Oxidative Phosphorylation Patterns in Pituitary Adenoma/Neuroendocrine Tumors

Oxidative Phosphorylation Patterns in Pituitary Adenoma/Neuroendocrine Tumors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oxidative Phosphorylation Patterns in Pituitary Adenoma/Neuroendocrine Tumors Maaia Margo Jentus, René Feichtinger, Willem Corver, Sara Huber, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8475859/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Mar, 2026 Read the published version in Pituitary → Version 1 posted 7 You are reading this latest preprint version Abstract Purpose Pituitary neuroendocrine tumors (PitNETs), also known as pituitary adenomas, exhibit marked lineage-specific heterogeneity. The underlying molecular biology of certain tumor types, particularly gonadotroph tumors (SF1-lineage) — which typically exhibit stable genomes — remains poorly understood. This study aimed to define oxidative phosphorylation (OXPHOS) system patterns across PitNET lineages. Methods Immunohistochemistry was performed in 43 previously molecularly and histologically classified PitNETs on tumor and normal adenohypophyseal tissue for VDAC1 (porin) to assess mitochondrial density and OXPHOS subunits of complexes I–V. Quantified staining intensity scores were used for statistical analyses, and mtDNA sequencing was successful in 21 tumors. Results Mitochondrial density was significantly increased in PitNETs compared with normal tissue. OXPHOS alterations were non-uniform: complex I deficiency was the most frequent abnormality, often associated with disruptive mtDNA mutations, particularly in genomically stable gonadotroph tumors. Two corticotroph tumors with near-haploid genomes also harboured disruptive complex I mutations. Alterations in other complexes were less common and typically occurred in combination. Staining heterogeneity was frequent (24/43 tumors), including focal expression loss, especially in SF1-lineage and all mtDNA-mutated tumors, but also present in tumors without mtDNA mutations. Conclusions PitNETs display lineage-specific and highly heterogeneous OXPHOS phenotypes. Complex I deficiency and mtDNA mutations occur not only in genomically stable gonadotroph tumors but also in highly disrupted corticotroph tumors with a near-haploid genome. Further studies including sequencing of nuclear-encoded OXPHOS-related genes are required to clarify the contribution of OXPHOS and mitochondrial pathways to PitNET biology and potential clinical applications. oxidative phosphorylation pituitary adenoma PitNET Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Pituitary Neuroendocrine Tumors (PitNETs), also known as pituitary adenomas, are increasingly diagnosed, with a prevalence of 78–116 cases per 100 000 people in the last 15 years [ 1 ]. While some PitNET types mostly treated with medication, surgical resection is performed for approximately a half of clinically diagnosed PitNETs. Among those > 40% are non-functional gonadotroph tumors (SF1 lineage), ~ 15% are corticotroph tumors (TPIT lineage), and ~ 30% are tumors of PIT1 lineage. Recently, we explored chromosomal alteration patterns in diverse PitNET subtypes due to relatively low occurrence of somatic mutation hallmarks in sporadic tumors, confirming previous observations [ 2 – 5 ]. Tumors with aggressive clinical behaviour showed massive chromosomal losses (TPIT-lineage) which are likely to be mutually exclusive with USP8 -mutations. Tumors of PIT1-lineage mostly showed complex patterns of chromosomal losses and gains, with the mechanisms still to be elucidated. Gonadotroph tumors (SF1-lineage) do not cause hormone excess and usually present as large tumors causing mass effects, often require multiple operations due to difficulty of complete initial surgical resection. These tumors mostly show stable genome and no somatic mutations, challenging the search for biomarkers and therapeutical targets. Prior to introduction of systematic staining for transcription factors, a subset of PitNETs was classified as pituitary oncocytoma/oncocytic null-cell adenomas (WHO Tumor Classification 2017 and prior), which most likely was represented by gonadotroph tumors [ 6 ]. In this historical tumor type, mtDNA mutations in complex I of oxidative phosphorylation (OXPHOS) were repeatedly reported [ 7 – 9 ]. Kurelac et al. reported a series of 48 pituitary adenomas, in which a correlation was found between CI disruptive mutations, the oncocytic phenotype and low number of chromosomal aberrations [ 8 ]. Complex II alterations are known drivers in subset of tumors, which occur mostly in the context of SDH-deficient tumour syndrome (hereditary phaeochromocytoma-paraganglioma syndromes), and are less detected sporadically [ 1 , 10 , 11 ]. Abnormal mitochondria and oncocytic phenotype are often described as going hand in hand [ 12 ]. It is worth to mention that oncocytic changes also occur in the normal adenohypophysis [ 13 ]. Furthermore, there is room for subjective interpretation in the definition of oncocytic phenotype. While oncocytes have big eosinophilic cytoplasm, not all cells with such cytoplasm are true oncocytes. The eosinophilic appearance in some lesions may be attributed to the accumulation of various cellular components (e.g. lysosomes, endoplasmic reticulum, neuroendocrine granules), rather than an increased number of deviant morphology of mitochondria [ 14 ]. Immunohistochemistry for subunits of the respiratory chain can be utilized to visualize mitochondria [ 12 ]. The oncocytic features with corresponding abnormalities in mitochondria are widely described in other endocrine and non-endocrine organs [ 12 , 15 , 16 ]. For PitNETs, the situation is likely to be more even complex, as the volume of mitochondria is different among different subtypes in pituitary adenomas; for example, the volume of mitochondria in prolactinomas is larger than those in growth hormone producing tumors [ 17 ]. Somatotroph tumors with oncocytic cells show similar cytokeratin patterns and higher proliferative activity, which is not correlated with local aggressiveness [ 18 ]. Acidophilic stem cell tumors are per definition oncocytic [ 1 ]. Anyways, there is growing consideration of mitochondria as probable therapeutic target in PitNETs as mitochondrial alterations have commonly been recognized in these tumors [ 19 – 21 ]. The OXPHOS system within mitochondria represent the main source of energy (ATP) under aerobic conditions in eukaryotic cells [ 22 ]. The core elements are five complexes (I-V) consisting of diverse amount of subunits and organized in supercomplexes, embedded in the inner mitochondrial membrane [ 23 ]. Even though mitochondrial DNA (mtDNA) codes only for 13 proteins all of which are subunits of OXPHOS complexes, the mtDNA is prone to mutations. Still, most of the subunits of the OXPHOS system are encoded via the nuclear genome and are imported into mitochondria where large multi-subunit complexes are assembled. Beside those, the whole orchestra of supporting proteins and signalling systems are necessary for proper function of OXPHOS system. Beyond the roles in energy production, participation in cell signalling, regulation of apoptosis, mitochondria continuously gain attention in the context of tumorigenesis. In this study, we explored mitochondrial density, mitochondrial DNA mutation status and expression patterns of OXPHOS system complexes in a previously molecularly defined and subtyped PitNET cohort [ 2 ]. MATERIALS AND METHODS Sample collection Material from 43 sporadic PitNETs previously described by our group was available for additional analyses, with case numbering consistent the earlier study [ 2 ]. Adjacent normal adenohypophyseal tissue was available in nine cases. All analyses were covered by the ethical approval of the original study (Medical Ethics Review Committee Leiden, G19.011). Assessment of oncocytic phenotype Haematoxylin and eosin-stained sections were independently evaluated for oncocytic features by two pathologists (MJ and HM). Tumors were categorized as non-oncocytic; with oncocytic-metaplasia like features, or oncocytic. Oncocytic features were defined by abundant, voluminous granular eosinophilic cytoplasm, sharp cytoplasmic borders, a low nuclear-to-cytoplasmic ratio, and round, centrally located nucleus with evenly distributed chromatin and prominent nucleolus. Oncocytic metaplasia-like features were assigned when tumor cells exhibited some, but not all oncocytic features, or when oncocytic features were present only focally. Non-oncocytic phenotype lacked the characteristic cytoplasmic and nuclear features of oncocytes. In cases of discrepant scoring, consensus was reached by joint review. Immunohistochemistry for mitochondrial density and subunits of OXPHOS system Materials and considerations Immunohistochemistry was performed for subunits of the OXPHOS system and for porin (VDAC1) as described previously [ 7 ]. Immunohistochemical staining of porin (VDAC1) was performed to explore potential alterations in mitochondrial biogenesis within PitNETs, both intratumorally and compared to