ABSTRACT

The pineal gland was described as the “Seat of the Soul” by Renee Descartes and it is located in the center of the brain. The main function of the pineal gland is to receive information about the state of the light-dark cycle from the environment and convey this information by the production and secretion of the hormone melatonin. Changing photoperiod is indicated by the duration of melatonin secretion and is used by photoperiodic species to time their seasonal physiology. The rhythmic production of melatonin, normally secreted only during the dark period of the day, is extensively used as a marker of the phase of the internal circadian clock. Melatonin itself is used as a therapy for certain sleep disorders related to circadian rhythm abnormalities such as delayed sleep phase syndrome, non-24h sleep wake disorder and jet lag. It might have more extensive therapeutic applications in the future, since multiple physiological roles have been attributed to melatonin. It exerts physiologic immediate effects during night or darkness and when suitably administered has prospective effects during daytime when melatonin levels are undetectable. In addition to its role in regulating seasonal physiology and influencing the circadian system and sleep patterns, melatonin is involved in cell protection, neuroprotection, and the reproductive system, among other possible functions. Pineal gland function and melatonin secretion can be impaired due to accidental and developmental conditions, such as pineal tumors, craniopharyngiomas, injuries affecting the sympathetic innervation of the pineal gland, and rare congenital disorders that alter melatonin secretion. This chapter summarizes the physiology and pathophysiology of the pineal gland and melatonin.

PINEAL PHYSIOLOGY

Pineal Anatomy and Structure

The pineal gland in humans is a small (100-150 mg), highly vascularized, and a secretory neuroendocrine organ (1). It is located in the mid-line of the brain, outside the blood-brain barrier and attached to the roof of the third ventricle by a short stalk. In humans, the pineal gland usually shows a degree of calcification with age providing a good imaging marker (for a speculative discussion of pineal calcification see reference(2)). The principal innervation is sympathetic, arising from the superior cervical ganglia (3). Arterial vascularization of the pineal gland is supplied by both the anterior and posterior circulation, being the main artery supplying the lateral pineal artery, which originates from the posterior circulation (4). In mammals, the main cell types are pinealocytes (95%) followed by scattered glial cells (astrocytic and phagocytic subtypes) (5). Pinealocytes are responsible for the synthesis and secretion of melatonin.

Main Function of the Pineal Gland

The main function of the pineal gland is to receive and convey information about the current light-dark cycle from the environment via the production and secretion of melatonin cyclically at night (dark period) (6, 7). Although in cold-blooded vertebrates (lower-vertebrate species), the pineal gland is photosensitive, this property is lost in higher vertebrates. In higher vertebrates, light is sensed by the inner retina (retinal ganglion cells) that send neural signals to the visual areas of the brain. However, a few retinal ganglion cells contain melanopsin and have an intrinsic photoreceptor capability that sends neural signals to non-image forming areas of the brain, including the pineal gland, through complex neuronal connections. The photic information from the retina is sent to the suprachiasmatic nucleus (SCN), the major rhythm-generating system or “clock” in mammals, and from there to other hypothalamic areas. When the light signal is positive, the SCN secretes gamma-amino butyric acid, responsible for the inhibition of the neurons that synapse in the paraventricular nucleus (PVN) of the hypothalamus, consequently the signal to the pineal gland is interrupted and melatonin is not synthesized. On the contrary, when there is no light (darkness), the SCN secretes glutamate, responsible for the PVN transmission of the signal along the pathway to the pineal gland. However, it is important to note that in continuous darkness the SCN continues to generate rhythmic output without light suppression since it functions as an endogenous oscillator (master pacemaker or clock). The rhythm deviates from 24h and ‘free-runs’ in the absence of the important light time cue. Light-dark cycles serve to synchronize the rhythm to 24h.

The PVN nucleus communicates with higher thoracic segments of the spinal column, conveying information to the superior cervical ganglion that transmits the final signal to the pineal gland through sympathetic postsynaptic fibers by releasing norepinephrine (NE).  NE is the trigger for the pinealocytes to produce melatonin by activating the transcription of the mRNA encoding the enzyme arylalkylamine N-acetyltransferase (AA-NAT), the first molecular step for melatonin synthesis (8) (Figure 1).

Figure 1. Melatonin synthesis in the pineal gland

MELATONIN SYNTHESIS AND METABOLISM

Melatonin Synthesis

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized within the pinealocytes from tryptophan, mostly occurring during the dark phase of the day, when there is a major increase in the activity of serotonin-N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT), responsible for the transformation of 5-hydroxytryptamine (5HT, serotonin) to N-acetylserotonin (NAS) (Figure 1). Finally, N-acetylserotonin is converted to melatonin by acetylserotonin O-methyltransferase. The rapid decline in the synthesis with light treatment at night appears to depend on proteasomal proteolysis (9). Both AA-NAT and serotonin availability play a role limiting melatonin production. AA-NAT mRNA is expressed mainly in the pineal gland, retina, and to a lesser extent in some other brain areas, pituitary, and testis. Melatonin synthesis is also described in many other sites. AA-NAT activation is triggered by the activation of β1 and α1b adrenergic receptors by NE (9). NE is the major transmitter via β-1 adrenoceptors with potentiation by α-1 stimulation. NE levels are higher at night, approximately 180 degrees out of phase with the serotonin rhythm. Both availability of NE and serotonin are stimulatory for melatonin synthesis. Pathological, surgical or traumatic sympathetic denervation of the pineal gland or administration of β-adrenergic antagonists abolishes the rhythmic synthesis of melatonin and the light-dark control of its production.

There is evidence that melatonin can be synthesized in other sites of the body (skin, gastrointestinal tract, retina, bone marrow, placenta and others) acting in an autocrine or paracrine manner (10). Very recently it has been demonstrated that in the mouse brain melatonin is exclusively synthesized in the mitochondrial matrix. It is released to the cytoplasm, thereby activating a mitochondrial MT1 signal-transduction pathway which inhibits stress-mediated cytochrome c release and caspase activation: these are preludes to cell death and inflammation. This is a new mechanism whereby locally synthesized melatonin may protect against neurodegeneration. It is referred to as automitocrine signaling (11). Except for the pineal gland, other structures contribute little to circulating concentrations in mammals, since after pinealectomy, melatonin levels are mostly undetectable (12). Importantly several other factors, summarized in Table 1, have been related to the secretion and production of melatonin (13, 14).

Abbreviations: A: antagonist, U: uptake, I: inhibitor, MAO: monoamine oxidase, OC: oral contraceptives, 5HT: 5-hydroxytryptamine.

Control of Melatonin Synthesis: A Darkness Hormone

The rhythm of melatonin production is internally generated and controlled by interacting networks of clock genes in the bilateral SCN (15). Damage to the SCN leads to a loss of the majority of circadian rhythms. The SCN rhythm is synchronized to 24 hours mainly by the light-dark cycle acting via the retina and the retinohypothalamic projection to the SCN; the longer the night the longer the duration of secretion is, and the ocular light serves to synchronize the rhythm to 24h and to suppress secretion at the end of the dark phase, as explained above. Light exposure is the most important factor related to pineal gland function and melatonin secretion. A single daily light pulse of suitable intensity and duration in otherwise constant darkness is enough to phase shift and to synchronize the melatonin rhythm to 24h (16). The amount of light required at night to suppress melatonin secretion varies across species. In humans, intensities of 2500 lux full spectrum light (domestic light is around 100 to 500 lux) or light preferably in the blue range (460 to 480 nm) are required to completely suppress melatonin at night, but lower intensities < 200 lux can suppress secretion and shift the rhythm (17-21). Previous photic history influences the response: living in dim light increases sensitivity. Furthermore, the degree of light perception between individuals is related to the incidence of circadian desynchrony; along these lines, blind people with no conscious or unconscious light perception show free-running or abnormally synchronized melatonin and other circadian rhythms (22-24). Some blind subjects retain an intact retinohypothalamic tract and therefore a normal melatonin response despite a lack of conscious light perception (25, 26). It seems clear that an intact innervated pineal gland is necessary for the response to photoperiod change (27). Melatonin functions as a paracrine signal within the retina, it enhances retinal function in low intensity light by inducing photomechanical changes and provides a closed-loop to the pineal-retina-SCN system. All together, they are the basic structures to perceive and transduce non-visual effects of light, and to generate the melatonin rhythm by a closed-loop negative feedback of genes (Clock, “Circadian locomotor output cycles kaput” and Bmal, “Brain and muscle ARNT-like” genes), positive stimulatory elements (Per, “period” and Cry, “Cryptochrome” genes), and negative elements (CCG, clock-controlled genes) of clock gene expression in the SCN (Figure 2).

Figure 2. Diagrammatic representation of the control of production and the functions of melatonin, regarding seasonal and circadian timing mechanisms. Abbreviations: SCN: suprachiasmatic nucleus, PVN: paraventricular nucleus, SCG: superior cervical ganglion, NA: norepinephrine (noradrenalin), RHT: retino-hypothalamic-tract, CCG: clock-controlled genes. Based on an original diagram by Dr Elisabeth Maywood, MRC Laboratory of Molecular Biology, Neurobiology Division, Hills Road Cambridge, CB2 2QH, UK.

Melatonin Metabolism

Once synthesized, melatonin is released directly into the peripheral circulation (bound to albumin) and to the CSF without being stored. In humans, melatonin’s half-life in blood is around 40 minutes and it is metabolized within the liver, converted to 6-hydroxymelatonin mainly by CYP1A2 and conjugated to 6-sulfatoxymelatonin (aMT6s) for subsequent urinary excretion. The measurement of urinary aMT6s is a good marker of melatonin secretion, since it follows the same pattern with an approximate 2-hours offset (28, 29). Overall, women have slightly higher values of plasma melatonin at night than men (30). On average, the maximum levels of plasma melatonin in adults occur between 02.00 and 04.00 hours and are on average about 60 to 70 pg/mL when measured with high-specificity assays. Concentrations in saliva, like other hormones, are three times lower than in plasma. Minimum concentrations detected are below 1 pg/mL. The plasma melatonin rhythm (timing and amplitude) strongly correlates with urinary aMT6s. Although there is a large variability in amplitude of the rhythm between subjects, the normal human melatonin rhythm is reproducible from day to day intraindividually (Figure 3 and 4).

Figure 3. Average concentrations of melatonin in human plasma (black, N=133), saliva (blue, N=28) and 6-sulphatoxymelatonin (aMT6s) in urine (red, N=88) using radioimmunoassay measurements. Diagrammatic representation of mean normal values (healthy men and women over 18 years old) from Dr. J. Arendt. Stockgrand Ltd., University of Surrey, UK.

Figure 4. Plasma melatonin and urinary aMT6s in hourly samples to show the delay in the rhythm of urinary aMT6s compared to plasma melatonin (mean/SEM, N=14). Red lines show typical urine sample collection times over 24-48h to determine timing of the rhythm in out-patient or field studies. Redrawn from R. Naidoo, Thesis, University of Surrey, UK, 1998.

