Serum prolactin level to tumor size ratio as a potential parameter for preoperative differentiation of prolactinomas from hyperprolactinemia-causing non-functional pituitary adenomas.

OBJECTIVE
Preoperative diagnosis of prolactinomas is critical because dopamine agonists have been regarded as a primary treatment. However, serum prolactin level alone is suboptimal for differentiating prolactinomas from hyperprolactinemia-causing non-functioning pituitary adenomas (NFPAs). By using the tumor size, the authors tried to investigate an effective parameter for the discrimination.


METHODS
We performed a retrospective review of patients who underwent trans-sphenoidal surgery for pituitary lesions in a single institute between January 2015 and May 2021. Using the receiver operating curve (ROC) analyses, we compared performances of serum prolactin levels (PRL), a ratio of serum PRL levels to maximal tumor diameter (MD) (PRL/MD; PDR1), and MD squared (PRL/[MD]2; PDR2) in preoperative diagnosis of prolactinomas.


RESULTS
223 patients with NFPAs (n=175) and prolactinomas (n=48) were included in the analysis. A moderate correlation was found between serum prolactin levels and MDs in prolactinomas (pearson's rprl=0.43, p=0.002), whereas a weak correlation was observed in NFPAs (pearson's rnfpa=0.17, p=0.028). Among diagnostic parameters, PDR2 exhibited the optimal diagnostic performance with the cutoff value of 0.83 [㎍/L]/mm2 (area under the curve [AUC] = 0.945), compared to the PDR1 (8.93 [㎍/L]/mm with AUC 0.938) and PRL (99.4 ㎍/L with AUC 0.910). In the external validation study, PDR2 maintained superior performance over PDR1 and PRL (Accuracy of 94.8%, 91.8%, and 75.8%, respectively).


CONCLUSIONS
PDR2 was a more effective indicator than prolactin alone in the preoperative differential diagnosis of prolactinomas and NFPAs, which may help select patients who need medical treatment first.


Introduction
Prolactinoma is the most common type of pituitary adenoma (PA), accounting for 32-66% of all pituitary tumors requiring treatment. Current guidelines suggest the use of dopamine agonists (DAs) as a primary treatment for almost all spectra of prolactinomas, from microadenomas to giant prolactinomas (maximal diameter [MD] > 40 mm) 1 . Surgical indicative of prolactinomas is usually the second option for the adenomas resistant to DAs at least 3 months after initiation or the patients who are intolerable to the complications of medication 2,3 . Thus the clinical diagnosis of prolactinomas before surgery is essential for treatment strategy.
However, the preoperative diagnosis of prolactinomas has been a matter of debate because of other pituitary pathologies accompanied by hyperprolactinemia. Hyperprolactinemia is defined as serum prolactin (PRL) levels above the upper reference limit (commonly >20 μg/L in men and >25 μg/L in women), with different physiological, pharmacological, and pathological causes [4][5][6] . Though prolactinoma is the most common cause of the prolactin hypersecretion in PAs, the "stalk section effect" of non-functional pituitary adenomas (NFPAs), the mechanical compression of the stalk blocks dopamine inhibition of lactotroph, makes it challenging to discriminate prolactinomas 4,[7][8][9] .
Previous endocrinology guidelines suggested several ranges of hyperprolactinemia to distinguish between these two pathologies. Regarding the lactotroph tumor cells secreting PRL, the level of PRL secretion in prolactinomas was associated with tumor size [10][11][12] . Serum PRL levels >500 μg/L are generally always indicative of prolactinomas 3,4 . Macroadenomas (MD larger than 10 mm) often exhibit serum PRL higher than 250 μg/L and even reach 20,000 μg/L or more, while microprolactinomas (MD less than 10 mm) commonly result in hyperprolactinemia with the range between 100-200 μg/L 4,10 . Hyperprolactinemia of less than 100 μg/L is often related to the diagnostic uncertainty. Up to 25% of microprolactinomas present with hyperprolactinemia < 100 μg/L 4,13 . Other studies have reported elevated PRL levels < 100 μg/L with a solid pituitary macroadenoma are highly suspicious of an NFPA 4,8 . These variations make serum PRL alone to be insufficient for discriminate prolactinomas from NFPA.
The authors conducted a retrospective study to investigate novel parameters to improve the preoperative differentiation of these two pathologies. We firstly examined the different relationships of serum PRL and MD in prolactinomas and NFPAs. Using this property, the diagnostic performance of potential diagnostic

Patient demographic data
A total of 223 patients were included in the final analyses, with 175 patients in the NFPA group and 48 patients in the prolactinoma group. The descriptive characteristics of the two groups are compared in Table   1. There was no significant difference in the proportion of sex between the NFPA and prolactinoma groups (25.7% versus 31.2%, respectively, p-value > 0.05). The median age of the NFPA group was higher than that of the prolactinoma group (43.0 years versus 30.0 years, respectively, p-value < 0.01).

Surgical indications of prolactinoma group
Nineteen of the 48 (39.6%) patients in the prolactinoma group underwent surgical resection of adenoma due to complications related to medical DA treatment. Among them, twelve (25.0%) patients experienced medication resistance, including persistent hyperprolactinemia (three cases, 6.2%) or its symptoms (four cases, 8.3%), or no reduction of adenoma size (five cases, 10.4%). The other seven cases (14.5%) showed medical intolerance before surgery, despite a change in the dopamine agonist regimen.
Five (10.4%) patients with prolactinomas were primarily treated with surgery due to preparation of pregnancy. This decision was based on the previous studies that DAs significantly increase the risk of pregnancy loss and of preterm birth [14][15][16] . Pituitary apoplexy with clinical symptoms were diagnosed in 4 patients (8.3%), and these patients were treated with steroid medication and urgent decompression surgery. 20 (41.6%) patients with prolactinomas were preoperatively diagnosed as NFPA by the physician's empirical determination from their size and serum prolactin levels.