normal adenohypophyseal tissue. Porin is a protein in the outer mitochondrial membrane that is frequently used as a marker for mitochondrial mass [ 24 – 26 ]. The following primary antibodies were used: complex I (rabbit anti-NDUFB8, Abcam, ab192878; 1:500, 1 h), complex II (mouse anti-SDHA, Abcam, ab14715; 1:2000, 1 h), complex III (mouse anti-UQCRC2), Abcam, ab14745; 1:1000, 1 h), complex IV (mouse anti-MT-CO1), Abcam, ab14705; 1:1000, 1 h), complex V (mouse anti-ATP5F1A, Abcam, ab14748; 1:2000, 1 h), and porin (mouse anti-VDAC1, Abcam, ab14734; 1:1000, 1 h). Scoring Scoring and statistical analyses were performed as previously described [ 26 , 27 ]. Immunohistochemical expression levels in tumor tissue were compared with adjacent pre-existent tissue when available, and otherwise with the mean expression levels observed in normal adenohypophyseal tissue across cases. Briefly, immunohistochemical scores were calculated by multiplying the staining intensity (range 0–3, with half-point increments) by the percentage of tissue area exhibiting that intensity. Quantification was performed independently by two observers (MJ, HM), and the mean interobserver score was used for all analyses. Assessment of heterogeneity Heterogeneous or patchy staining patterns and focal loss of expression were frequently observed in tumors. To semi-quantify intratumoral heterogeneity, variability in staining intensity was first assessed in normal adenohypophyseal tissue by calculating the 95% confidence interval (CI) of the mean score for each marker. Tumors with optically variable staining were classified as heterogenous for a given staining when the intratumoral range of staining scores — defined as the maximum minus the minimum observed score — exceeded the width of the 95% CI derived from normal adenohypophyseal tissue. Definition of altered expression Increased or decreased immunohistochemical expression in tumor tissue was defined as a relative change of 25%, 50%, or 75% in the score value compared with the mean score of normal tissue or the corresponding normal tissue, when available. In tumors with detected mtDNA mutations and heterogeneous staining patterns, the tumor area corresponding to the observed heteroplasmy percentage of mutation. For these cases, the ratio was calculated between expression levels in the mutation-associated region and adjacent tumor tissue. Additional molecular studies DNA extraction Fresh-frozen material was available for 21 samples, from which DNA was isolated using ten 20-µm cryosections per case. For an additional 21 tumors, as well as two cases with suboptimal fresh-frozen material on visual inspection, previously extracted DNA from formalin-fixed, paraffin-embedded tissue was used, as described previously [ 2 ]. mtDNA sequencing Long-range PCR amplification MtDNA was amplified from proteinase K-digested fresh-frozen tumor tissue using long-range PCR. For each sample, two PCR reactions were performed using primer sets (Microsynth): primer set A: (forward 5’-CACCAGCCTAACCAGATTTCA-3’; reverse 5’-TGGTACCCAAATCTGCTTCC-3’) and primer set B (forward 5’-GGCTCACATCACCCCATAAA-3’; reverse 5’-CGTGTGGGCTATTTAGGCTTT-3’). Each PCR reaction contained 1 µl of digested tumor sample, 1x LongAmp-Taq (New England Biolabs, NEB), and primers at a final concentration of 0,4 µM. Initial heating to 94°C for 30 seconds, was followed by 40 cycles of 94°C for 30 seconds, 58°C for 60 seconds, and 68°C for 13 minutes. PCR products were purified using the Monarch® PCR & DNA cleanup Kit 5 µg (New England Biolabs, NEB) according to the manufacturer’s instructions. Long-read mtDNA sequencing Long-read mtDNA sequencing library was prepared using the Oxford Nanopore Technologies Native Barcoding Kit (SQK-NBD114.24). For each sample, 500 ng of each long-range PCR product were mixed with 0,875 µl NEBNext Ultra II End-prep Reaction Buffer (NEB), 0,75 µl NEBNext Ultra II End-prep Enzyme Mix (NEB) and nuclease-free water to a total volume of 15 µl. The sample was incubated at 20°C for 5 minutes followed by 65°C for 5 minutes. Each sample was mixed with 1x AMPure XP Beads and incubated for 5 minutes on a rotator mixer. The beads were washed twice with 200 µl of 80% ethanol and the sample was eluted in 10 µl nuclease-free water. 7.5 µl of each end-prepped sample were mixed with a unique barcode and 10 µl Blunt/TA Ligase Master Mix (NEB). The reaction was incubated for 20 minutes at RT and stopped by adding 4 µl of 0.25 M EDTA. Barcoded samples were pooled, and 0.4x AMPure XP Beads were added. The sample was incubated for 10 minutes on a rotator mixer and 700 µl of 80% ethanol were used for washing the beads twice. To elute the DNA from the beads, 35 µl nuclease-free water were added and the beads were incubated at 37°C for 10 minutes and every 2 minutes the beads were gently mixed. The beads were pelleted on a magnet and 35 µl of barcoded sequencing library removed. Sequencing adapters were ligated by mixing 30 µl of the barcoded sequencing library with 5 µl Native Adapter, 10 µl NEBNext Quick Ligation Reaction Buffer (NEB) and 5 µl Quick T4 DNA Ligase (NEB) and incubation for 20 minutes at RT. The library was purified by adding 0.4x AMPure XP beads and incubating the reaction for 10 minutes on a rotator mixer at RT. The beads were washed twice by resuspending in 125 µl Long Fragment Buffer, pelleting the beads and removing the supernatant. Finally, the sequencing library was eluted in 35 µl Elution Buffer by incubation for 10 minutes at 37°C and flicking the beads every 2 minutes to mix. Sequencing was performed on a P2 Solo sequencing device using FLO-PRO114M flow cells. Basecalling, demultiplexing, and alignment against human reference genome (GRCh38) were performed in real time using MinKNOW (v.24.02.6). BAM files were sorted and indexed using SAMtools (v.1.18) [ 28 ], and variant calling was performed with freebayes (v.1.3.6) [ 29 ]. Evaluation of pathogenicity of detected mtDNA variants The pathogenicity of detected mtDNA variants was evaluated based on population frequency and existing annotations in the MitoMap database ( https://www.mitomap.org ), which integrates data from gnomAD, GenBank, and Helix. MitoMap annotations were used to identify previously reported variants and their known or suggested pathogenic relevance. For novel mtDNA variants, in silico pathogenicity prediction was performed using multiple tools, including Apogee2, Hmtvar, AlphaMissense, BayesDel_addAF, DEOGEN2, LIST_S2, MutationAssessor, PhyloP100, PROVEAN, Sift4G, GERP RS, and Varity_R. Statistical methods All statistical analyses were performed using GraphPad Prism version 10.2.3 (GraphPad Software, USA). Given the limited sample size, the study was exploratory in nature and primarily relied on descriptive statistics, with inferential analyses interpreted cautiously. Normality of staining intensity distributions in grouped samples was assessed using the Shapiro–Wilk test. For comparisons between groups, Welch’s t-test was applied when data approximated a normal distribution, whereas the Mann–Whitney U test was used for non-normally distributed data. Associations between VDAC1 expression, oncocytic phenotype, and mtDNA mutation status were explored using Spearman rank correlation, simple linear regression, and logistic regression, as appropriate. All tests were two-sided, and p values < 0.05 were considered statistically significant. RESULTS An overview of the study cohort, including immunohistochemistry and summarized molecular data, is presented in Fig. 1 . Immunohistochemistry for VDAC1 and OXPHOS system subunits was successful on 43 tumors, comprising 20 PIT1-lineage, 13 SF1 lineage, 9 TPIT-lineage, and one multilineage tumor (SF1/PIT1-lineages). Additional mtDNA analysis was successful in 21 cases, of which nine harboured mtDNA mutations (Table 1 ). Mitochondrial density Mitochondrial density, assessed by porin (VDAC1) expression, was significantly higher in tumor tissue (162.3 ± 29.2) compared with normal adenohypophyseal tissue (111.1 ± 22.1; p < 0.0001). Thirty-eight of 43 tumors (88.4%) showed mean intratumoral VDAC1 staining intensity above the normal range. Heterogenous/patchy staining Of 43 tumors, 24 showed heterogenous/patchy staining pattern (Fig. 2 ), whereas 17 showed homogenous staining. Two samples were too small to assess heterogeneity (1xSF1 and 1xTPIT lineage). Among 24 tumors with heterogeneous staining, 23 were patchy in 2 or more stainings, and four tumors demonstrated a heterogeneous pattern in all six stainings. Heterogenous staining was most frequently observed for SDHA (CII, 19/24), followed by NDUFB8 (CI, 17/24), ATP5F1A (CV, 16/24), VDAC1 (porin, 15/24), MT-CO1 (CIV, 9/24), and UQCRC2 (CIII, 5/24). Staining heterogeneity was most prevalent in SF1-lineage tumors (11/12). Three of eight TPIT-lineage tumors showed heterogeneous staining. In contrast, approximately half of PIT1-lineage tumors (12/23) showed a homogenous staining. (a) Case 34 (b) (SF1-lineage), m.11038del (91%) detected. (c) Case 41 (d) (SF1-lineage), no mtDNA mutations detected. Magnification x10. Complex I (subunit