Melatonin Production During Development and Across Life

At birth, melatonin levels are almost undetectable, the only fetal source of melatonin being via the placental circulation. Melatonin levels in fetal umbilical circulation reflect the day-night difference as seen in the maternal circulation. Maternal melatonin sends a temporal circadian signal to the fetus (called “maternal photoperiodic adaptative programming”), preparing the CNS to properly deal with environmental day/night fluctuations after birth. A melatonin rhythm appears around 2 to 3 months of life (31), levels increasing exponentially until a lifetime peak on average in prepubertal children; melatonin concentrations in children are associated with Tanner stages of puberty (32). Thereafter, a steady decrease occurs reaching mean adult concentrations in late teens (33, 34). Values are stable until 35 to 40 years, followed by a decline in amplitude of melatonin rhythm and lower levels with ageing, associated with fragmented sleep-wake patterns (35). In people >90 years, melatonin levels are less than 20% of young adult concentrations (36). The decline in age-related melatonin production is attributed to different reasons; calcification of the pineal gland starting early in life and an impairment in the noradrenergic innervation to the gland or light detection capacity (ocular mydriases, cataracts) (2, 37). Interestingly, pinealectomy accelerates the aging process and several reports suggest that melatonin has anti-aging properties (38).

MELATONIN’S MECHANISMS OF ACTION

Melatonin Target Sites and Receptors

Melatonin’s target sites are both central and peripheral. Binding sites have been found in many areas of the brain, including the pars tuberalis and hypothalamus, but also in the cells of the immune system, gonads, kidney, and the cardiovascular system (39, 40). Melatonin binding sites in the brain might vary according to species. Melatonin exerts both non-receptor and receptor-mediated actions. Non-receptor-mediated actions are due to amphipathic properties (of both lipo- and hydrophilic structure) that allows melatonin to freely cross the cell and nuclear membranes. It can be easily detected in the nucleus of several cells in the brain and peripheral organs (41). Antioxidant properties are one example of non-receptor-mediated actions of melatonin. On the other hand, melatonin has receptor-mediated actions.

Two types of a new family of G protein coupled melatonin receptors (MT) have been cloned in mammals (42). Melatonin receptors are widely expressed, often with overlapping distributions. MT1 is primarily expressed in the pars tuberalis, the SCN together with other hypothalamic areas, pituitary, hippocampus, and adrenal glands, suggesting that circadian and reproductive effects are mediated through this receptor. MT2 is mainly expressed in the SCN, retina, pituitary and the other brain areas, and is associated with phase shifting (43). The affinity of melatonin is five-fold greater for MT1 than MT2. Melatonin administration induces (1) an acute suppression of neuronal firing in the SCN via the MT1 receptor, and (2) a phase-shifting of SCN activity through the MT2 receptor. However, agonists that exclusively act on one or another receptor have not been identified as yet, and the understanding of the role of each receptor in most of the tissues in which both receptors are present is difficult. Also, melatonin receptors are transiently expressed in neuroendocrine tissues during development, suggesting that melatonin has a role as a neuroendocrine synchronizer in developmental physiology (44).

Chronobiotic Effects of Melatonin

Figure 5. Phase shifting of circadian rhythms. From data in Middleton B. et al., J. Sleep Res, 2002, 11, Suppl 1, p 154, Melatonin phase shifts all measured rhythms abstr. No. 309. Rajaratnam SW, et al., J Clin Endocrinol Metab 2003;88:4303-9. Rajaratnam SW, et al., J Physiol 2004; 561:339-351.

Phase shifting of circadian rhythms by melatonin (Figure 5) was first described in the 1980s (45). Melatonin has chronobiotic effects and is able to synchronize and reset biological oscillations. Melatonin acts on oscillators according to a well-defined phase-response curve (PRC). PRC is characterized by phase-advance zone (early in the evening before the beginning of the nocturnal melatonin production), phase-delay zone (in the late night/early morning hours), and a non-responsive dead.  zone (when melatonin levels are high) (46). The melatonin PRC is useful for the clinical administration of melatonin as a chronobiotic agent and treatment of sleep circadian and mood  disorders (47). In addition to the circadian chronobiotic effects, melatonin importantly has chronobiotic seasonal effects and acts as a circannual synchronizer, one season determined by increasing duration of the nocturnal melatonin production (in the direction of the winter solstice) and the other season defined by the reduction of nocturnal melatonin production (in the direction of the summer solstice) (8, 48). Interestingly, most of the clock genes are expressed in the pars tuberalis with a 24h rhythmicity different from their expression in the SCN (49), and these clock genes are influenced by melatonin with numerous potential seasonal effects. However, whether melatonin modulates the activity of the SCN via regulation of clock genes is unclear. Also, a central clock, independent of the SCN, can be entrained by food availability, temperature variations, forced activity and rest, drugs (melatonin itself), and timing (50).

MELATONIN PHYSIOLOGY AND PATHOPHYSIOLOGY

As previously stated, melatonin can act through several mechanisms and at almost all levels of the organism. Therefore, it has multiple and diversified actions with immediate (endogenous melatonin, during the night) or prospective effects (exogenous melatonin, during the previous day).

Hypomelatoninemia is more common, and it can be due to factors that affect directly the pineal gland, innervation, melatonin synthesis as a result of congenital disease; or secondary as a consequence of environmental factors and/or medications (shift work, spinal cord cervical transection, sympathectomy, aging, neurodegenerative diseases, genetic diseases, β-blockers, calcium channel blockers, ACE inhibitors). Hypermelatoninemia is less common, and except for pharmacological effects, few conditions have been associated with high melatonin production: spontaneous hypothermia, hyperhidrosis syndrome, polycystic ovary syndrome, hypogonadotropic hypogonadism, anorexia nervosa, and Rabson-Mendenhall syndrome that induces pineal hyperplasia.

Melatonin During Puberty, Menstrual Cycle and Reproductive Function

Neuroendocrine control of sexual maturation is influenced by the pattern of melatonin secretion as a consequence of the light-dark cycle, and in some species the photoperiod via melatonin secretion determines the timing of puberty (51). In vitro studies, in cultured prepuberal rat pituitary gland, showed that melatonin inhibits gonadotrophin-releasing hormone and therefore luteinizing hormone release, providing evidence for a potential causal role of melatonin in the timing of developmental stages (52) (Figure 2). The mechanism by which melatonin inhibits GnRH secretion is not clear; recently, kisspeptin was suggested to mediate this effect. Lower melatonin levels have been associated with precocious puberty and higher levels in delayed puberty and hypothalamic amenorrhea compared to age-matched controls; however, a causal role of melatonin in pubertal development has not been described (53, 54). Data on circulating melatonin, and its variation during the menstrual cycle, are inconsistent (55). In males, high melatonin doses (100 mg daily) potentiate testosterone-induced LH suppression, and a negative correlation between nocturnal serum LH and melatonin have been reported (56, 57). Nevertheless, attempts to develop melatonin as a contraceptive pill in combination with progestin have not been successful (58). Interestingly, people living near the Arctic circle present lower conception rates during winter darkness, when melatonin levels are high, than in summer. In summary, the overall perception in human studies is that melatonin is inhibitory to human reproductive function.

Melatonin and Core Body Temperature

Melatonin plays a role in circadian thermoregulation. In particular, the melatonin peak is associated with the nadir in body temperature, together with maximum tiredness, lowest alertness and performance (59) (Figure 6). Exogenous administration of melatonin during the daytime reproduces this association, increasing fatigue and sleepiness and decreasing body temperature, especially if the subject is seated (posture dependent effect) (59). Along these lines, the rise in temperature during the ovulatory phase of the menstrual cycle is associated with a decline in the amplitude of melatonin.

Figure 6. Relationship of plasma melatonin to other major circadian rhythms driven by the internal clock. Abbreviations: VAS: visual analogue scale. Reproduced from Rajaratnam SMW and Arendt J. Lancet 358:999-1005, 2001 by permission.

Melatonin and Energy Metabolism and Glucose Homeostasis

Melatonin is an important player in the regulation of energy metabolism and glucose homeostasis. It is responsible for the daily distribution of energy metabolism functions (daily phase of high insulin sensitivity, glycogen synthesis, and lipogenesis and a sleep phase associated with the usage of stored energy) (60). Interestingly, administration of melatonin in post-menopausal women induced a reduction in fat mass and increase in lean mass compared to placebo (61). Nocturnal melatonin secretion facilitates diurnal insulin sensitivity and preservation of beta cell mass and function (62) and low melatonin secretion has independently been associated to a higher risk for type 2 diabetes (T2DM) (63). In short sleepers or when there is a misalignment between waking time and melatonin secretion (melatonin secretion is not interrupted), insulin resistance and hyperglycemia can be observed. Melatonin administration can result in iatrogenic insulin resistance and hyperglycemia in the morning, depending when it is administered and on the metabolizing characteristics of the subject. Moreover, recently, some variants of the gene encoding for the MT-1B have been associated with reduced beta-cell function and increased risk for T2DM (64). Several cardiovascular effects have also been attributed to melatonin, such as, antihypertensive properties, regulation of heart rate, and vascular resistance (65, 66).

Melatonin, Antioxidant Properties and Cancer

Antioxidant and anti-aging properties have also been attributed to melatonin. Melatonin acts as a potent free radical scavenger and antioxidant in vitro, independently of the presence of the receptor (38, 67, 68) protecting lipids, protein and DNA from oxidative damage. It is more effective than glutathione in reducing oxidative stress under many circumstances, being highly concentrated in the mitochondria. Although most of these effects in humans have been observed in supraphysiological doses of melatonin, the quantity of exogenous melatonin required to generate relevant antioxidant activity is not well established. Moreover, the clinical benefits of antioxidant supplements are not clear.  Quote “research has not shown antioxidant supplements to be beneficial in preventing diseases”, https://www.nccih.nih.gov/health/antioxidants-in-depth#:~:text=Antioxidants%20are%20man%2Dmade%20or,be%20beneficial%20in%20preventing%20diseases.

Also, there is growing recent evidence for anti-tumoral activity of melatonin (69, 70). Melatonin seems to be useful as an oncostatic agent at the cellular level and slows the progression of cancer (most of the studies are focused on breast and prostate cancer). Interestingly, in rats when a carcinogen is given at night during the highest levels of melatonin, DNA damage is significantly lower (20%) than in rats that receive a carcinogen exposure during the day (71). Pinealectomy stimulates cancer initiation or growth in animals, and pineal calcification, that leads to a decline in melatonin production, have been associated with an increase in pediatric primary brain tumors (72).

An ’Umbrella review’, whereby the results of numerous meta-analyses are combined, has provided important data on which of the numerous claims for melatonin therapeutic benefit stand up to scrutiny. A simplified version is provided here (see below).

As mentioned previously, exposure to light during the “biological night” can suppress melatonin production, and it is also associated to a deleterious effect on health (i.e., increased risk of cancer in most epidemiology studies in night shift workers) (70, 73). Night-shift workers present a higher incidence of hormone-dependent cancer which has been related to light-induced melatonin suppression which consequently increases estrogen production. A 50% increased risk to develop breast cancer in nurses exposed to rotating shift work has been documented (74). In contrast, a reduced risk for breast cancer was observed in blind women, with potentially higher levels of melatonin throughout all day (although there is no evidence that blind people produce more melatonin than sighted people) However, the mechanisms by which melatonin exerts any of oncostatic effects remains to be established.

Miscellaneous

The circadian annual rhythm of prolactin secretion depends on the circadian melatonin signal. Melatonin regulates pars tuberalis timer cells, and coordinates prolactin-secreting cells which together function as an intrapituitary pacemaker timer system. Interestingly, several clock genes are expressed within the pituitary with a circadian rhythm independent of the SCN (75).