Prolactinoma groups
Serum PRL and the maximal tumor diameter (MD) also showed significant differences between the two groups ( Table 1) We further compared the serum PRL and MD parameters according to tumor size, particularly in the prolactinoma group (Table 2). Macroprolactinomas showed larger scales of both serum PRL levels and tumor MD than those of microprolactinomas, whereas the median value of both PDR1and PDR2 parameters was no significant difference between the two groups (p-value=0.11).

Correlation between serum PRL and MD in prolactinoma or NFPA groups
We conducted a correlation test between serum PRL levels and MD in each histological group. Pearson's correlation coefficients were used to estimate the linear relationship between the two variables. In the prolactinoma group, moderate strength of linear correlation between serum PRL and MD was confirmed in the positive direction (Pearson's r=0.43, p=0.002) < 0.05). In comparison, a low correlation was confirmed (Pearson's r=0.17, p=0.028) in the NFPA group ( Figure. 1).

Diagnostic performance of PRL, PDR1, and PDR2 in the study group
The receiver operating characteristic (ROC) curve and the diagnostic power of PRL, PDR1, and PDR1 were calculated ( Figure 2). The optimal cutoff for indicating prolactinomas was determined using the Youden index, which maximizes the sum of sensitivity and specificity. The statistical difference between three AUCs were examined by the DeLong's test. The AUCs of PDR1 and PDR2 were significantly higher than PRL (PRL versus PDR1; p=0.012 and PRL versus PDR2; p=0.037). In contrast, there was no significant difference between the AUCs of PDR1 and PDR2 (p=0.339).
These results suggest that the ratio of serum prolactin to adenoma size showed better diagnostic powers than serum prolactin level alone in the study group.

Optimal parameter for preoperative diagnosis of prolactinomas: validation with the test group
We conducted external validation with an independent cohort of 50 patients to select the optimal parameters among three cutoffs from ROC analyses. The test group consisted of 13 patients with prolactinoma and 37 patients with NFPA.

Discussion
This study investigated the predictive value of PRL and PDR for preoperative differentiation of prolactinomas and NFPAs. Distinguishing these two pathologies is critical given the satisfactory response to DAs in prolactinomas and the need for surgical resection in large NFPAs. The European Endocrine Society suggested serum PRL levels >250 μg/L in macroadenomas (diameter > 1 cm) as a clinical diagnostic threshold for prolactinomas 3  Previous studies reported that hormonal symptoms were much more prevalent than mass effects in prolactinomas and vice versa in NFPAs 10,19 . We found similar findings that hyperprolactinemic symptoms such as amenorrhea, galactorrhea was more common in prolactinomas (62.6% in prolactinomas vs 25.1% in NFPAs, p < 0.001) and tumor mass effects with headache or visual disturbance were more prevalent in In this study, we examined and validated the diagnostic performance of the novel parameters, the ratio of PRL to MD (PDR1) and MD squared (PDR2), to that of PRL alone. The optimal cutoff values were The limitation of this study includes the bias from its retrospective nature and sample sizes susceptible to outlier effects. One of the major limitations is selection bias; data were obtained only from surgical cases, causing relatively low "prevalence" of prolactinomas compared to NFPAs in our clinical setting. In the future, multi-center and prospective clinical studies are required to improve the accuracy and further elucidate the role of PDR in the differential diagnosis of prolactinomas from hyperprolactinemia-causing other pituitary pathologies.
In conclusion, our study demonstrated the effectiveness of serum PRL to tumor size ratio as a potential parameter for preoperative differentiation of prolactinomas and NFPAs. Based on the positive correlation between serum PRL and tumor MD in prolactinomas, contrary to the weak relationship observed in NFPAs, we examined and validated the diagnostic value of the PDR parameters compared to PRL alone. The optimal thresholds of the PRL to MD squared ratio may contribute to preoperative diagnosis of prolactinomas from other conditions of PAs, hence improving a treatment strategy whether administration of DA agonist or surgical resection should be recommended.

Patient enrollment
We performed a retrospective review of patients who underwent trans-sphenoidal surgery (TSS) for pituitary lesions between January 2015 and May 2021. This study was approved by the Samsung Medical Center institutional review board (IRB number: 2021-06-028). The informed consent was waived by Samsung Medical Center institutional review board, due to retrospective analyses. All medical performance in this study were conducted according to current diagnostic or treatment guidelines.
Among 997 consecutive patients with PAs who underwent TSS in this period, hyperprolactinemia, defined as a serum PRL level > 25 μg/L, was confirmed at the time of initial diagnosis in 242 patients. We

Declarations
Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflicts of interest/Competing interests:
The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.    Comparison of the ROC curves and diagnostic performance of serum prolactin level (PRL) and a ratio of serum prolactin to adenoma maximal diameter (PRL/MD, PDR1) and to diameter squared (PRL/[MD]2, PDR2) for differentiating prolactinomas from non-functioning pituitary adenomas.

Figure 3
Validation of PRL, PDR1, and PDR2 classi cation models using confusion matrix. A classi cation of prolactinomas (prl) and non-functional pituitary adenoma (nfpa) by clinicopatholgical diagnosis is the reference. Predictors are de ned as classi cation by PRL, PDR1, and PDR2. The p-values of McNemar's test were described beside each matrix.