NDUFB8) Reduced NDUFB8 expression was observed in 12 tumors (8xSF1, 2xTPIT, 1xPIT1, 1xSF1/PIT1). In eight cases, the deficiency was isolated, whereas four tumors showed combined deficiencies involving other OXPHOS complexes (CII in two cases, CIII in two, CIV in four, and CV in one). Heterogeneous staining was present in 10/12 tumors with reduced NDUFB8 expression. Among the eight tumors harbouring mtDNA mutations affecting complex I, six showed markedly reduced NDUFB8 expression (≤ 50% of normal tissue), while two displayed a moderate reduction (25–49%). Reduced NDUFB8 expression was also observed in two tumors without detectable mtDNA mutations and in two tumors for which mtDNA sequencing was unsuccessful. No tumor showed increased NDUFB8 expression. Complex II (subunit SDHA) Isolated SDHA deficiency was observed in two tumors (1xPIT1, 1xSF1), while two tumors showed combined deficiencies (1xSF1, 1xSF1/PIT1); all four demonstrated heterogeneous SDHA staining. In addition, heterogeneous SDHA staining with overall intensity comparable to normal tissue was observed in 15 additional tumors across all lineages. Upregulated SDHA expression was found in 4 tumors (3xTPIT, 1xSF1). Ot these, three showed no deficiencies in other OXPHOS complexes, whereas one (case 30, mtDNA-mutated SF1-lineage tumor) exhibited CI-deficiency and co-upregulation of CIII-expression. Complex III (subunit UQCRC2) Reduced UQCRC2 expression occurred only in tumors with combined deficiencies, as described above. Upregulated UQCRC2 expression was identified in eight tumors, two of which with co-occurrent CI-deficiency. Heterogeneous staining with overall intensity comparable to normal tissue was observed in four tumors (3xSF1, 1xTPIT), none in PIT1-lineage. Complex IV (subunit MT-CO1) Reduced MT-CO1 expression was observed in three tumors (2xSF1, 1xPIT1), exclusively in combination with CI-deficiency. In two of these cases, co-occurring with heterogeneous staining pattern and deficiencies of additional OXPHOS complexes. No tumor demonstrated increased MT-CO1-expression. Heterogeneous staining with overall intensity comparable to normal tissue was observed in seven tumors (4xSF1, 3xTPIT) and was absent in PIT1-lineage. Complex V (subunit ATP5F1A) Reduced ATP5F1A expression was observed in a single tumor (case 10, SF1-lineage), occurring as a part of a combined deficiency involving CI, CIII, and CIV and accompanied by heterogeneity across all stainings. Nine tumors (5xPIT1, 3xTPIT, 1xSF1) showed increased ATP5F1A expression. Heterogeneous staining with overall intensity comparable to normal tissue was observed in 14 tumors, across all lineages. In multiple linear regression analysis, SDHA (p = 0.004) and ATP5F1A expression (p = 0.03) were independently associated with higher VDAC1 expression, reflecting increased mitochondrial density. In contrast, NDUFB8 (p = 0.02) and MT-CO1 (p = 0.02) showed inverse associations. Lineage-specific expression patterns and the overall distribution of immunohistochemical staining intensities are illustrated in Fig. 3 A, showing tumor-to-normal expression ratios for subunits of CI-CV and VDAC1. Tumor-to-normal staining intensity ratios stratified by lineage. The dashed line at 1.0 indicates parity with normal tissue. A lineage-specific trend toward reduced NDUFB8 expression (CI) is observed in SF1-lineage tumors, whereas VDAC1 expression, reflecting mitochondrial density, is increased across all lineages. Intratumoral staining heterogeneity in seven tumors harbouring mtDNA mutations affecting complex I. Ratios represent staining intensity in regions corresponding to the heteroplasmic mutation relative to other regions within the same tumor. mtDNA sequencing mtDNA sequencing was successful in 21 tumors, of which nine harboured mtDNA mutations (Table 1 ). Two novel missense and one novel loss-of-function variant were identified. The MT-ND1 m.3631T > C variant results in substitution of a highly conserved serine residue within a transmembrane domain (p.Ser109Pro), spanning amino acid residues 100–120. The variant was absent from gnomAD 4.1 and MitoMap. According to ACMG criteria, it was classified as a variant of uncertain significance (PM2 moderate, PP3 supporting). Multiple in silico prediction tools support a pathogenic effect (Apogee2, Pathogenic, 0.87; Hmtvar, Pathogenic, 0.84; AlphaMissense, Pathogenic, 0.90; BayesDel_addAF; Uncertain; 0.057, T; DEOGEN2, Benign, 0.23, T; LIST_S2, Uncertain, 0.95, D; MutationAssessor, Pathogenic, 4.3, H; PhyloP100, 4.8; PROVEAN, Pathogenic, -4.6, D; Sift4G, Pathogenic, 0.0010, D; GERP RS, 4.5; Varity_R, 0.95). The MT-ND4 m.11484G > A variant was detected in one tumor and affects a highly conserved glycine residue within a transmembrane domain (p.Gly242Asp), spanning residues 224–244. This variant has been reported at relatively low heteroplasmy (25%) in one individual in gnomAD but was absent in gnomAD 4.1 and MitoMap. In the tumor sample, heteroplasmy reached 84%. According to ACMG criteria, this variant was also classified as of uncertain of uncertain significance (PM2 moderate, PP3 supporting), with the majority of in silico tools (7/11) predicting a damaging or pathogenic effect (Apogee2, Pathogenic, 0.74; Hmtvar, Pathogenic, 0.88; AlphaMissense, Pathogenic, 1.0; BayesDel_addAF, Benign, -0.19, T; DEOGEN2, Uncertain, 0.44, T; LIST_S2, Uncertain, 0.93, D; MutationAssessor; Pathogenic, 5.2, H; PhyloP100, 9.4; PROVEAN, Pathogenic, -6.0, D; Sift, Pathogenic, 0.0, D; Sift4G, Pathogenic, 0.0, D; GERP RS, 5.1; Varity_R, 0.97). The novel loss-of-function variant in MT-ND2 (m.5366_5367del; c.896_897del) causes a frameshift leading to a premature stop codon (p.Ser299TyrfsTer10). The variant was absent from gnomAD and MitoMap. The remaining pathogenic mtDNA variants identified in this cohort had been previously reported (Table 1 ). An overview of all 161 detected mtDNA variants across the 21 PitNETs is provided in Supplemental Material. Table 1 All detected mtDNA mutations. Case (Lineage) mtDNA variant Mutant load (%) Effect Affected gene(s) OXPHOS system complex Reference/prior report 8 (SF1) m.10158T > C 46 p.Ser34Pro MT-ND3 C I Pathogenic in neurological disease [ 30 , 31 ] 9 (SF1) m.4412G > A 38 tRNA DHU stem MT-TM C I, C III, C IV, C V Pathogenic in neurological disease [ 32 , 33 ] 10 (SF1) m.12923G > A 31 Stop MT-ND5 C I Likely pathogenic in neurological disease [ 34 ] 30 (SF1) m.11484G > A 84 p.Gly242Asp MT-ND4 C I https://reg.genome.network/redmine/projects/registry/genboree_registry/alleles?refseq=NC_012920.1&begin=11481&end=11581&skip=0&limit=50 32 (SF1) m.4436_16463del 41 Deletion MT-ND1, MT-ND2, MT-ND3, MT-ND4L, MT-ND4, MT-ND5, MT-CYB, MT-CO1, MT-CO2, MT-CO3, MT-ATP6, MT-ATP8 C I, C III, C IV, C V Large deletions reported in human cancers [ 35 , 36 ]. m.4974G > A 36 Stop MT-ND2 CI Variant of uncertain significance [ 37 ] or pathogenic [ 38 ] 34 (SF1) m.11038del 91 Frameshift MT-ND4 CI Kidney oncocytoma [ 39 ], oncocytic thyroid carcinoma [ 40 ], adrenal oncocytoma [ 7 ] 51 (SF1) m.12425del 90 Frameshift MT-ND5 CI IHC-negative oncocytic pituitary adenoma [ 8 ], nasopharyngeal oncocytic tumor [ 41 ] 52 (TPIT) m.3631T > C 23 p.Ser109Pro MT-ND1 CI dbSNP: rs1603219053 56 (TPIT) m.5366_5367del 42 Frameshift MT-ND2 CI This study All tumors with detected mtDNA mutations showed regions of reduced NDUFB8 expression by immunohistochemistry, accompanied by a complementary increase in mitochondrial density (Fig. 4 ). Intratumoral staining variability in tumors harbouring complex I mtDNA mutations with pronounced staining heterogeneity is visualised in Fig. 3 B. SF1-lineage, a-g: (a) Tumor 8, stable genome. The tumor tissue mostly showed preserved expression of NDUFB8 with focal deficiency not corresponding with 46% of mutational load (heteroplasmy), probably due to intratumoral variation in tissue obtained for DNA-isolation and stained tissue. The evident focal CI-deficiency with increased mitochondrial density in VDAC1 is shown. (b) Tumor 9, stable genome. The only tumor with mutation affecting MT-TM (mitochondrial tRNA required for OXPHOS complexes I, III, IV, and V). This mutation has been reported to induce a pattern of OXPHOS system multicomplex deficiency [ 32 ], as also observed in this tumor tissue. (c) Tumor 10 with chromosomal imbalance due to copy-number gains on 7p and 7q. (d) Tumor 30, stable genome. (e) Tumor 32, stable genome. (f) Tumor 34, stable genome. (g) Tumor 51, stable genome. TPIT-lineage, h-i: (h) Tumor 52 with near-haploid genome with LOH on multiple chromosomes due to whole chromosome loss and expected endoreduplication. (i) Tumor 56 with near-haploid genome with LOH on multiple chromosomes due to whole chromosome loss and detected endoreduplication. Oncocytic phenotype Among the 43 tumors, ten were classified as oncocytic, 23 showed oncocytic metaplasia-like features, and 10 lacked oncocytic features. Neither correlation nor regression analyses demonstrated association between oncocytic morphology and mitochondrial density as assessed by VDAC1 expression. Lineage-specific analyses likewise revealed no significant