Melatonin might act on the adrenal glands as an endogenous pacemaker. Glucocorticoids levels are low after the onset of darkness and rise after the middle of the night concomitantly with a decrease in melatonin levels. This is consistent with an inhibitory effect of melatonin through MT1 on glucocorticoid production. Interestingly, many antidepressant drugs increase the availability of the precursors (tryptophan and serotonin) and the major pineal neurotransmitter NE and therefore stimulate melatonin secretion. A melatonin agonist has been developed as an antidepressant through its actions on serotonin 2C receptors (76). If there is a link between this increase in melatonin production and the efficacy of antidepressant medications this needs further evaluation.

Several conditions (congenital or acquired) might induce a dysregulation of the melatonin synthesis/signaling and affect rhythmic melatonin production:

Pathological or traumatic sympathetic denervation (i.e., injury to the spinal cord) of the pineal gland or administration of β-adrenergic antagonists abolishes the rhythmic synthesis of melatonin and the light-dark control of its production (77, 78). NE is the major transmitter via β1 adrenoceptors with potentiation by α1 stimulation. NE levels are higher at night, approximately 180 degrees out of phase with the serotonin rhythm. Both availability of NE and serotonin are stimulatory for melatonin synthesis. However, several other factors have been related to the secretion and production of melatonin (13) (Table 1). In humans, administration of atenolol suppresses melatonin and enhances the magnitude of light-induced phase shift (79). In fact, several related observations suggest that endogenous melatonin acts to counter undesirable abrupt changes in phase. Pinealectomy i.e., abolishment of the melatonin rhythm, leads to a more rapid circadian adaptation to phase shift in rats (80).

Craniopharyngioma patients often display low melatonin secretion as a result of SCN impairment associated with alterations in sleep pattern (81).

Smith-Magenis syndrome (a congenital disorder due to a haplo-insufficiency of the retinoic acid-induced 1 gene, involved in the regulation of the expression of circadian genes) patients present with an inverted rhythmic melatonin secretion and sleep difficulties that can be managed with melatonin administration in the evening and β-adrenergic blockers during the day to reduce melatonin secretion (82). Low levels of melatonin have been consistently associated with autism spectrum disorders (ASD) (83). The last enzyme in the synthesis of melatonin, acetylserotonin-O-methyltransferase, has been associated with susceptibility for ASD which could be an explanation for low melatonin levels in ASD (84). Interestingly, administration of melatonin has shown to be efficacious for insomnia in children with ASD (85).

MELATONIN, CLINICAL APPLICATION AND THERAPEUTIC USE

Literature on the clinical use of melatonin is extensive and has increased exponentially over the last decade. Melatonin effects on sleep are the most well-known; however, the finding of increased cancer risk in shift workers and that patients with neurodegenerative diseases, autism or depression present abnormal melatonin rhythms, have recently increased attention on the role of melatonin.

Melatonin and Sleep

Numerous factors and comorbidities are associated with chronic insomnia in the adult population, different from the circadian sleep-wake rhythm disorders. Some of these risk factors and comorbidities involve psychiatric conditions (i.e. depression, anxiety, posttraumatic stress disorder), medical conditions (i.e. pulmonary diseases, chronic pain, heart failure, hyperthyroidism, nocturia, gastroesophageal reflux, cancer, pregnancy, pruritus, HIV infection, obstructive sleep apnea syndrome), neurological conditions (i.e. Alzheimer’s, Parkinson’s disease, peripheral neuropathies, strokes, brain tumors, headache syndromes), and medications (i.e. central nervous system stimulants or depressants, bronchodilators, antidepressants, diuretics, glucocorticoids, caffeine, alcohol). All these conditions should be ruled out before establishing the suspicion of a circadian sleep-wake rhythm disorder. The importance of melatonin levels for human sleep was apparently demonstrated by studies on pinealectomized subjects. These patients presented a disrupted 24h circadian rhythm, and a reduction of sleep time and quality, that were reversed after the administration of melatonin (86, 87). However, another careful, prospective study using polysomnography before and following pinealectomy found no effect on sleep and similar data are reported in rats (88-90). Thus, the question is not resolved. Clearly it is not a sleep hormone since in nocturnal animals it is secreted during the active periods. Overall, exogenous melatonin has been shown consistently to reduce sleep latency, and less consistently increase total sleep time, reduce night awakenings, and ultimately improve sleep quality (91). The most obvious action is to optimize sleep timing with respect to the circadian clock: we sleep better when melatonin production (and thus the circadian clock) and sleep are correctly aligned. Diagnostic criteria of every circadian sleep-wake rhythm disorder were fully described by the American Academy of Sleep Medicine (2014) (92).

CIRCADIAN SLEEP WAKE-RHYTHM DISORDERS EVALUATION

The origin of a circadian rhythm disorder can be related either to an intrinsic abnormality in the circadian system itself (misalignment of the intrinsic circadian timing with the desired sleep schedule) or to external factors, as when individuals must be awake at times that are not synchronized with their intrinsic rhythms. Overall, sleep disorders result in clinically significant symptoms of insomnia, daytime sleepiness and impaired physical, neurocognitive (concentration, processing speed, memory), emotional and social functioning, as well as impaired functioning at work or school (92). Age and associated comorbidities can help in the differential diagnoses. Symptoms across sleeping disorders are non-specific; the key to properly identify them is the recognition of abnormal sleep-wake patterns using sleep diaries beyond the complaint of insomnia or daytime sleepiness (93). Time domain is very important for melatonin actions (immediate or prospective effects, chronobiotic, or seasonal effects); therefore, adequate melatonin measurement and time of administration are crucial. Actigraphy can supplement self-reported sleep diary information or be useful in situations of neurodevelopmental disorders where a sleep diary cannot be completed.

Melatonin concentrations typically increase 90 to 120 minutes prior to the habitual bedtime if bright light (>10 lux) is absent. Preferentially frequent blood sampling every 30 to 60 minutes from six hours prior to and one hour following habitual bedtime is collected to assess the individual’s dim light melatonin onset (DLMO) and circadian phase. Samples should be collected in a dark environment (red light <10 lux) without interfering with sleep. In research studies a DLMO protocol is used as a valid and reliable indicator of circadian phase position, according to the time at which melatonin levels rise, and is extremely helpful to guide the timing for melatonin therapy in each individual; however, this is rarely feasible for routine use in clinical settings (94). Alternatively, urinary excretion of 6-sulfatoxymelatonin is a good index of nocturnal melatonin production and it should be collected as sequential urine samples for 48h in order to calculate the timing of the peak or acrophase. Also, it is possible to measure melatonin in saliva, during the evening before sleep and this approach has been frequently used. But, in the case of for example very delayed melatonin rise the time of onset can be missed during sleep. Importantly, melatonin values vary individually and according to age and sex. Although, endogenous melatonin production is closely related with the onset and offset of sleep, few associations have been found between melatonin production and sleep stages (95), and sleep deprivation does not abolish the melatonin rhythm.

CIRCADIAN SLEEP WAKE-RHYTHM DISORDERS

The primary goal of treatment of circadian sleep-wake rhythm disorders is to realign the circadian timing of sleep and wake with the required sleep-wake period. Appropriately timed and dosed melatonin, melatonin receptor agonists, and light therapy seem to be useful approaches (96, 97). Following the melatonin PRC and taking the individual DLMO as the reference phase to decide the most suitable time of melatonin administration, according to the desirable effect, seem the most appropriate guide to decide when to start chronic melatonin therapy (98-100).

Delayed and advanced sleep-wake phase disorders, non-24 hour, and irregular sleep-wake rhythm disorders are considered intrinsic circadian disorders; in contrast, jet lag and shift work disorders are due to an environmentally imposed misalignment. The most potent phase shifting factor (zeitgeber) is the environmental light-dark cycle (101).

Light exposure during the last hours of the usual sleep period moves the circadian rhythm forward (phase advanced). On the contrary, light exposure in the evening and the first half of the usual sleep period moves the circadian rhythm back (phase delayed) (Figure 7) (96). Advanced sleep-wake phase disorder is partially due to the physiologic advance that occurs with aging together with the weakening of circadian rhythms observed with aging. It can be genetically determined in some families as well. Delayed sleep-wake phase disorder is more often seen in children with some neurodevelopmental disorders; these patients cannot sleep during the dark time and delay sleep onset until the early hours of the morning, sleeping much of the day. In these situations both bright light in the early morning and evening melatonin 5 hours before endogenous melatonin onset secretion (0.5-5 mg) has been shown to advance sleep time significantly (102), the magnitude of the advanced shift being dose-dependent. In contrast early “biological morning” administration of melatonin induces delayed shifts. The non-24-hour sleep-wake rhythm disorder is most commonly seen in blindness, since the light-dark cycle is the most powerful cue for synchronizing the hypothalamic pacemaker to the 24-hour day (103). Therefore, this disorder is characterized by a failure to maintain stable alignment to the 24-h day, and a “free-running” circadian rhythm system which usually shifts to a later and later phase position. Timed melatonin treatment has been successful in entraining free-running blind patients.

The irregular sleep-wake rhythm disorder is seen in patients with dementia, which a neurodegenerative process might induce a disruption of the circadian system with a consequent loss of modulatory influence on sleep and wakefulness (104).

Figure 7. Representation of a simplified diagram of phase shifts of the circadian system, as evidenced by changes in melatonin rhythm itself, following oral treatment with fast release melatonin at different times. Biological night” is the time of endogenous melatonin secretion and defines “circadian time” which is independent of clock time.

Jet lag occurs when crossing time zones, and one needs to sleep and be awake at times that are not aligned with one’s own circadian system. It is more severe when more time zones are crossed and if the direction of the travel is eastbound, as it is more difficult to advance than delay the natural circadian cycle. Melatonin accelerates the phase shift if given at the appropriate time prior to bedtime at destination, and the benefits of melatonin administration in the alleviation of jet lag seems to be greater with larger numbers of time zones (105). If melatonin is appropriately time-administered, self-rated jet lag can be reduced by 50 percent (106). The maximum advance shift obtainable with a single treatment of oral melatonin (3-5 mg) is approximately 1-1.5 h.

For night shift workers wakefulness is required at the time that melatonin secretion is rising, and alertness is dissipating, and the opposite for the next day when melatonin secretion decreases, and circadian rhythms promote alertness. While some studies suggested that the use of melatonin improved sleep and increase daytime alertness in night shift workers compared to placebo (107), other studies have not shown beneficial effects (108). More data are needed before recommendations about melatonin as a therapy for shift workers can be made. However anecdotal evidence suggests that it is widely used for daytime sleep.

Figure 8. Diagram to illustrate free-running of the sleep wake cycle and other circadian rhythms in non-24h sleep-wake disorder, frequently seen in totally blind people. Melatonin, usually in doses of 0.5 – 5mg daily can synchronize sleep and the circadian system to the 24h day with benefits for sleep, and daytime alertness.

