from a variety of factors. Studies evaluating the relationship between SSTR expression and SSA response have included diverse experimental designs, have used different criteria for pharmacological response and have variably included patients with and without preoperative treatment with SSA.17–23 IHC studies have used different antibodies against the SSTRs and immunoreactivity has been graded by different methods.19–23 Although we did not use the recently introduced monoclonal antibodies against SSTR2, the IHC technique was rigorously controlled and validated. Furthermore, the quantification of the individual immunostaining scores in the TMA was established by an automated technique which is less susceptible to the inherent observer bias than the visual interpretation performed by one or more pathologists. The antibodies we used for SSTR2 and SSTR5 IHC are targeted against the carboxyl terminus of the molecule, which is located in the cytoplasm. This is in contrast to other studies that use antibodies that predominantly detect membrane-bound receptor. Since these antibodies may be tracking receptors downstream from signaling, perhaps in the process of being recycled, they may not reflect the actual pool of receptors to bind to SSA.

In regards to the criteria for pharmacological response, one study has reported SSTR2 mRNA expression to correlate with IGF-1 decrement upon SSA treatment, but not with the achievement of GH control target; no data are provided concerning receptor expression and achievement of combined GH-IGF-1 therapeutic goals.18 Among the three most recently published studies evaluating SSTR2 protein expression with a highly specific monoclonal antibody,21–23 only one evaluates SSA response defined according to combined GH and IGF-1 targets.22 This study by Wildenberg et al. reports that among the 54 patients with high to moderate immunostaining scores for SSTR2, only 21 [38%] met the Cortina Consensus criteria for biochemical control.22 In our work, considering biochemical response as the achievement of both IGF-1 and GH targets, we could not find a difference in the SSTR2 or SSTR5 mRNA level between octreotide responsive and octreotide resistant patients. SSTR2 was the predominantly expressed receptor subtype by IHC in almost one-third of the octreotide responsive patients, while in none of the octreotide resistant patients. SSTR5 mRNA and protein expression tended to predominate more in non-responders than in responders; however, this did not reach statistical significance.

A consistent finding among studies evaluating SSTRs in pituitary adenomas by RT/PCR, including our own, has been the wide variability of mRNA expression, even after correction with a normalization factor derived from simultaneously amplifying different house keeping genes.17–18,22 In this regard, it must be emphasized that RT/PCR yields only partially quantitative results of the mRNA copy number. Thus, the absence of statistically significant correlations between IHC and RT/PCR data is not surprising.21,27 Among the 13 patients in our study in whom both IHC and RT/PCR data were available, concordance rate between the two methods was 77%. To our knowledge, only one study has evaluated SSTR2 expression at both mRNA and protein levels in patients with GH-secreting adenomas21; it reports a high concordance between the two methods but does not provide a concordance rate. Furthermore, only 3 of the 7 patients in this study who normalized IGF-1 after octreotide treatment had their SSTR2 mRNA expression analyzed.21

The response to SSA by GH-secreting adenomas depends on a complex array of mechanisms, and not only on the density of receptor expression at the cell membrane.24,28 A low Ki-67 proliferative index has been associated with a favorable response to SSA29–30 and, not unexpectedly, appears to be related to a densely granulated tumor phenotype as determined by both electron microscopy31 and CAM5.2 cytokeratin immunostaining.30,32 Somatostatin receptors are known to homo- and heterodimerize, and this phenomenon affects receptor signaling, internalization and recycling.28,33–34 Elegant in vitro studies using confocal microscopy have shown that although both SSTR2 and SSTR5 bind octreotide with high affinity, the internalization of the octreotide-SSTR2 complex is more rapid and efficient than that of the octreotide-SSTR5 complex.35 The latter is paralleled by a greater biological effect in terms of cAMP inhibition and ERK activation.35 The existence of two splice variants of the SSTR5 gene, encoding truncated but functional proteins with 4 and 5 transmembrane domains (SSTR5TMD4 and SSTR5TMD5, respectively), has been demonstrated.36 Both variants appear to be located at intracellular compartments, and although they are notoriously absent in normal pituitary tissue, SSTR5TMD4 is abundantly expressed in pituitary tumors, particularly somatotrophinomas and non-functioning pituitary adenomas.36 In somatotrophinomas SSTR5TMD4 expression positively correlates with SSTR5 but not with SSTR2 expression. The finding of a statistically significant negative correlation between the expression of SSTR5TMD4 and the GH response to octreotide in vivo and with the in vitro GH inhibitory response to a SSTR5-selective agonist suggests that the concomitant presence of SSTR5 and its truncated splice variant could interfere with the pharmacological response to SSA.37 The possibility that the SSTR5 antibody used in our study could be detecting SSTR5TMD4 is practically excluded since it is directed against carboxy terminus epitopes located distally from the truncation site, and therefore identifies only the full length SSTR5. Based on this in vitro evidence and the available in vivo data, we could speculate that SSTR5 acting in concert with its truncated splice variant may exert a regulatory role modulating the effect of somatostatin and SSA, sometimes inhibiting and sometimes promoting signaling and biological effect.38 Although D2R expression has not been positively associated with the pharmacological response to SSA, D2R/SSTR2 heterodimers have been shown in vitro to potentiate the GH inhibitory effect of SSA, perhaps explaining the benefit of adding cabergoline to SSA-resistant patients in reducing GH secretion.28,38

In conclusion, our results underscore the complexity of the interaction between SSA and its receptors. The mere expression of these receptors does not guarantee an adequate pharmacological response; rather, other not fully characterized functional aspects of this interaction, such as receptor homo- and heterodimerization, and the resulting signaling cascade, probably play a role in determining whether a patient with acromegaly will respond or not to these agents.

The authors have nothing to declare.

Fondo de Investigación en Salud, Instituto Mexicano del Seguro Social número 3601.