relationship between VDAC1 staining intensity and oncocytic features. The presence of mtDNA mutations was also not associated with oncocytic morphology or VDAC1-defined mitochondrial density. Given the absence of correlation between oncocytic appearance and mitochondrial mass, representative cases are shown to illustrate this discrepancy (Fig. 5 ). Both, non-oncocytic and tumors with oncocytic features showed variable VDAC1 intensity. These examples highlight that oncocytic morphology is an unreliable surrogate for mitochondrial density, and vice versa. Additionally, no significant association was observed between genome stability (absence of chromosomal alterations) and oncocytic features or mitochondrial density. (e) Tumor 41 with oncocytic features, VDAC1 intensity ranging from 175 to 200 (shown). No mtDNA mutation detected. DISCUSSION Previously, we explored chromosomal alteration patterns across all lineages of pituitary neuroendocrine tumors (PitNETs), also known as pituitary adenomas[ 2 ]. Gonadotroph tumors (SF1-lineage) – typically occurring in older patients and presenting as larger, invasive tumors – showed almost no chromosomal alterations and were characterized by a stable genome or only chromosomal imbalances due to copy-number gains, suggesting biological characteristics distinct from other PitNET lineages. Although several PitNETs displayed oncocytic features, no clear relationship with chromosomal alteration patterns could be established, in contrast to oncocytic thyroid lesions [ 42 ]. Kurelac at al. previously investigated pituitary adenomas in the context of oxidative phosphorylation (OXPHOS) system, reporting stable genome or copy-number gains in tumors with oncocytic phenotype (formerly termed pituitary oncocytoma/oncocytic adenoma), together with frequent disruptive mtDNA mutations affecting complex I [ 8 ]. Following the revision of PitNET nomenclature [ 1 ] by introduction of transcription factors, the entity of pituitary oncocytic adenoma was abandoned, and reassessment of these cases allows only limited extrapolation to current lineage definitions. These earlier findings motivated us to investigate OXPHOS system alterations in a molecularly defined and contemporary subtyped PitNET cohort. We found that PitNETs commonly show increased mitochondrial density, as reflected by elevated VDAC1 expression, compared to normal adenohypophyseal tissue. However, this increase was not accompanied by uniform up- or downregulation of OXPHOS system components. Instead, increased SDHA (subunit of CII) and ATP5F1A (subunit of CV) expression was positively associated with mitochondrial mass, whereas NDUFB8 (subunit of CI) and MT-CO1 (subunit of CIV) correlated inversely. This pattern suggests that mitochondrial expansion in PitNETs may reflect compensatory metabolic adaptation, with induction of complexes II and V in the context of impaired or depleted complexes I and IV. Complex I deficiency and mtDNA mutations were observed predominantly in SF1-lineage tumors, followed by TPIT-lineage tumors, whereas no mtDNA mutations were detected in PIT1-lineage – likely reflecting selection bias. Kurelac et al. reported disruptive CI mtDNA mutation (m.11832G > A) in an oncocytic tumor with TSH/PRL-expression (likely PIT1-lineage under current classification) accompanied by copy-number losses along chromosome 13 [ 8 ]. Another oncocytic tumor without hormone expression harboured a different disruptive complex I mtDNA mutation (m.11873insC), also co-occurring with copy-number losses along chromosome 13. A third oncocytic growth hormone-expressing tumor (again, likely PIT1-lineage) showed copy-number losses across chromosomes 10, 13, and 16, harboured GNAS mutation (c.2530C > T; p.Arg844Cys), and lacked mtDNA mutations. Together, these cases illustrate that the relationship between oncocytic morphology, genome stability, and mitochondrial mutations is not straightforward. Our findings extend these observations by identifying disruptive complex I mtDNA mutations in two TPIT-lineage tumors, which show highly unstable near-haploid genome extensive loss of heterozygosity of multiple chromosomes due to whole chromosome loss. This observation underscores the biological diversity of PitNETs and indicates that mitochondrial dysfunction is not restricted to genomically stable tumors. Despite the frequent presence of oncocytic features across all lineages, neither mitochondrial mass nor mtDNA mutation status correlated with oncocytic morphology, and we therefore could not confirm a previously assumed association between mtDNA mutations and the oncocytic phenotype [ 9 ]. OXPHOS system abnormalities showed a strikingly non-uniform pattern. While complex I deficiency — often associated with mtDNA mutations — was the most frequent alteration, deficiencies of complexes II–V were uncommon and typically occurred as part of combined multi-complex defects. Reduced MT-CO1 (subunit CIV) expression was rare and consistently accompanied complex I deficiency. Upregulation of SDHA (subunit CII), UQCRC2 (subunit CIII), and especially ATP5F1A (subunit CV) was observed in subsets of tumors across all lineages. To our knowledge, this study provides the first detailed description of extensive intratumoral heterogeneity of OXPHOS system complex expression in PitNETs. Heterogeneous immunohistochemical staining — previously described in papillary thyroid carcinoma and non-neoplastic tissue [ 25 , 43 ] — was common in our cohort, particularly in SF1-lineage tumors, and was present in all tumors harbouring mtDNA mutations. Similar heterogeneity and/or reduced expression was also observed in multiple tumors without detectable mtDNA mutations, suggesting a potential contribution of nuclear-encoded OXPHOS-related genes (not assessed). The coexistence of strongly and weakly stained tumor regions supports clonal metabolic divergence and likely reflects variable heteroplasmy. Whether this heterogeneity affects hormone secretion, proliferation, or treatment response in PitNETs remains to be determined. In summary, PitNETs demonstrate lineage-specific and highly heterogeneous OXPHOS system patterns. Genomically stable SF1-lineage tumors frequently shown complex I deficiency and mtDNA mutations, while similar alterations may also occur in TPIT-lineage tumors with highly disrupted near-haploid genomes. These finding highlight the biological complexity of PitNETs and suggest that mitochondrial dysfunction represents an additional layer of heterogeneity beyond lineages and chromosomal alteration patterns. Further studies in larger cohorts, including analysis of nuclear-encoded OXPHOS-related genes, are needed to clarify the role of mitochondrial dysfunction in PitNET biology and its potential clinical relevance. Declarations Author Contribution Conceptualization: M.M.J., W.E.C., R.G.F., H.M; Methodology: R.G.F., J.A.M., H.M.; Formal analysis and investigation: M.M.J., R.G.F., S.H., L.E., J.A.M., H.M.; Writing - original draft preparation: M.M. J., R.G.F., J.A.M.; Writing - review and editing: all authors; Resources: I.P., L.B., W.F., M.V., N.B.; Supervision: H.M. 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04:05:04","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138049,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8475859/v1/80d0aceb88018bd01c70939c.html"},{"id":99575027,"identity":"f7714c8c-ebae-455f-bfb4-689607fefff1","added_by":"auto","created_at":"2026-01-06 04:05:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":184632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the study cohort. \u003c/strong\u003eSelected clinical characteristics of the patients, PitNET subtypes (grouped by lineage), chromosomal alteration patterns, immunohistochemistry for porin (VDAC1) and subunits of OXPHOS complexes I-V, and mtDNA mutation status (where available) are shown.\u003cstrong\u003e \u003c/strong\u003eHeterogeneous or patchy immunohistochemical staining was observed across all lineages and stainings. No mtDNA mutations were observed in tumors of PIT1-lineage. In contrast, the majority of SF1-lineage tumors harboured disruptive mtDNA mutations in complex I, which were associated with reduced immunohistochemical expression of NDUFB8 (complex I subunit).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8475859/v1/e9e91d480da808d1c9687d2f.png"},{"id":99792767,"identity":"ced03e0f-8963-47bb-8f9b-fa25db12a927","added_by":"auto","created_at":"2026-01-08 13:25:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12211083,"visible":true,"origin":"","legend":"\u003cp\u003eExamples of heterogenous/patchy intratumoral staining\u003c/p\u003e\n\u003cp\u003e(a) Case 34 (SF1-lineage), m.11038del (91%) detected.\u003c/p\u003e\n\u003cp\u003e(b) \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eCase 41 (SF1-lineage), no mtDNA mutations detected. Magnification x10.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8475859/v1/fd7924641e569fb2f2a59af1.png"},{"id":99575030,"identity":"6083c5bc-e92d-453a-a744-210a8cd188dc","added_by":"auto","created_at":"2026-01-06 04:05:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84918,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining intensity ratios for subunits of OXPHOS system complexes I–V and VDAC1 in pituitary adenomas/PitNETs.