Melatonin and the CNS

Individuals with neurodegenerative disorders (i.e., Alzheimer’s and Parkinson’s disease, Huntington’s disease, autism) had significant flattened and attenuated melatonin rhythms compared to age-matched controls (37, 109). Melatonin showed efficacy in managing the insomnia in elderly people or improving cognitive function associated with neurodegenerative disorders (110). It has reported effects on anxiety, cognitive function and memory. Knowing the wide distribution of melatonin receptors in the CNS, melatonin seems one of the promising neuroprotective agents to be tested in humans. However other studies have found deleterious effects of melatonin in elderly demented patients (111).

Melatonin: Therapeutic Use

Almost 200 randomized clinical trials on the use of melatonin and evaluation of clinical effects have been published. Moreover, several patents have been registered in relation to the therapeutic applications of melatonin and melatonin analogues (sleep disorders, neuroprotection, cancer). The American Academy of Sleep Science recommends the use of melatonin for jet lag, delayed sleep phase syndrome, and non-24h sleep wake disorder syndrome seen in blind people; however, there were few consensuses acknowledging its therapeutic benefits (112) until an ‘Umbrella’ review of meta-analyses identified consistent therapeutic targets (91). Interestingly, at least in animal models, appropriate melatonin dose administration can reverse most of the effects after a pinealectomy.

Combined meta-analyses of studies meeting strict criteria (simplified from Posadzki PP et al. BMC Med (2018) 16: 18.)

Several melatonin receptor agonists have been synthesized, some of them have higher affinity for the receptor than endogenous melatonin. Agomelatine (activity at the serotonin-2C receptor) functions as an anti-depressant, ramelteon (selective MT1/MT2 agonist) is marketed for use in sleep onset insomnia, tasimelteon (MT1/MT2 agonist), is commercialized for the treatment of circadian rhythm disorders particularly non-24h sleep wake disorder (113, 114). Also, slow-release formulations of the natural biologic melatonin have been developed.

The time of melatonin administration is critical, especially in chronic treatments, and the melatonin profile shows large interindividual variation; however, the profile of an individual is highly reproducible from day to day. Also, absorption, metabolism and excretion of melatonin vary between individuals and should be considered to get the desired clinical efficacy of the therapy. Ideally, although not feasible in a daily clinical practice, DLMO should be determined for each individual, and used as a timing reference for the prescription of melatonin. Alternatively, the time each individual goes to sleep could determine the time of administration of melatonin; it is advisable to take oral melatonin around 45 minutes to 1 hour before the usual bedtime (time to reach maximal plasma concentrations following oral immediate-release formulations). Moreover, the duration of the pharmacological profile should last until the usual wake time of the patient; thus, the type of pharmaceutical formulation (slow or fast release) is also an important consideration. However, there is little evidence for greater benefit of slow-release preparations. Route of administration, as well as age, liver function and potential drug interactions (since melatonin is metabolized in the liver), may influence plasma melatonin levels and should be taken into account (115). Sensitivities and pharmacokinetics of melatonin vary between individuals, and a lower dose of 0.3-0.5 mg might be more effective than higher doses in many subjects.

There is no general consensus regarding dosage. A wide range of dose formulations are available, and the usage varies depending on the clinical application. The usual advice is to start with the lowest dose available. Low doses 0.1 to 0.3 mg/d that produce near physiological melatonin concentrations can be used for central clock synchronization; doses ranging from 0.6 to 5 mg/d for sleep disorders, or doses as high as 300 mg/d for neurodegenerative disorders (amyotrophic lateral sclerosis) (116, 117). There is a current tendency to recommend high pharmacological doses for ‘protective’ or antioxidant effects. What consequences these might have for the circadian aspects of melatonin function is not known. It should not be forgotten that melatonin has profound effects on the reproductive function of photoperiodic seasonal breeders and there is good evidence for residual photoperiodicity in humans. However in most clinical trials or studies using melatonin, it is well accepted that in general melatonin lacks toxic adverse events, and it is a safe drug at most of the usual tested doses from 0.5 to 5 mg/d (118). Whether dosage should be changed in chronic melatonin treatment according to the annual season requires further investigation; further studies are needed on the undesirable consequences of melatonin suppression in the long term (for example by beta blocking drugs or shiftwork).

To summarize, benefits of melatonin and its analogues on circadian sleep disorders are consistently reported but for more generalized “insomnia” are often of low strength. The dose must be timed and individually adjusted as optimization will vary among individuals. More studies are required to develop treatment guidelines for different conditions. The long-term consequences of taking high dose melatonin especially in pediatrics need careful evaluation. Melatonin mechanisms of action and effects need much further work, to better understand the therapeutic value of melatonin and its potential applications.

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ABSTRACT

Pituitary adenomas comprise approximately 10-20% of intracranial tumors. Non-functioning pituitary adenomas (NFPAs) are benign adenohypophyseal tumors not associated with clinical evidence of hormonal hypersecretion. NFPAs comprise different histological subtypes, classified according to their immunostaining to different adenohypophyseal hormones and transcription factors. The silent gonadotroph adenoma is the most common subtype, followed by corticotroph, PIT1 (POU1F1) gene lineage, and null cell tumors. Patients with NFPAs usually come to medical attention as a result of “mass effects” symptoms such as headaches, visual disorders, and/or cranial nerve dysfunction caused by lesions large enough to damage surrounding structures. Hypopituitarism, caused by the compression of the normal anterior pituitary, and hyperprolactinemia due to pituitary stalk deviation can also be present. Some cases may be diagnosed incidentally through imaging studies performed for other purposes. Patients with NFPAs should undergo hormonal, clinical, and laboratory evaluation to rule out hyper and hypopituitarism. Assessment of prolactin and IGF-1 levels have been recommended in all patients whereas screening for cortisol excess is suggested in the presence of clinical symptoms. Impairment of pituitary function should be assessed by baseline hormonal measurements and/or stimulatory tests, if needed. Patients in whom the tumor abuts the optic chiasm should be submitted to visual field perimetry. Surgical resection is the primary treatment for symptomatic patients with NFPAs, i.e., those with neuro-ophthalmologic complaints and/or tumors affecting the optic pathway. Visual deficits and, less commonly, hormone deficiencies may improve following surgical treatment although new hormone deficiencies may also occasionally develop after a surgical approach. For patients with residual NFPAs following transsphenoidal surgery, a therapeutic attempt using cabergoline can be made according to clinical judgment in individual cases. Radiotherapy in the postoperative period is not consensual and is generally reserved for cases of tumors not completely resected by surgery, those cases that present progressive tumor growth during follow-up, or for patients who, at diagnosis, already have tumors with aggressive features. Highly aggressive tumors need special care during follow-up, including temozolomide with or without radiotherapy complementation or new potential emerging treatments. For patients with asymptomatic NFPAs a “watch and wait” option is reasonable. Follow up is individualized and should consider tumor size, prior treatments, and clinical symptoms.

INTRODUCTION

Pituitary adenomas are common, predominantly indolent neoplasms comprising approximately 10-20% of intracranial tumors (1,2). Non-functioning pituitary adenomas (NFPAs) are benign neoplasms that originate from the adenohypophyseal cells and are not associated with clinical evidence of hormonal hypersecretion (3). They comprise a large and heterogeneous group, representing a sizeable proportion (22% to 54% in different series) of all pituitary adenomas (4-7). Malignant transformation in a non-functioning pituitary adenoma (pituitary carcinoma) is extremely rare and is characterized by craniospinal or systemic dissemination (8).

NFPAs can be further classified according to their pituitary hormone and transcription factor profile, as defined by the 2017 World Health Organization (WHO) classification for endocrine tumors (9). Tumors that express one or more anterior pituitary hormones or their transcription factors with immunohistochemistry (IHC) but do not secrete hormones at a clinically relevant level can be referred to as silent pituitary adenomas (SPAs) (10,11). As a consequence, the definition of “null cell adenoma” is now limited to an exceptionally rare primary adenohypophyseal tumor that shows immunonegativity for all adenohypophyseal hormones as well as for cell-type specific transcription factors (12) - Figure 1.

Figure 1 - Classification of non-functioning pituitary adenomas rely upon clinical features and histopathological data. Reprinted with permission from Drummond et al., 2019

The term “totally silent” has been proposed to be used when a patient with an NFPA presents basal and stimulated serum concentrations of the correspondent hormones within the normal range and there are no clinical signs or symptoms that can be attributed to hormone excess (13). The term “clinically silent” may be used when NFPAs secrete hormonal products that cause an elevation of the serum concentration but do not result in clinical signs or symptoms of hormonal hypersecretion (13). Some cases are referred to as “whispering” adenomas with borderline, mild, often overlooked, clinical symptoms and signs (14,15).

EPIDEMIOLOGY

The prevalence of NFPAs is variable and is often based upon autopsy or magnetic resonance imaging (MRI) series. Data from Europe, North and South America have estimated that the prevalence of clinically relevant NFPAs is 7–41.3 cases per 100,000 of population (16). This is likely an underestimate of the true prevalence, as many NFPAs go undiagnosed until they are large enough to cause mass effect or are accidentally discovered. Data are discordant about gender predominance and the peak occurrence is from the fourth to the eighth decade. A recent population-based study from South Korea showed an annual incidence of 3.5 cases/ 100,000 population for NFPAs, which is noticeably higher than the annual incidences previously reported in other countries, such as Sweden, Finland, and Argentina (17). These differences may be related to genetic and environmental factors in Asian populations or inter-study heterogeneity and may also reflect the good local access to diagnostic evaluations using magnetic resonance imaging and computed tomography.

CLINICAL PRESENTATION

The absence of clinical manifestations of hormonal hypersecretion usually results in significant diagnostic delay and therefore NFPAs may not be diagnosed until they cause mass effects to surrounding structures (3), causing symptoms such as headaches, visual disorders, and/or cranial nerve dysfunction. Other manifestations are hormone deficiencies or hyperprolactinemia due to pituitary stalk deviation and, less frequently, pituitary apoplexy (18,19). Additionally, some cases may be diagnosed incidentally through imaging studies performed for other purposes, the so-called pituitary incidentaloma.

Neurologic Manifestations

VISUAL IMPAIRMENT 

Impaired vision, caused by suprasellar extension of the adenoma that compresses the optic chiasm, is the most common neuro-ophthalmological symptom (19). Different types of visual defects depend on the degree and site of optic nerve compression. Both eyes are usually affected, although a significant proportion of patients may have unilateral or altitudinal problems in 33 and 16% of the cases, respectively (20). Diplopia, induced by oculomotor nerve compression resulting from parasellar expansion of the adenoma may occur, and the fourth, fifth and sixth cranial nerves may also be occasionally involved when there is parasellar expansion (16). Nevertheless, the typical visual field defect associated with pituitary tumors is bitemporal hemianopia, reported in approximately 40% of the patients.

HEADACHE

Headaches, the second most common neurologic symptom, occur in 19-75% of patients with pituitary tumors, regardless of size (21). In a retrospective case series of incidentally-discovered NFPAs, headache was present in approximately 20% of the cases (22). In a recent prospective series, 138 out of 269 patients with NFPAs complained of tumor-related symptoms and, among them, 23% presented with headaches (23). Although it is not always clear whether the presenting headache is related to the tumor, proposed mechanisms for headache include increased intrasellar pressure, stretching of dural membrane pain receptors, and activation of trigeminal pain pathways (16,24). Cerebrospinal fluid (CSF) rhinorrhea, associated or not with headache, can occur in cases where the tumor causes erosion of the sellar floor and extends inferiorly to the sphenoid sinus.

PITUITARY APOPLEXY

Pituitary apoplexy (sudden hemorrhage into a pituitary adenoma) is rare. According to a retrospective case series of 485 Mexican patients with NFPAs, pituitary apoplexy was the initial presentation in 8% of the cases (25). It causes acute onset of a severe headache associated with visual disturbances and can occur in all types of pituitary tumors, although some series suggest pituitary apoplexy might be more common in NFPAs than other adenomas subtypes (26). It has been suggested that the combination of the high metabolism of pituitary adenomas combined with their special blood supply would make them more prone to vascular events (27). Pituitary apoplexy may occur without an identified risk factor, but it has also been reported as being related to pregnancy, use of anticoagulants, surgical procedures, as well as in association with dynamic tests, such as thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), and insulin-tolerance stimulation tests (28).

The actual long-term risk of apoplexy in NFPAs has not been clearly defined (29). In a Japanese prospective cohort study of 42 asymptomatic patients with NFPAs (mean initial tumor size of 18.3 mm) followed-up for approximately four years, pituitary apoplexy was reported in 9.5% of the cases (30). A systematic review and meta-analysis evaluating the outcomes of patients with NFPAs and pituitary incidentalomas who were treated conservatively and followed-up for mean period of 3.9 years showed that the development of pituitary apoplexy was rare (0.2/100 patients-year), although a trend for a greater incidence of pituitary apoplexy was seen in macroadenomas compared with microadenomas, with a reported incidence of apoplexy of 1.1% per year (31). These findings are in line with two recent retrospective studies including a Korean series of 197 patients with NFPAs without mass effect symptoms or pituitary apoplexy at baseline who were followed-up for a median of 37 months (32) and an Italian series of 296 patients with incidentally discovered NFPAs (macroadenomas in 49% of the cases) who we followed-up for approximately three years (33). Five out of 169 patients with macroadenomas developed pituitary apoplexy with an overall incidence of 0.83 per 100 patients-years in the Korean series while no cases of pituitary apoplexy were reported in the Italian series.

Endocrine Manifestations

HORMONAL DEFICIENCIES

Most patients with nonfunctioning pituitary macroadenomas present with deficiency of at least one pituitary hormone resulting from the compression of the normal anterior pituitary and/or pituitary stalk, preventing the stimulation of pituitary cells by hypothalamic factors. Hypogonadism can result from either a direct compressive effect on gonadotropic cells or by stalk compression-induced hyperprolactinemia that inhibits the pulsatile secretion of gonadotropin releasing hormone via interfering with hypothalamic kisspeptin-secreting cells (34). This “disconnection hyperprolactinemia” usually <2000mIU/L (95 ng/mL) (35) is characterized by compression of the pituitary stalk, which prevents the arrival of dopamine to the anterior pituitary, the main inhibitor of prolactin (stalk effect). GH and gonadotroph axes are most commonly affected, followed by adrenal insufficiency and central hypothyroidism (16,23,32).

HORMONAL EXCESS

Gonadotroph adenomas are usually considered to be "nonfunctioning" although they can secrete intact gonadotropins, as they do not generally result in a clinical syndrome. Occasionally, gonadotroph adenomas secrete primarily FSH but also LH in quantities high enough to raise serum gonadotropin levels, which in turn, may lead to the development of some specific symptoms, such as ovarian hyperstimulation in young women (36-38) or, more rarely, precocious puberty or testicular enlargement in men. In addition, low serum LH:FSH ratios (usually < 1.0) have been described in clinically-secreting gonadotroph adenomas (39). Measurement of α-subunit may also contribute to a pre-operative diagnosis in clinically silent but biochemically-secreting NFPAs, as it may be the sole biochemical marker of the gonadotroph subtype in a number of cases. In addition, circulating FSH, LH and α-subunit levels can help the post-operative surveillance of these patients (39).

HISTOPATHOLOGICAL CLASSIFICATION

The 2017 WHO classification for endocrine tumors (9) defines pituitary adenomas, including NFPAs, according to their pituitary hormone and transcription factor profile. It is noteworthy that in this classification system, IHC forms the basis of the new categorization, in which an adenohypophyseal cell lineage designation of the pituitary adenoma has replaced the concept of a “hormone-producing pituitary adenoma” (40) -Table 1.

Adapted from Mete et al, 2017.

Indeed, the prevalence of the different histological subtypes of NFPAs is dependent on the extent of the IHC profile. According to a large retrospective case series (11), the gonadotroph adenoma is the most common subtype, followed by corticotroph, PIT1 (GH/ Prolactin/TSH) lineage and null cell - Figure 2.

Figure 2 - Prevalence of silent pituitary adenoma subtypes according to immunochemistry for anterior pituitary hormones and transcription factors. Adapted from Nishioka et al., 2015

Pituitary hormones gene expression analysis using real-time quantitative PCR (RT-qPCR) has been shown to complement the IHC classification of pituitary tumors, according to a large observational, cross-sectional study evaluating 268 patients (41). Addition of RT-qPCR analysis reduced the proportion of null-cell subtype in this series from 36% to 19%. Lower specific adenohypophyseal hormone gene expression was observed in some SPA variants when compared with their secreting counterparts, including somatotroph, lactotroph, mixed somatotroph-lactotroph and plurihormonal adenomas, which could contribute to the absence of endocrine manifestations.

The European Pituitary Pathology Group has recently proposed a standardized report for the diagnosis of pituitary adenomas which includes a multi-step approach, comprising the summary of clinical and neuroimaging features, IHC for hormones and transcription factors, assessment of proliferation and, when indicated, the use of markers predictive of treatment response (42). This proposition highlights the role of the pituitary pathologist as part of a multidisciplinary pituitary team helping the clinician define the most appropriate post-operative strategy for each patient, including appropriate follow-up and early recognition and treatment of potentially aggressive tumors (43).

Subtypes of NFPAs

NULL CELL ADENOMAS AND SILENT GONADOTROPH ADENOMAS

Null cell adenomas and silent gonadotroph adenomas (SGAs) were previously misunderstood to be the same type of tumor. The addition of immunostaining for steroidogenic factor 1 (SF1) as a tool in the diagnosis of pituitary lesions has shown that many LH/FSH immunonegative adenomas are in fact SGAs (44), which comprise almost 80% of resected NFPAs. The distinction between SGAs and null cell adenomas is of clinical relevance because true null cell adenomas are rare and likely to be more invasive and aggressive than SGAs (45). In addition, these rare cases of ‘null cell adenomas’ may require a broad panel of immunohistochemistry in order to exclude a sellar metastasis of a neuroendocrine tumor (NET) from another organ. Several markers, including TTF1, serotonin, ATRX, DAXX and CDX2 may be useful in the differential diagnosis between an NFPA and a metastatic NET in the sellar regionData from a retrospective case series of 516 patients with NFPAs have shown that from the 23% of the tumors initially classified as null cell adenomas by using only classical pituitary hormone IHC, only 5% of them remained as true null cell type. Indeed, immunostaining using lineage-specific markers, namely, PIT-1 (coded by the POU class 1 homeobox 1 (POU1F1) gene), SF1, TPIT (coded by the T-Box transcription factor 19 (TBX19) gene) and estrogen receptor-α (ERα) provided tumor reclassification in 95% of cases (11).

SILENT CORTICOTROPH ADENOMAS

Silent corticotroph adenomas (SCAs) are characterized by the absence of clinical features of Cushing syndrome, along with normal circadian cortisol secretion (totally silent) or elevated ACTH (clinically silent) (5,47-49). They currently account for approximately 15% of NFPAs, an underestimated proportion, since IHC for the transcription factor TPIT, a marker of corticotroph differentiation which regulates the proopiomelanocortin (POMC) lineage giving origin to the corticotrophs, is still not widely available (50). For instance, in a recent retrospective series, inclusion of IHC for TPIT allowed identification of eight SCAs out of 18 (44%) pituitary tumors previously classified as null cell adenomas (51).

SCAs often present themselves as macroadenomas associated with mass-related symptoms. In comparison with SGAs, SCAs show female preponderance, are more frequently giant adenomas, and are more often associated with marked cavernous sinus invasion (49). Importantly, the presence of multiple microcysts in T2-weighted pituitary MRI sequences in a NFPA has a high specificity (> 90%) for the corticotroph subtype. The first study showed that multiple microcysts were present in 76% (13/17) of SCAs as opposed to 5% (3/60) of SGAs (52) while a more recent study showed comparable results, as multiple microcysts were present in 58% (7/12) SCAs but in only 9.5% of SGAs (53).Histologically, SCAs can be further divided into type 1 (densely granulated), type 2 (sparsely granulated) and Crooke-cell adenoma. Type 1 SCAs show strong ACTH immunoreactivity whereas type 2 SCAs resemble the rare chromophobe corticotroph adenoma and show weak and focal ACTH immunoreactivity. Type 2 SCAs seem to be more common and are likely to display higher expression of migration and proliferation factors compared with type 1 SCAs (5,11,40). Clinically silent Crooke-cell adenoma is a rare yet highly aggressive subtype as it carries significant risk of morbidity (54). Since SCAs are nonfunctional, an important point to note is the absence of Crooke’s hyalinization in the normal surrounding pituitary gland, as there is no exposure to high circulating glucocorticoid levels to promote hyaline deposits of cytokeratin filaments in the cytoplasm of normal corticotrophs (49).

The transformation of a silent corticotroph tumor into Cushing disease has been described, although the mechanism involved in this phenomenon is not yet well understood (55,56). It has been proposed that the clinical manifestations of Cushing disease are dependent on the processing of the pro-hormone POMC in corticotrophs. The pro-hormone convertase 1/3 (PC1/3) is involved in the post-translational processing of POMC into mature and biologically active ACTH. SCAs show a decrease in PC1/3 expression associated with a down-regulation of PC1/3 genes compared with corticotroph adenomas associated with Cushing disease (47). An attractive and more likely hypothesis involves epigenetic mechanisms: DNA hypermethylation of regulatory regions could lead to reduced expression of POMCtranscripts impairing the production of secreted ACTH. Differences in methylation status were observed when comparing ACTH-secreting pituitary tumors and SCAs: the second promoter was highly methylated in SCAs, partially demethylated in normal pituitary tissue and highly demethylated in pituitary and ectopic ACTH-secreting tumors (57).

SILENT SOMATOTROPH ADENOMAS

Silent somatotroph adenomas are PIT1 and GH-immunoreactive tumors without clinical and biological signs of acromegaly. They represent approximately 2-4% of all pituitary adenomas in surgical case series (58). Patients with silent somatotroph adenomas usually present with normal pre-operative GH and IGF-1 levels but there have been few reports of "clinically silent" cases, with non-suppressible serum GH and elevated IGF-1 levels (59).

Similar to secreting somatotroph adenomas, silent somatotroph adenomas are classified into densely granulated and sparsely granulated types, based on the presence and pattern of the low molecular weight cytokeratin staining. More than 50% of silent somatotroph adenoma cases constitute mixed GH–PRL adenomas, a proportion twofold higher than somatotroph adenomas causing acromegaly (60). Unlike clinically functioning somatotroph tumors, the silent ones are more frequently sparsely granulated with more aggressive behavior and a lower response to somatostatin analog therapy (61). Furthermore, silent somatotroph adenomas are more frequent in females, present at a younger age, are larger, more invasive, and recur earlier and more frequently than their secreting counterparts (62).