\u003c/p\u003e\n\u003cp\u003e(a) Tumor-to-normal staining intensity ratios stratified by lineage. The dashed line at 1.0 indicates parity with normal tissue. A lineage-specific trend toward reduced NDUFB8 expression (CI) is observed in SF1-lineage tumors, whereas VDAC1 expression, reflecting mitochondrial density, is increased across all lineages.\u003c/p\u003e\n\u003cp\u003e(b) Intratumoral staining heterogeneity in seven tumors harbouring mtDNA mutations affecting complex I. Ratios represent staining intensity in regions corresponding to the heteroplasmic mutation relative to other regions within the same tumor.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8475859/v1/3c78f4a8e0e0d5a83f8dcf1b.png"},{"id":99575035,"identity":"7af63783-e26e-4d67-a701-198c945b0cc1","added_by":"auto","created_at":"2026-01-06 04:05:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6506238,"visible":true,"origin":"","legend":"\u003cp\u003ePitNETs with detected mtDNA mutations. Representative HE-sections and immunohistochemical stainings for VDAC1 (porin) and NDUFB8 (CI subunit), x20.\u003c/p\u003e\n\u003cp\u003eSF1-lineage, a-g:\u003c/p\u003e\n\u003cp\u003e(a) Tumor 8, stable genome. The tumor tissue mostly showed preserved expression of NDUFB8 with focal deficiency not corresponding with 46 % of mutational load (heteroplasmy), probably due to intratumoral variation in tissue obtained for DNA-isolation and stained tissue. The evident focal CI-deficiency with increased mitochondrial density in VDAC1 is shown.\u003c/p\u003e\n\u003cp\u003e(b) Tumor 9, stable genome. The only tumor with mutation affecting MT-TM (mitochondrial tRNA required for OXPHOS complexes I, III, IV, and V). This mutation has been reported to induce a pattern of OXPHOS system multicomplex deficiency [32], as also observed in this tumor tissue.\u003c/p\u003e\n\u003cp\u003e(c)\u003cstrong\u003e \u003c/strong\u003eTumor\u003cstrong\u003e \u003c/strong\u003e10 with chromosomal imbalance due to copy-number gains on 7p and 7q.\u003c/p\u003e\n\u003cp\u003e(d) Tumor 30, stable genome.\u003c/p\u003e\n\u003cp\u003e(e) Tumor 32, stable genome.\u003c/p\u003e\n\u003cp\u003e(f) Tumor 34, stable genome.\u003c/p\u003e\n\u003cp\u003e(g) Tumor 51, stable genome.\u003c/p\u003e\n\u003cp\u003eTPIT-lineage, h-i:\u003c/p\u003e\n\u003cp\u003e(h) Tumor 52 with near-haploid genome with LOH on multiple chromosomes due to whole chromosome loss and expected endoreduplication. (i) Tumor 56 with near-haploid genome with LOH on multiple chromosomes due to whole chromosome loss and detected endoreduplication.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8475859/v1/abed961dd653f70f22869b8b.png"},{"id":99575034,"identity":"a55b5791-1dfe-4be4-b8c5-bedf77524628","added_by":"auto","created_at":"2026-01-06 04:05:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7162893,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative examples of PitNETs with or without oncocytic features and corresponding immunohistochemical VDAC1-expression, x20. (a) Tumor 16, no oncocytic features, VDAC1 intensity 100. (b) Tumor 12, no oncocytic features, VDAC1 intensity 150. (c) Tumor 38, no oncocytic features, overall VDAC1 intensity 130, with areas of reaching staining intensity 250 (shown). No mtDNA mutation detected. (d) Tumor 49 with oncocytic features, VDAC1 intensity varying from 100 (shown) to 150. No mtDNA mutation detected.\u003c/p\u003e\n\u003cp\u003e(e) Tumor 41 with oncocytic features, VDAC1 intensity ranging from 175 to 200 (shown). No mtDNA mutation detected.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8475859/v1/8e3ba99b32d268609e1c309d.png"},{"id":104739327,"identity":"34c2191e-61b4-4c85-b55d-254cecb1f2de","added_by":"auto","created_at":"2026-03-16 16:02:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23658985,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8475859/v1/4fe534cb-08ed-4796-b0f8-cb6c6e74749c.pdf"},{"id":99575043,"identity":"9251295b-3a9b-45c3-b1cf-9b0558d4bcf7","added_by":"auto","created_at":"2026-01-06 04:05:04","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17534,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8475859/v1/4d68a75502a1291bb1d6e95a.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oxidative Phosphorylation Patterns in Pituitary Adenoma/Neuroendocrine Tumors ","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePituitary Neuroendocrine Tumors (PitNETs), also known as pituitary adenomas, are increasingly diagnosed, with a prevalence of 78\u0026ndash;116 cases per 100 000 people in the last 15 years [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While some PitNET types mostly treated with medication, surgical resection is performed for approximately a half of clinically diagnosed PitNETs. Among those\u0026thinsp;\u0026gt;\u0026thinsp;40% are non-functional gonadotroph tumors (SF1 lineage), ~\u0026thinsp;15% are corticotroph tumors (TPIT lineage), and ~\u0026thinsp;30% are tumors of PIT1 lineage. Recently, we explored chromosomal alteration patterns in diverse PitNET subtypes due to relatively low occurrence of somatic mutation hallmarks in sporadic tumors, confirming previous observations [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Tumors with aggressive clinical behaviour showed massive chromosomal losses (TPIT-lineage) which are likely to be mutually exclusive with \u003cem\u003eUSP8\u003c/em\u003e-mutations. Tumors of PIT1-lineage mostly showed complex patterns of chromosomal losses and gains, with the mechanisms still to be elucidated. Gonadotroph tumors (SF1-lineage) do not cause hormone excess and usually present as large tumors causing mass effects, often require multiple operations due to difficulty of complete initial surgical resection. These tumors mostly show stable genome and no somatic mutations, challenging the search for biomarkers and therapeutical targets. Prior to introduction of systematic staining for transcription factors, a subset of PitNETs was classified as pituitary oncocytoma/oncocytic null-cell adenomas (WHO Tumor Classification 2017 and prior), which most likely was represented by gonadotroph tumors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this historical tumor type, mtDNA mutations in complex I of oxidative phosphorylation (OXPHOS) were repeatedly reported [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Kurelac et al. reported a series of 48 pituitary adenomas, in which a correlation was found between CI disruptive mutations, the oncocytic phenotype and low number of chromosomal aberrations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Complex II alterations are known drivers in subset of tumors, which occur mostly in the context of SDH-deficient tumour syndrome (hereditary phaeochromocytoma-paraganglioma syndromes), and are less detected sporadically [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAbnormal mitochondria and oncocytic phenotype are often described as going hand in hand [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It is worth to mention that oncocytic changes also occur in the normal adenohypophysis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, there is room for subjective interpretation in the definition of oncocytic phenotype. While oncocytes have big eosinophilic cytoplasm, not all cells with such cytoplasm are true oncocytes. The eosinophilic appearance in some lesions may be attributed to the accumulation of various cellular components (e.g. lysosomes, endoplasmic reticulum, neuroendocrine granules), rather than an increased number of deviant morphology of mitochondria [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Immunohistochemistry for subunits of the respiratory chain can be utilized to visualize mitochondria [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The oncocytic features with corresponding abnormalities in mitochondria are widely described in other endocrine and non-endocrine organs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For PitNETs, the situation is likely to be more even complex, as the volume of mitochondria is different among different subtypes in pituitary adenomas; for example, the volume of mitochondria in prolactinomas is larger than those in growth hormone producing tumors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Somatotroph tumors with oncocytic cells show similar cytokeratin patterns and higher proliferative activity, which is not correlated with local aggressiveness [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Acidophilic stem cell tumors are per definition oncocytic [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Anyways, there is growing consideration of mitochondria as probable therapeutic target in PitNETs as mitochondrial alterations have commonly been recognized in these tumors [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The OXPHOS system within mitochondria represent the main source of energy (ATP) under aerobic conditions in eukaryotic cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The core elements are five complexes (I-V) consisting of diverse amount of subunits and organized in supercomplexes, embedded in the inner mitochondrial membrane [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Even though mitochondrial DNA (mtDNA) codes only for 13 proteins all of which are subunits of OXPHOS complexes, the mtDNA is prone to mutations. Still, most of the subunits of the OXPHOS system are encoded via the nuclear genome and are imported into mitochondria where large multi-subunit complexes are assembled. Beside those, the whole orchestra of supporting proteins and signalling systems are necessary for proper function of OXPHOS system. Beyond the roles in energy production, participation in cell signalling, regulation of apoptosis, mitochondria continuously gain attention in the context of tumorigenesis.