SILENT THYROTROPH ADENOMAS

Silent thyrotroph adenomas usually present TSHβ, α-subunit, and PIT-1 expression on IHC in a variable manner (40). They are more frequent when compared with their functioning counterparts and seem to behave similarly regarding treatment outcomes and recurrence rates (63). In a recent retrospective series of 20 patients with silent thyrotroph adenomas who underwent transsphenoidal surgery, 95% were macroadenomas and 85% showed extrasellar growth. Gross total tumor resection was achieved in 45% while major tumor progression or recurrence occurred in 10% of the patients over a median follow-up period of 18.5 months (64). These results show that, although silent thyrotroph adenomas tend to be large and invasive tumors, good overall outcomes with low complication rates can be achieved.

SILENT LACTOTROPH ADENOMAS

Clinically silent lactotroph adenomas are rare. The positive prolactin staining by IHC with no clinical signs of hyperprolactinemia is usually encountered concomitantly with

GH positive staining (silent mixed somatotroph-lactotroph adenoma) (61). They can also express ERα on immunostaining (9).

PLURIHORMONAL ADENOMAS

According to the WHO 2017 classification, plurihormonal adenomas can be classified on the basis of their transcription factor expression into two groups: 1) “PIT1-positive adenomas” (previously known as silent subtype 3 pituitary adenoma); and 2) plurihormonal with more than one transcription factor, termed “plurihormonal adenomas with unusual immunohistochemical combinations” (PAWUC) (9,40). In a recent large retrospective series comprising 665 patients who underwent endoscopic endonasal transsphenoidal surgery, plurihormonal adenomas were identified in 4% of the cases (18 patients had PAWUC and 9 patients were diagnosed with PIT-1 positive adenomas); 24 (89%) were macroadenomas (including 6 giant adenomas) and cavernous sinus invasion was found in 12 patients (44%) (65).

PIT1-positive plurihormonal adenomas are a distinct entity, with reportedly aggressive behavior. A single-center retrospective case series reported a prevalence of 0.9% for ‘silent adenomas type 3’ among resected pituitary tumors over a period of 13 years. All tumors were macroadenomas, aggressive, invasive, with a high rate of persistent/recurrent disease (66). Interestingly, these tumors may present clinical symptoms of hormone excess, such as acromegaly, hyperthyroidism or marked hyperprolactinemia (67) and the diagnosis of a PIT-1 positive plurihormonal adenoma should be kept in mind in case of large and invasive NFPA tumors in young patients, particularly those showing TSH and often minor reactivities for PRL and GH as well as cytologic atypia in conjunction with an elevated Ki-67 labelling index (66).

PAWUC are also characterized by aggressive behavior and higher rates of cavernous sinus invasion. A recent single-center retrospective series comparing clinical characteristics, outcome parameters and rate of invasiveness of 22 PAWUC to 51 SGAs showed that patients with PAWUC were younger, were more likely to present with intraoperatively signs of tumor invasion and less likely to achieve gross-total resection than patients with SGAs, suggesting a more aggressive behavior of NFPAs if additional transcription factors other than SF1, GATA2/3 and ER α are expressed within the tumor cells (68).

EVALUATION

According to current practice guidelines on incidentally discovered sellar masses, all patients, including those without symptoms, should undergo hormonal, clinical, and laboratory evaluation for hyper and hypopituitarism (69). The extent of the evaluation is still debatable and has commonly relied on clinical experience - assessment of prolactin and IGF-1 levels have been recommended, whereas screening for cortisol excess (i.e., overnight 1mg dexamethasone and/or late-evening salivary cortisol and/or urinary-free cortisol) is suggested in the presence of clinical symptoms, while measurement of ACTH levels is not routinely recommended. (69).

Patients with macroadenoma, especially those >3 cm and with normal or slightly elevated prolactin, must have their serum prolactin diluted in order to exclude the "hook effect" (70,71). This effect occurs when excessively high levels of this hormone interfere with the formation of the complex antibody-antigen-antibody sandwich, and a prolactinoma might be mistaken for a NFPA (72).

It is also suggested to check for pituitary hormone deficiencies in patients with non-functioning tumors, irrespective of symptoms. Macroadenomas can cause impairment of pituitary function by the involvement of the normal gland or pituitary stalk compression, and the risk of hypopituitarism is directly related to tumor volume. Microadenomas eventually lead to pituitary dysfunction, particularly if larger than 5mm (32,69). A large retrospective cohort of 218 patients with NFPAs found at least one pituitary deficiency at diagnosis in 33.3% of microadenomas (73) although other studies have failed to identify hormonal deficiencies in microadenomas. An interesting and cost-effective approach for microincidentalomas could be no hormonal evaluation for cystic and solid lesions smaller than 5 mm. For solid microadenomas (between 6-9 mm), it is suggested a simple investigation with measurements of PRL and IGF1 (74).

Concerning macroadenomas, current guidelines suggest that serum cortisol levels at 8-9 AM should be done as the first-line test for diagnosing central adrenal insufficiency (75). Despite well recognized limitations of most commercial cortisol immunoassays (76), baseline cortisol levels <3 µg/dL (83 nmol/L) are suggestive of central adrenal insufficiency, while baseline cortisol levels >15 µg/dL (414 nmol/L) likely exclude it, and levels between 3 and 15 µg/dL require a corticotropin/insulin stimulation test. Central hypothyroidism is assessed by measuring serum TSH and free T4 (fT4), and fT4 level below the laboratory reference range in conjunction with a low, normal, or mildly elevated TSH in the setting of pituitary disease usually confirms the diagnosis (75). Diagnosing GH deficiency (GHD) is relatively straightforward in patients with IGF1 concentration lower than the gender- and age-specific lower limit of normal and structural pituitary disease. Low IGF1 in patients with non-functioning tumors who have at least three pituitary hormone deficiencies is also suggestive of GHD (29). Nevertheless, some adults with suspected GHD may have normal IGF1, in such cases GH stimulation testing may be useful. Central hypogonadism in males, manifests with low serum testosterone, low or inappropriately normal FSH/LH and features of testosterone deficiency. For females, pre-menopausal women with oligomenorrhea or amenorrhea should be screened with serum E2, FSH, and LH. In postmenopausal women, if the patient is not in hormonal therapy, the absence of high serum FSH and LH is sufficient for a diagnosis of central hypogonadism (75).

Sellar MRI with gadolinium is the best imaging study for suspected adenomas because it provides images of high resolution of the mass as well as its relationship with surrounding structures. Pituitary adenomas usually appear hypo or isointense compared to normal pituitary tissue in T1 images on MRI. In addition, while the normal pituitary tissue enhances earlier with contrast, pituitary adenomas commonly present with delayed contrast uptake (77). Patients whose tumor abuts the optic chiasm should have visual field perimetry, preferably by the Goldmann method, to assess for visual deficits (69).

Functional (FDG- and/or SSTR- positron emission tomography (PET) imaging studies should only be considered in aggressive pituitary adenomas/carcinomas in the setting of site-specific symptoms (neck/back pain or neurological complaints), and/or where laboratory measures are discordant with known visible extent of disease (78). Recently, 37 patients diagnosed with NFPAs enrolled in a clinical trial underwent ⁶⁸Ga-DOTATATE PET/computed tomography (CT) of the head. ⁶⁸Ga-DOTATATE uptake was positive in 34/37 patients (92%), demonstrating in vivo SSTR expression in NFPAs and ultimately showing superior sensitivity of PET imaging when compared with ¹¹¹In-DTPA-octreotide scintigraphy (79).

The pre-operative differential diagnosis of NFPAs includes several additional primary non-hormonal-secreting lesions (80). This distinction is challenging in many cases because the clinical presentations and radiological aspects may be similar between adenomas and other pituitary lesions. The presence of diabetes insipidus (DI) is common in tumors of non-pituitary origin and indicates that the sellar mass is most likely not a pituitary adenoma (81). An increase in α subunit of the glycoprotein hormones (elevated in 30% of NFPA patients) can help in the differential diagnosis although normal values do not rule them out (82).

TREATMENT

In spite of current knowledge of definite NFPAs pathologic subtypes, initial treatment strategies are similar regardless of the subset. A small number of studies have reported treatment outcome results taking into consideration adenohypophyseal hormone immuno-profile (82-84) and, for the future, precise pathologic characterization utilizing adenohypophyseal hormones and transcription factors may potentially aid in predicting the response to specific adjunctive therapies.

Surgery

Surgical resection is the primary treatment for symptomatic patients with NFPAs (85), i.e., those with neuro-ophthalmologic complaints and/or tumors affecting the optic pathway. Surgery is also urgently indicated for patients with apoplexy who develop neuro-ophthalmologic complaints. In some experts’ opinion, tumors larger than 2cm should also be considered for surgery due to their propensity for growth (86). Treatment for hypopituitarism, primarily central adrenal insufficiency, and central hypothyroidism, should be commenced prior to surgical resection. Mortality rate is low (<1%) and reported surgical complication rates are acceptable. Postoperative complications such as CSF leakage, fistula, meningitis, vascular injury, persistent diabetes insipidus (DI), or new visual field defect occurred in ≤ 5% of patients, as reported by a systematic review and metanalysis (87). Accomplishment of total or near-total resection can be challenging and varies in different series, ranging from 20% to 80% (88,89). According to a recent large retrospective series evaluating 254 patients who underwent endoscopic endonasal surgery for a macroadenoma (including 72 patients with NFPAs), cavernous sinus invasion as assessed by the modified Knosp classification (90)effectively predicted surgical outcomes. Gross total resection was negatively correlated with tumor grade and success rates of gross-total resection were between 30% and 56% for tumors extending beyond the internal carotid artery and into the cavernous sinus compartments (grades 3A and 3B) (91).

A new “shape grading system” (Figure 3) has been recently proposed for predicting outcomes in patients with NFPAs operated by transsphenoidal surgery. In a retrospective single center study, 191 NFPAs were assessed according to different radiological growth patterns: spherical (Shape I), oval (Shape II), dumbbell (Shape III), mushroom (Shape IV), and polylobulated (Shape V) (92). Gross total resection was achieved in 53% of the patients, with decreasing likelihood of accomplishment in higher shape grades - shape I (82%), shape II (74%), shape III (24%), shape IV (17%) and shape 5 (0%). Furthermore, shape grades predicted resection rate better than Knosp grades. During a mean follow-up of 59 months, tumor recurrence or regrowth was observed in 6% and 12% of the patients, respectively. Higher shape grades also correlated with higher risk of tumor recurrence/regrowth as well as the need for reoperation and/or radiotherapy. Of note, SCAs more often grew as shape IV or V, while silent somatotroph adenomas were more often detected in the shape V group. Thus, the shape grading system seems to be a complimentary tool to better predict NFPA surgical outcomes, however, these findings need validation in other cohorts.

Figure 3 - The Shape grading system. Abbreviations: OC, optic chiasm; PS, pituitary stalk. From Berkaman et al Acta Neuropathologica 2021 (92)

Visual field deficits and, less commonly, hormone deficiencies may improve following surgical treatment although new hormone deficiencies may also occasionally develop after a surgical approach (87,93). The incidence of DI after endoscopic transsphenoidal surgery to remove NFPAs was recently investigated in 168 patients. Seventy-seven (45.8%) patients experienced postoperative DI and 10 (6.0%) patients suffered from permanent DI. A large cephalocaudal tumor diameter (cut off value-of 2.7 cm) was predictive of postoperative DI in such patients (94). Moreover, younger age and the absence or intrasellar location of the bright spot (as opposed to suprasellar location) on preoperative T1-weighted MRI were further factors predicting postoperative DI in a Japanese series of 333 patients with NFPAs undergoing transsphenoidal surgery (95).

Pituitary function should be reassessed one to three months after surgery and treatment of hypopituitarism introduced according to hormone deficiencies. Whether or not GH deficiency should be replaced requires a thoughtful and individualized evaluation of risks and benefits (96,97). Current data supports the absence of any stimulation of remnant or induction of recurrence by growth hormone replacement in patients with NFPAs solely treated by surgical removal (98). In a retrospective series, tumor regrowth occurred in 38/107 (36%) of non-GH treated subjects and in 8/23 (35%) of GH-treated subjects, followed up for a mean period of 6.8 years. The Cox regression analysis showed that after adjusting for sex and age at tumor diagnosis, cavernous sinus invasion at diagnosis, and type of tumor removal, GH treatment was not a significant independent predictor of recurrence.

A sellar MRI should be obtained three to six months after surgery to assess the extent of tumor resection. Also, as a significant number of patients with NFPAs may develop tumor re-growth, long-term imaging surveillance is recommended. In a retrospective analysis of 155 patients with NFPAs treated solely by surgery and followed-up for a mean period of six years (twenty-nine were followed up for more than 10 years), re-growth was reported in 34.8% of the cases, with 20% of relapse/re-growth occurring after 10 years (88). Likewise, in patients with NFPAs who present with classical apoplexy, tumor re-growth rate is not negligible. In a retrospective series of thirty-two patients with NFPAs who underwent surgery for pituitary apoplexy, tumor re-growth was reported in 11% of the cases at just over five years (99). Therefore, it is advisable that patients with NFPAs, particularly those with post-operative tumor remnants, be closely monitored following surgery, and follow-up surveillance needs to certainly be continued for more than 10 years.

Radiation Therapy

Radiation therapy (RT) has been shown to be effective as an adjunct to surgical resection in cases of post-operative residual tumor or recurrence. However, it carries a major long-term risk of hypopituitarism (85,100,101). Stereotactic techniques such as stereotactic radiosurgery or fractionated stereotactic radiotherapy have been developed with the purpose of delivering more localized irradiation and reducing long-term side-effects. Both techniques provide excellent tumor control in patients with NFPAs, ranging from 85% to 95% at five to ten years (102). However, at the present time, there is no consensus concerning the systematic use of RT in the postoperative period for patients with incompletely resected NFPAs (29) and whether an earlier approach would be preferable over conservative management. In general, RT is reserved for cases with large tumor remnants and for those cases that present progressive tumor growth during follow-up (85). Adjuvant RT may also be considered for patients who, at diagnosis, already present aggressive tumors, such as those invading parasellar structures or with extensive positive immunostaining for Ki-67, a proliferative index significantly associated with recurrence in NFPAs (9,103). Furthermore, NFPA subtype may be a relevant factor in the expected response to radiotherapy. A retrospective multicenter study demonstrated that overall tumor control rate after radiosurgery was lower in SCAs compared to other NFPA subtypes (104) suggesting that, in SCAs, an elevated margin dose may be considered in order to achieve a better chance of tumor control. In line with these findings, a recent and large retrospective surgical series showed a significantly lower progression-free survival in patients with SCAs compared to other NFPAs (24.5 vs 51.1 months) (105). Among the SCA cohort, progression was noted despite the use of adjuvant radiosurgery in one third of the patients. Notwithstanding, a recent systematic review and meta-analysis showed no evidence supporting higher recurrence rate after primary treatment of SCAs compared to other NFPAs (106), however the evaluated study samples included only a small number of patients who had been offered adjuvant radiotherapy.

Primary treatment of NFPAs with medical therapy is not currently recommended (15,85). Small series have demonstrated significant tumor volume stabilization under medical treatment in non-operated patients with NFPAs due to contraindications or refusal (110), whereas other studies have revealed tumor volume progression in the long term (111). The main medical agents that have been evaluated in NFPAs are dopamine agonists (DA) and somatostatin analogues (SAs), mainly in patients with residual tumor after transsphenoidal surgery. Figure 4 shows a suggested algorithm for the management of patients with NFPAs.

Figure 4 - Suggested algorithm for the management of patients with NFPAs. CBA: cabergoline; NFPA: non-functioning pituitary adenoma, MRI: magnetic resonance imaging; PRRT: peptide receptor radionuclide therapy RT: radiotherapy, *potential option, less evidenced-based approach; SSTR: somatostatin receptor

DOPAMINE AGONISTS

Dopamine receptor type 2 (D2R) expression has been demonstrated in patients with NFPAs. In a small series of 18 patients with hormone-negative NFPAs, two thirds of them (12/18) expressed D2R by real-time polymerase chain reaction. Patients who presented residual tumor (9/18) were treated with cabergoline up to 3mg per week. After 12 months of treatment, tumor shrinkage was observed in 56% of the patients and tumor reduction was significantly greater in those expressing D2R (112). A historical cohort analysis on the adjunctive role of DA in adult patients with GH and ACTH negative NFPAs showed favorable results (83). The treatment group consisted of patients who were either initiated on DA upon post-surgical residual tumor detection or when tumor growth was subsequently detected on follow-up, while the control group received no medication after surgical treatment. Tumor control was significantly superior in patients who were treated upon detection of post-operative residual tumor, compared to those who were treated after presenting tumor progression or the control group, 87% vs. 58% vs. 47%, respectively. The requirement for additional treatment (surgery and/or radiotherapy) during follow-up was significantly decreased from 47% to 16% with adjunctive DA therapy. In this series, there were no correlations between clinical response to DA and D2R expression, the isoform type or their expression levels.

A randomized open-label clinical trial was recently conducted aiming to compare cabergoline with non-intervention in 116 Brazilian patients with residual NFPA after transsphenoidal surgery followed-up for over two years. NFPAs were classified solely based on IHC for anterior pituitary hormones and included 59% hormone-negative adenomas and 34% silent gonadotroph adenomas. Silent corticotroph adenomas were excluded from this study. By the end of the study, residual tumor shrinkage was significantly more frequently observed in the medical-therapy group compared to patients in the control group (28.8% vs. 10.5%). Tumor response was not associated with D2R expression (113). Thus, although cabergoline has not been a consensual treatment for patients with NFPAs, a therapeutic attempt can be made according to clinical judgment in individual cases; particularly in patients with large extrasellar remnants after surgery and a high probability of tumor progression (114). On the other hand, most studies published so far included only patients with hormone-negative or silent gonadotroph adenomas, such that this proposition should not be extended to silent corticotroph adenomas or the other rarer silent tumors. It is also important to bear in mind the natural history of untreated NFPAs, which may show a spontaneous decrease in tumor volume in up to 30% of them(115).

Cabergoline doses are variable, but usually started at 0.5 mg weekly, increasing 0.5 mg each week until a maximum dose of 3.0 mg/week is reached. For these patients, tumor shrinkage is not the major target although it can occur in 38% of them; otherwise, prevention of tumor growth is the main treatment goal. Tumor progression can be assessed by a sellar MRI every six months for the first two years, and annually thereafter. Once stability is achieved without significant side effects, the drug can be maintained indefinitely, taking into account individual cost-benefits (82,116).

SOMATOSTATIN RECEPTOR LIGANDS 

The finding of somatostatin receptors (SSTRs) expression by NFPAs has raised the possibility that the use of somatostatin receptor ligands (SRLs) could be an effective treatment strategy (117,118). The SRL octreotide which binds with high affinity to SSTR2 was not effective in controlling tumor size or improving visual field in a small group of patients with NFPAs (119). A case-control study evaluated the results of long-acting octreotide in patients harboring post-surgical NFPA residues and it demonstrated tumor remnant stabilization in 81% (21/26) of patients in the treated group compared to 47% (6/13) of patients in the control group after a mean follow-up of 37 months (120). However, neither visual field nor pituitary function changed in any of the groups. This cohort of 39 NFPAs consisted of a heterogeneous group as IHC revealed positivity for pituitary hormones in 28 cases (20 of those showing positivity for FSH and/or LH) whereas the remaining 11 cases were negative for adenohypophyseal hormones. SSTR5 was the predominant SSTR expressed (84%), followed by SSTR3 (61%), while SSTR2 was expressed in 46% of the cases.

The expression of SSTRs and zinc finger protein 1 (ZAC1), a protein regulating apoptosis and cell cycle arrest, were assessed in a group of NFPAs (SGAs and hormone-negative adenomas), active somatotroph adenomas, and normal pituitary. SSTR2 and ZAC1 expression was reduced whereas SSTR3 expression was increased in SGAs compared to active somatotroph adenomas and normal pituitary (121). Likewise, other studies have suggested that SSTR3 is the predominant SSTR expressed in most NFPAs, both by IHC studies (118,122) and mRNA levels (11,122). However, a few other studies have demonstrated higher SSTR2 expression than SSTR3 or SSTR5 expression in SGAs and hormone-negative adenomas (103,123). Regarding corticotroph pituitary tumors, SSTR5 expression was significantly less frequently found in SCAs as compared to their secreting counterparts (2/23 vs. 24/39) (124). Furthermore, amongst the 23 SCAs, somatic ubiquitin-specific protease 8 (USP8) mutation was detected in only two tumors, in line with previous reports showing a significantly lower prevalence of USP8 mutations in SCAs compared to functioning corticotroph tumors (125,126).

There is an ongoing multicenter, randomized, double-blind, placebo-controlled trial (GALANT study) evaluating the effectiveness of first generation SRLs in patients with NFPAs and suprasellar extension, either surgery-naive or with a postoperative remnant. Forty-four patients with positive results on 68Ga-DOTATATE PET are being randomized to treatment with either the SRL lanreotide or placebo (127).

Pasireotide is a universal SRL with action on SSTR1, SSTR2, SSTR3 and STR5 subtypes and, therefore, seems more attractive than first-generation SRLs as an alternative medical treatment for NFPAs. A head-to-head comparison of octreotide and pasireotide in MENX-affected rat harboring a mutation I the gene encoding p27, an in vivo model of NFPAs, was recently performed. Pasireotide showed superior anti-tumor effect vs. octreotide, especially in females, which also showed higher SSTR3 expression (128). There are two phase 2 clinical trials evaluating the safety and efficacy of pasireotide for the treatment of NFPAs. Both trials have been recently completed but the results are not yet available. Passion 1 (NCT01283542) is an open-label single arm study evaluating tumor volume response to pasireotide in naive patients with NFPAs >1cm. Another phase 2 clinical trial (NCT01620138) is currently comparing the response of patients with NFPAs and surgical remnants to cabergoline vs. pasireotide. Notwithstanding, until further data are available, the use of SSRLs is not currently recommended for the treatment of patients with NFPAs.