\u003c/p\u003e \u003cp\u003eIn this study, we explored mitochondrial density, mitochondrial DNA mutation status and expression patterns of OXPHOS system complexes in a previously molecularly defined and subtyped PitNET cohort [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eMaterial from 43 sporadic PitNETs previously described by our group was available for additional analyses, with case numbering consistent the earlier study [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Adjacent normal adenohypophyseal tissue was available in nine cases. All analyses were covered by the ethical approval of the original study (Medical Ethics Review Committee Leiden, G19.011).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAssessment of oncocytic phenotype\u003c/h3\u003e\n\u003cp\u003eHaematoxylin and eosin-stained sections were independently evaluated for oncocytic features by two pathologists (MJ and HM). Tumors were categorized as non-oncocytic; with oncocytic-metaplasia like features, or oncocytic.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOncocytic\u003c/em\u003e features were defined by abundant, voluminous granular eosinophilic cytoplasm, sharp cytoplasmic borders, a low nuclear-to-cytoplasmic ratio, and round, centrally located nucleus with evenly distributed chromatin and prominent nucleolus.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOncocytic metaplasia-like\u003c/em\u003e features were assigned when tumor cells exhibited some, but not all oncocytic features, or when oncocytic features were present only focally.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNon-oncocytic\u003c/em\u003e phenotype lacked the characteristic cytoplasmic and nuclear features of oncocytes.\u003c/p\u003e \u003cp\u003eIn cases of discrepant scoring, consensus was reached by joint review.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry for mitochondrial density and subunits of OXPHOS system\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMaterials and considerations\u003c/h2\u003e \u003cp\u003eImmunohistochemistry was performed for subunits of the OXPHOS system and for porin (VDAC1) as described previously [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Immunohistochemical staining of porin (VDAC1) was performed to explore potential alterations in mitochondrial biogenesis within PitNETs, both intratumorally and compared to normal adenohypophyseal tissue. Porin is a protein in the outer mitochondrial membrane that is frequently used as a marker for mitochondrial mass [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The following primary antibodies were used: complex I (rabbit anti-NDUFB8, Abcam, ab192878; 1:500, 1 h), complex II (mouse anti-SDHA, Abcam, ab14715; 1:2000, 1 h), complex III (mouse anti-UQCRC2), Abcam, ab14745; 1:1000, 1 h), complex IV (mouse anti-MT-CO1), Abcam, ab14705; 1:1000, 1 h), complex V (mouse anti-ATP5F1A, Abcam, ab14748; 1:2000, 1 h), and porin (mouse anti-VDAC1, Abcam, ab14734; 1:1000, 1 h).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eScoring\u003c/h3\u003e\n\u003cp\u003eScoring and statistical analyses were performed as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Immunohistochemical expression levels in tumor tissue were compared with adjacent pre-existent tissue when available, and otherwise with the mean expression levels observed in normal adenohypophyseal tissue across cases.\u003c/p\u003e \u003cp\u003eBriefly, immunohistochemical scores were calculated by multiplying the staining intensity (range 0\u0026ndash;3, with half-point increments) by the percentage of tissue area exhibiting that intensity. Quantification was performed independently by two observers (MJ, HM), and the mean interobserver score was used for all analyses.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of heterogeneity\u003c/h2\u003e \u003cp\u003eHeterogeneous or patchy staining patterns and focal loss of expression were frequently observed in tumors. To semi-quantify intratumoral heterogeneity, variability in staining intensity was first assessed in normal adenohypophyseal tissue by calculating the 95% confidence interval (CI) of the mean score for each marker. Tumors with optically variable staining were classified as \u003cem\u003eheterogenous\u003c/em\u003e for a given staining when the intratumoral range of staining scores \u0026mdash; defined as the maximum minus the minimum observed score \u0026mdash; exceeded the width of the 95% CI derived from normal adenohypophyseal tissue.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDefinition of altered expression\u003c/h3\u003e\n\u003cp\u003eIncreased or decreased immunohistochemical expression in tumor tissue was defined as a relative change of 25%, 50%, or 75% in the score value compared with the mean score of normal tissue or the corresponding normal tissue, when available.\u003c/p\u003e \u003cp\u003eIn tumors with detected mtDNA mutations and heterogeneous staining patterns, the tumor area corresponding to the observed heteroplasmy percentage of mutation. For these cases, the ratio was calculated between expression levels in the mutation-associated region and adjacent tumor tissue.\u003c/p\u003e\n\u003ch3\u003eAdditional molecular studies\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction\u003c/h2\u003e \u003cp\u003eFresh-frozen material was available for 21 samples, from which DNA was isolated using ten 20-\u0026micro;m cryosections per case. For an additional 21 tumors, as well as two cases with suboptimal fresh-frozen material on visual inspection, previously extracted DNA from formalin-fixed, paraffin-embedded tissue was used, as described previously [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003emtDNA sequencing\u003c/h2\u003e \u003cp\u003eLong-range PCR amplification\u003c/p\u003e \u003cp\u003eMtDNA was amplified from proteinase K-digested fresh-frozen tumor tissue using long-range PCR. For each sample, two PCR reactions were performed using primer sets (Microsynth): primer set A: (forward 5\u0026rsquo;-CACCAGCCTAACCAGATTTCA-3\u0026rsquo;; reverse 5\u0026rsquo;-TGGTACCCAAATCTGCTTCC-3\u0026rsquo;) and primer set B (forward 5\u0026rsquo;-GGCTCACATCACCCCATAAA-3\u0026rsquo;; reverse 5\u0026rsquo;-CGTGTGGGCTATTTAGGCTTT-3\u0026rsquo;).\u003c/p\u003e \u003cp\u003eEach PCR reaction contained 1 \u0026micro;l of digested tumor sample, 1x LongAmp-Taq (New England Biolabs, NEB), and primers at a final concentration of 0,4 \u0026micro;M. Initial heating to 94\u0026deg;C for 30 seconds, was followed by 40 cycles of 94\u0026deg;C for 30 seconds, 58\u0026deg;C for 60 seconds, and 68\u0026deg;C for 13 minutes. PCR products were purified using the Monarch\u0026reg; PCR \u0026amp; DNA cleanup Kit 5 \u0026micro;g (New England Biolabs, NEB) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eLong-read mtDNA sequencing\u003c/p\u003e \u003cp\u003eLong-read mtDNA sequencing library was prepared using the Oxford Nanopore Technologies Native Barcoding Kit (SQK-NBD114.24). For each sample, 500 ng of each long-range PCR product were mixed with 0,875 \u0026micro;l NEBNext Ultra II End-prep Reaction Buffer (NEB), 0,75 \u0026micro;l NEBNext Ultra II End-prep Enzyme Mix (NEB) and nuclease-free water to a total volume of 15 \u0026micro;l. The sample was incubated at 20\u0026deg;C for 5 minutes followed by 65\u0026deg;C for 5 minutes. Each sample was mixed with 1x AMPure XP Beads and incubated for 5 minutes on a rotator mixer. The beads were washed twice with 200 \u0026micro;l of 80% ethanol and the sample was eluted in 10 \u0026micro;l nuclease-free water. 