Treatment of NFPAs with dopastatin, i.e., chimeric compound with dopamine and somatostatin agonist activity, has also been under investigation. BIM-23A760, a compound with potent agonist activity both at D2R and SSTR2 effectively inhibited cell proliferation in primary cultures of NFPAs (129). Interestingly, TBR-760 (formerly BIM-23A760)was recently tested in a mouse model of aggressive NFPA and resulted in nearly complete inhibition of tumor growth (130). A one year, randomized, double-blind, placebo-controlled phase 2 study of TBR-760 in adult patients with residual NFPAs > 1cm after transsphenoidal surgery (NCT04335357) is being conducted and is expected to be completed by 2023.

TEMOZOLOMIDE

Temozolomide (TMZ) was the first alkylating chemotherapeutic drug to show significant response rates in aggressive pituitary tumors (131). Notwithstanding, TMZ seems to be less effective in NFPAs compared to their secreting counterparts as 45% of 110 clinically functioning pituitary tumors showed regression on first-line TMZ while only 17% of 47 NFPAs did (132).

Responsiveness to TMZ is probably dependent on the immuno-expression of O (6)-methylguanine DNA methyltransferase (MGMT), a DNA repair protein that acts by removing the alkyl group and therefore is associated with TMZ resistance (133). Low immuno-expression of MGMT by pituitary tumors has been associated with higher response rates to TMZ (131). MGMT immuno-expression was assessed in a group of 45 NFPAs and the degree of expression was correlated with tumor aggressiveness (133). Low MGMT expression was observed in 50% of the aggressive NFPAs compared to 24% of the non-aggressive NFPAs. These findings suggest that aggressive NFPAs with low MGMT expression could be potential candidates for treatment with TMZ.

Regarding dosing regimens and indications, current guidelines suggest using TMZ (150-200 mg/m²daily for 5/28 days) as an alternative treatment for patients with aggressive NFPAs presenting tumor progression despite radiotherapy and other therapeutic measures, and for exceptional cases of pituitary carcinomas (29,132). Furthermore, in the case of rapid tumor growth in patients who have not previously reached maximal doses of radiotherapy, the use of the STUPP protocol (six weeks of concomitant fractionated radiotherapy and TMZ 75 mg/m² daily, followed by TMZ alone 150-200 mg/m² daily for 5/28 days) has been suggested (132). Studies on long-term administration of TMZ are scarce but clinical observations indicate that TMZ should be continued for as long as it is effective and well tolerated. Some authors suggest continuing TMZ at standard dosage for two years and later reducing to half-dose (134). Patients receiving TMZ chemotherapy are at risk for hematologic toxicity, occurring in approximately 15-20% of the cases. The most common non-hematologic side effects of TMZ are nausea, anorexia, and fatigue (135).

PEPTIDE RECEPTOR RADIONUCLIDE THERAPY

In vivo SSTR expression by NFPAs has been previously demonstrated both by positive uptake on somatostatin receptor scintigraphy (120) and on 68Gallium DOTATATE PET/ CT (79) providing a rationale for the administration of PRRT in these patients. So far, PRRT has been studied in few patients with aggressive pituitary tumors (clinically functioning and NFPAs) with different response patterns (136,137). Among the six patients with nonfunctioning pituitary adenoma or carcinoma, three patients (not previously treated with TMZ) showed stable disease (138-140), two patients had progressive disease (138,141) and one patient died in the following months while information on tumor volume was lacking (142).

QUALITY OF LIFE AND LONG-TERM MORBIDITY AND MORTALITY

A substantial number of patients with NFPAs suffer from morbidities related to the tumor itself, as well as to the treatments offered. Standardized mortality ratios in these patients seem to be higher than that of the general population with deaths associated mainly with circulatory, respiratory, and infectious causes (143). Until now, there was no consensus on predictive factors of mortality but those most consistently described are older age at diagnosis (144,145) and high doses of glucocorticoid replacement therapy (146). A retrospective series of 546 patients operated on for a macro NFPA between 1963 and 2011 and followed up for a median period of 8 years reported a standardized mortality ratio of 3.6 (95% CI, 2.9–4.5) (144). After adjustment for factors proven to be significant in univariate analysis - radiotherapy, tumor regrowth, and untreated growth hormone deficiency - age at diagnosis was an independent predictor of mortality, with shorter survival observed in older patients.

Following NFPA treatment, patient-reported health-related quality of life (HR-QoL) substantially improves.evertheless, there are conflicting findings about HR-QoL normalization, which may also be related to the lack of a disease-specific HR-QoL questionnaire for NFPAs (147). Some studies have described a persistent decreased HR-QoL compared to healthy controls and reference data (148,149), while others have not (150). In the latest study, HR-QoL was evaluated in 193 consecutive patients with NFPAs followed up in a tertiary endocrine referral center (150). The overall health-related quality of life and perception of subjective health in patients with NFPAs was not compromised although specific groups, such as females, patients with tumor recurrences, and with visual defects, were shown to be affected in various dimensions. Altered sleep-wake rhythmicity has also been described in a cohort of 69 patients with NFPA in long term remission after transsphenoidal surgery on stable replacement treatment for hypopituitarism (151). NFPA patients reported severely impaired QoL, sleep quality, and increased daytime sleepiness. Preoperative visual field defects were associated with sleep-wake rhythm fragmentation and vasopressin deficiency was associated with decreased sleep efficiency, independent of age, hypopituitarism, or radiotherapy.

More recently, the use of the Wilson–Cleary model, a conceptual biopsychosocial model of HR-QoL, suggested that elements at each stage of this model could be contributing to the impairment in HR-QoL observed in patients with a NFPA (147). The authors concluded that currently available biomedical treatments, i.e., surgery, radiotherapy, and hormone replacement therapy, are clearly not sufficient for achievement of good HR-QoL in patients with a NFPA, and further improvement should be supported by a pituitary specific care trajectory, targeting not only biological and physiological variables, but also psychosocial care.

PROGNOSTIC FACTORS AND FUTURE PERSPECTIVES

There has been a search to identify reliable factors related to aggressiveness and the risk of recurrence in NFPAs. A single-center retrospective study which evaluated 108 surgically-resected NFPAs followed for up to 15 years (152)showed that 22% of the patients required further treatment, either second surgery or radiotherapy. Factors determining recurrence were the presence of residual tumor, tumor growth rate (>80 mm³/year), and suprasellar extension (152). According to another retrospective case series evaluating patients with NFPAs who presented tumor regrowth after primary treatment, the NFPA subtype, categorized by anterior pituitary hormone immunostaining, was not a predictive factor for the requirement of secondary treatment or tumor regrowth. Significant risk factors were female gender and treatment approach (monitoring vs. interventions); secondary progression was significantly higher in those patients who were followed conservatively (63%) as compared to those who received surgery (36%), radiotherapy (13%), and surgery/adjuvant radiotherapy (13%) (84). In patients with SGAs, ERα seems to be a prognostic factor for re-intervention (reoperation or radiation) in males - the combination of the absence of ERα expression and young age served as good predictive markers of aggressiveness (122).

The role of cellular markers, associated with cell proliferation and apoptosis, in predicting the recurrence of NFPAs has also been investigated (153). Proliferative indexes such as a high Ki-67 index, assessed by IHC, were significantly associated with a tumor size greater than 3 cm, as well as with tumor recurrence (103). Evaluation of tumor proliferation by using Ki-67 IHC is widely available and is recommended as part of the assessment of NFPAs (40). According to a large retrospective series evaluating 601 patients with surgically resected pituitary tumors, including approximately 30% NFPAs, the optimal cut-off points of the Ki-67 proliferation index that predicted recurrence was 2.5% with 84.6% sensitivity and 47.4% specificity (154). Moreover, a recent retrospective analysis of 120 patients operated on for an NFPA showed that invasive and proliferative tumors, i.e., grade 2b tumors. according to the clinicopathological classification of Trouillas et al. (155) showed an overall likelihood of recurrence that was approximately 9 times greater than those of grade 1a, i.e., non-invasive and non-proliferative tumors (156). Further risk factors associated with an increased risk of recurrence in this cohort were a younger age and the presence of residual tumor.

Minichromosome maintenance 7 (MCM7), a cell-cycle regulator protein, has been recently proposed as a marker of tumor progression in NFPAs. In a cohort study of 97 surgically treated NFPAs, the probability for reintervention within 6 years for patients harboring residual tumors with high MCM7 expression was 93% (157). Ki-67 expression >3%, age ≤55 years and mitotic index≥1, but not tumor subtype, were also associated with reintervention. Attempts to develop clinical nomograms to predict post-operative recurrence of NFPAs have recently been demonstrated based on age, tumor size, cavernous sinus invasion, sphenoid sinus invasion, and surgery extension. The nomogram model proposed by Lyu et al. (158) showed an area under the ROC curve of 0.953 and correlation analyses indicated that sphenoid sinus invasion, cavernous sinus invasion and tumor size could promote the recurrence of NFPA while advanced age and gross total resection could effectively inhibit it.

Genetic and epigenetic mechanisms underlying the development and aggressiveness of NFPAs have not been fully elucidated (159). The mutational landscape of a cohort of pituitary tumors including 37 NFPAs was recently published (160). Aside from demonstrating gonadotroph signatures in seven out of eight SCAs, retrieving the question whether a subset of SCAs arise from a specific pituitary lineage distinct from other corticotroph (161), this study showed that most NFPAs displayed no functional somatic variant and no chromosome alterations. Further characterization of these tumors with techniques such as whole genome sequencing coupled to chromatin structure analysis may disclose mutations in non-coding regions affecting chromatin opening and/or the binding of specific transcription factors. allowing for the development of novel therapeutic strategies.

An adverse pituitary adenoma phenotype is defined not only by the intrinsic activity of tumor cells but also by the infiltrated immune cells in the tumor microenvironment (162). The study of the immune profile of pituitary tumors for predicting immunotherapy responsiveness is an evolving field. Wang et al (163) have recently proposed classification of these tumors on three clusters based on tumor-infiltrating immune cells and the expression of immune checkpoint molecules This study cohort included 57 “unspecified“ NFPAs since they were not pathologically classified with pituitary transcription factors. The majority of them exhibited a “hot” immune microenvironment and were predicted to exhibit higher immunotherapy responsiveness.

CONCLUSION

NFPAs are frequent in endocrine practice. Clinically they range from being completely asymptomatic (incidental findings on head MRI or computed tomography scans) to causing significant hypothalamic/pituitary dysfunction and visual symptoms due to mass effect., For microadenomas and asymptomatic/relatively small macroadenomas (1–2 cm), a “watch and wait” option is reasonable. If tumor growth, development of visual field defects, or progressive pituitary dysfunction is detected during follow up, then surgery is indicated. It is now recommended that NFPAs are classified according to their pituitary hormone and transcription factor profiles along with proliferation markers such as Ki-67. This refined stratification may potentially aid in predicting disease course and in selecting adjunctive therapies. Radiotherapy is an effective treatment, although usually reserved for those patients with aggressive tumors and significant tumor remnant after pituitary surgery as considerable side effects may occur. Medical treatment with dopamine agonists stands as an alternative in selected cases, despite the lack of placebo-controlled trials. Highly aggressive tumors need special care during follow-up, including treatment with TMZ with or without RT complementation or new potential emerging treatments. Close collaboration of a multidisciplinary pituitary team is crucial to better serve this challenging group of patients.

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