7.5 \u0026micro;l of each end-prepped sample were mixed with a unique barcode and 10 \u0026micro;l Blunt/TA Ligase Master Mix (NEB). The reaction was incubated for 20 minutes at RT and stopped by adding 4 \u0026micro;l of 0.25 M EDTA. Barcoded samples were pooled, and 0.4x AMPure XP Beads were added. The sample was incubated for 10 minutes on a rotator mixer and 700 \u0026micro;l of 80% ethanol were used for washing the beads twice. To elute the DNA from the beads, 35 \u0026micro;l nuclease-free water were added and the beads were incubated at 37\u0026deg;C for 10 minutes and every 2 minutes the beads were gently mixed. The beads were pelleted on a magnet and 35 \u0026micro;l of barcoded sequencing library removed. Sequencing adapters were ligated by mixing 30 \u0026micro;l of the barcoded sequencing library with 5 \u0026micro;l Native Adapter, 10 \u0026micro;l NEBNext Quick Ligation Reaction Buffer (NEB) and 5 \u0026micro;l Quick T4 DNA Ligase (NEB) and incubation for 20 minutes at RT. The library was purified by adding 0.4x AMPure XP beads and incubating the reaction for 10 minutes on a rotator mixer at RT. The beads were washed twice by resuspending in 125 \u0026micro;l Long Fragment Buffer, pelleting the beads and removing the supernatant. Finally, the sequencing library was eluted in 35 \u0026micro;l Elution Buffer by incubation for 10 minutes at 37\u0026deg;C and flicking the beads every 2 minutes to mix.\u003c/p\u003e \u003cp\u003eSequencing was performed on a P2 Solo sequencing device using FLO-PRO114M flow cells. Basecalling, demultiplexing, and alignment against human reference genome (GRCh38) were performed in real time using MinKNOW (v.24.02.6). BAM files were sorted and indexed using SAMtools (v.1.18) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and variant calling was performed with freebayes (v.1.3.6) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of pathogenicity of detected mtDNA variants\u003c/h2\u003e \u003cp\u003eThe pathogenicity of detected mtDNA variants was evaluated based on population frequency and existing annotations in the MitoMap database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mitomap.org\u003c/span\u003e\u003cspan address=\"https://www.mitomap.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which integrates data from gnomAD, GenBank, and Helix. MitoMap annotations were used to identify previously reported variants and their known or suggested pathogenic relevance.\u003c/p\u003e \u003cp\u003eFor novel mtDNA variants, in silico pathogenicity prediction was performed using multiple tools, including Apogee2, Hmtvar, AlphaMissense, BayesDel_addAF, DEOGEN2, LIST_S2, MutationAssessor, PhyloP100, PROVEAN, Sift4G, GERP RS, and Varity_R.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical methods\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism version 10.2.3 (GraphPad Software, USA). Given the limited sample size, the study was exploratory in nature and primarily relied on descriptive statistics, with inferential analyses interpreted cautiously. Normality of staining intensity distributions in grouped samples was assessed using the Shapiro\u0026ndash;Wilk test. For comparisons between groups, Welch\u0026rsquo;s t-test was applied when data approximated a normal distribution, whereas the Mann\u0026ndash;Whitney U test was used for non-normally distributed data. Associations between VDAC1 expression, oncocytic phenotype, and mtDNA mutation status were explored using Spearman rank correlation, simple linear regression, and logistic regression, as appropriate. All tests were two-sided, and p values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eAn overview of the study cohort, including immunohistochemistry and summarized molecular data, is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Immunohistochemistry for VDAC1 and OXPHOS system subunits was successful on 43 tumors, comprising 20 PIT1-lineage, 13 SF1 lineage, 9 TPIT-lineage, and one multilineage tumor (SF1/PIT1-lineages). Additional mtDNA analysis was successful in 21 cases, of which nine harboured mtDNA mutations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial density\u003c/h2\u003e \u003cp\u003eMitochondrial density, assessed by porin (VDAC1) expression, was significantly higher in tumor tissue (162.3\u0026thinsp;\u0026plusmn;\u0026thinsp;29.2) compared with normal adenohypophyseal tissue (111.1\u0026thinsp;\u0026plusmn;\u0026thinsp;22.1; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Thirty-eight of 43 tumors (88.4%) showed mean intratumoral VDAC1 staining intensity above the normal range.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHeterogenous/patchy staining\u003c/h2\u003e \u003cp\u003eOf 43 tumors, 24 showed heterogenous/patchy staining pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), whereas 17 showed homogenous staining. Two samples were too small to assess heterogeneity (1xSF1 and 1xTPIT lineage).\u003c/p\u003e \u003cp\u003eAmong 24 tumors with heterogeneous staining, 23 were patchy in 2 or more stainings, and four tumors demonstrated a heterogeneous pattern in all six stainings. Heterogenous staining was most frequently observed for SDHA (CII, 19/24), followed by NDUFB8 (CI, 17/24), ATP5F1A (CV, 16/24), VDAC1 (porin, 15/24), MT-CO1 (CIV, 9/24), and UQCRC2 (CIII, 5/24).\u003c/p\u003e \u003cp\u003eStaining heterogeneity was most prevalent in SF1-lineage tumors (11/12). Three of eight TPIT-lineage tumors showed heterogeneous staining. In contrast, approximately half of PIT1-lineage tumors (12/23) showed a homogenous staining.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e(a) Case 34\u003c/strong\u003e \u003cp\u003e(b) (SF1-lineage), m.11038del (91%) detected.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e(c) Case 41\u003c/strong\u003e \u003cp\u003e(d) (SF1-lineage), no mtDNA mutations detected. Magnification x10.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eComplex I (subunit NDUFB8)\u003c/p\u003e \u003cp\u003eReduced NDUFB8 expression was observed in 12 tumors (8xSF1, 2xTPIT, 1xPIT1, 1xSF1/PIT1). In eight cases, the deficiency was isolated, whereas four tumors showed combined deficiencies involving other OXPHOS complexes (CII in two cases, CIII in two, CIV in four, and CV in one). Heterogeneous staining was present in 10/12 tumors with reduced NDUFB8 expression. Among the eight tumors harbouring mtDNA mutations affecting complex I, six showed markedly reduced NDUFB8 expression (\u0026le;\u0026thinsp;50% of normal tissue), while two displayed a moderate reduction (25\u0026ndash;49%). Reduced NDUFB8 expression was also observed in two tumors without detectable mtDNA mutations and in two tumors for which mtDNA sequencing was unsuccessful. No tumor showed increased NDUFB8 expression.\u003c/p\u003e \u003cp\u003eComplex II (subunit SDHA)\u003c/p\u003e \u003cp\u003eIsolated SDHA deficiency was observed in two tumors (1xPIT1, 1xSF1), while two tumors showed combined deficiencies (1xSF1, 1xSF1/PIT1); all four demonstrated heterogeneous SDHA staining. In addition, heterogeneous SDHA staining with overall intensity comparable to normal tissue was observed in 15 additional tumors across all lineages. Upregulated SDHA expression was found in 4 tumors (3xTPIT, 1xSF1). Ot these, three showed no deficiencies in other OXPHOS complexes, whereas one (case 30, mtDNA-mutated SF1-lineage tumor) exhibited CI-deficiency and co-upregulation of CIII-expression.\u003c/p\u003e \u003cp\u003eComplex III (subunit UQCRC2)\u003c/p\u003e \u003cp\u003eReduced UQCRC2 expression occurred only in tumors with combined deficiencies, as described above. Upregulated UQCRC2 expression was identified in eight tumors, two of which with co-occurrent CI-deficiency. Heterogeneous staining with overall intensity comparable to normal tissue was observed in four tumors (3xSF1, 1xTPIT), none in PIT1-lineage.\u003c/p\u003e \u003cp\u003eComplex IV (subunit MT-CO1)\u003c/p\u003e \u003cp\u003eReduced MT-CO1 expression was observed in three tumors (2xSF1, 1xPIT1), exclusively in combination with CI-deficiency. In two of these cases, co-occurring with heterogeneous staining pattern and deficiencies of additional OXPHOS complexes. No tumor demonstrated increased MT-CO1-expression. Heterogeneous staining with overall intensity comparable to normal tissue was observed in seven tumors (4xSF1, 3xTPIT) and was absent in PIT1-lineage.\u003c/p\u003e \u003cp\u003eComplex V (subunit ATP5F1A)\u003c/p\u003e \u003cp\u003eReduced ATP5F1A expression was observed in a single tumor (case 10, SF1-lineage), occurring as a part of a combined deficiency involving CI, CIII, and CIV and accompanied by heterogeneity across all stainings. Nine tumors (5xPIT1, 3xTPIT, 1xSF1) showed increased ATP5F1A expression. Heterogeneous staining with overall intensity comparable to normal tissue was observed in 14 tumors, across all lineages.\u003c/p\u003e \u003cp\u003eIn multiple linear regression analysis, SDHA (p\u0026thinsp;=\u0026thinsp;0.004) and ATP5F1A expression (p\u0026thinsp;=\u0026thinsp;0.03) were independently associated with higher VDAC1 expression, reflecting increased mitochondrial density. In contrast, NDUFB8 (p\u0026thinsp;=\u0026thinsp;0.02) and MT-CO1 (p\u0026thinsp;=\u0026thinsp;0.02) showed inverse associations. Lineage-specific expression patterns and the overall distribution of immunohistochemical staining intensities are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, showing tumor-to-normal expression ratios for subunits of CI-CV and VDAC1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTumor-to-normal staining intensity ratios stratified by lineage. The dashed line at 1.0 indicates parity with normal tissue. A lineage-specific trend toward reduced NDUFB8 expression (CI) is observed in SF1-lineage tumors, whereas VDAC1 expression, reflecting mitochondrial density, is increased across all lineages.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eIntratumoral staining heterogeneity in seven tumors harbouring mtDNA mutations affecting complex I. Ratios represent staining intensity in regions corresponding to the heteroplasmic mutation relative to other regions within the same tumor.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003emtDNA sequencing\u003c/h2\u003e \u003cp\u003emtDNA sequencing was successful in 21 tumors, of which nine harboured mtDNA mutations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two novel missense and one novel loss-of-function variant were identified.\u003c/p\u003e \u003cp\u003eThe MT-ND1 m.3631T\u0026thinsp;\u0026gt;\u0026thinsp;C variant results in substitution of a highly conserved serine residue within a transmembrane domain (p.Ser109Pro), spanning amino acid residues 100\u0026ndash;120. The variant was absent from gnomAD 4.1 and MitoMap. According to ACMG criteria, it was classified as a variant of uncertain significance (PM2 moderate, PP3 supporting). Multiple in silico prediction tools support a pathogenic effect (Apogee2, Pathogenic, 0.87; Hmtvar, Pathogenic, 0.84; AlphaMissense, Pathogenic, 0.90; BayesDel_addAF; Uncertain; 0.057, T; DEOGEN2, Benign, 0.23, T; LIST_S2, Uncertain, 0.95, D; MutationAssessor, Pathogenic, 4.3, H; PhyloP100, 4.8; PROVEAN, Pathogenic, -4.6, D; Sift4G, Pathogenic, 0.0010, D; GERP RS, 4.5; Varity_R, 0.95).\u003c/p\u003e \u003cp\u003eThe MT-ND4 m.11484G\u0026thinsp;\u0026gt;\u0026thinsp;A variant was detected in one tumor and affects a highly conserved glycine residue within a transmembrane domain (p.Gly242Asp), spanning residues 224\u0026ndash;244. This variant has been reported at relatively low heteroplasmy (25%) in one individual in gnomAD but was absent in gnomAD 4.1 and MitoMap. In the tumor sample, heteroplasmy reached 84%. According to ACMG criteria, this variant was also classified as of uncertain of uncertain significance (PM2 moderate, PP3 supporting), with the majority of in silico tools (7/11) predicting a damaging or pathogenic effect (Apogee2, Pathogenic, 0.74; Hmtvar, Pathogenic, 0.88; AlphaMissense, Pathogenic, 1.0; BayesDel_addAF, Benign, -0.19, T; DEOGEN2, Uncertain, 0.44, T; LIST_S2, Uncertain, 0.93, D; MutationAssessor; Pathogenic, 5.2, H; PhyloP100, 9.4; PROVEAN, Pathogenic, -6.0, D; Sift, Pathogenic, 0.0, D; Sift4G, Pathogenic, 0.0, D; GERP RS, 5.1; Varity_R, 0.97).\u003c/p\u003e \u003cp\u003eThe novel loss-of-function variant in MT-ND2 (m.5366_5367del; c.896_897del) causes a frameshift leading to a premature stop codon (p.Ser299TyrfsTer10). The variant was absent from gnomAD and MitoMap.\u003c/p\u003e \u003cp\u003eThe remaining pathogenic mtDNA variants identified in this cohort had been previously reported (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). An overview of all 161 detected mtDNA variants across the 21 PitNETs is provided in Supplemental Material.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAll detected mtDNA mutations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase (Lineage)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003emtDNA variant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMutant load (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEffect\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAffected gene(s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOXPHOS system complex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReference/prior report\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8 (SF1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003em.10158T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep.Ser34Pro\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT-ND3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePathogenic in neurological disease [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9 (SF1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003em.4412G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003etRNA DHU stem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT-TM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC I, C III, C IV, C V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePathogenic in neurological disease [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 (SF1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003em.12923G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStop\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT-ND5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLikely pathogenic in neurological disease [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30 (SF1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003em.11484G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep.Gly242Asp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT-ND4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://reg.genome.network/redmine/projects/registry/genboree_registry/alleles?refseq=NC_012920.1\u0026amp;begin=11481\u0026amp;end=11581\u0026amp;skip=0\u0026amp;limit=50\u003c/span\u003e\u003cspan address=\"https://reg.genome.network/redmine/projects/registry/genboree_registry/alleles?refseq=NC_012920.1\u0026amp;begin=11481\u0026amp;end=11581\u0026amp;skip=0\u0026amp;limit=50\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e32 (SF1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003em.4436_16463del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT-ND1, MT-ND2, MT-ND3, MT-ND4L, MT-ND4, MT-ND5, MT-CYB, MT-CO1, MT-CO2, MT-CO3, MT-ATP6, MT-ATP8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC I, C III, C IV, C V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLarge deletions reported in human cancers [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003em.4974G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStop\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT-ND2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eVariant of uncertain significance [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] or pathogenic [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e34 (SF1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003em.11038del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFrameshift\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT-ND4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eKidney oncocytoma [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e],\u003c/p\u003e \u003cp\u003eoncocytic thyroid carcinoma [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], adrenal oncocytoma [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e51 (SF1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003em.12425del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFrameshift\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT-ND5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIHC-negative oncocytic pituitary adenoma [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], nasopharyngeal oncocytic tumor [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e52 (TPIT)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003em.3631T\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep.Ser109Pro\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT-ND1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edbSNP: rs1603219053\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e56 (TPIT)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003em.5366_5367del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFrameshift\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMT-ND2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll tumors with detected mtDNA mutations showed regions of reduced NDUFB8 expression by immunohistochemistry, accompanied by a complementary increase in mitochondrial density (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Intratumoral staining variability in tumors harbouring complex I mtDNA mutations with pronounced staining heterogeneity is visualised in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSF1-lineage, a-g:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(a) Tumor 8, stable genome. The tumor tissue mostly showed preserved expression of NDUFB8 with focal deficiency not corresponding with 46% of mutational load (heteroplasmy), probably due to intratumoral variation in tissue obtained for DNA-isolation and stained tissue. The evident focal CI-deficiency with increased mitochondrial density in VDAC1 is shown.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(b) Tumor 9, stable genome. The only tumor with mutation affecting MT-TM (mitochondrial tRNA required for OXPHOS complexes I, III, IV, and V). This mutation has been reported to induce a pattern of OXPHOS system multicomplex deficiency [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], as also observed in this tumor tissue.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(c) Tumor 10 with chromosomal imbalance due to copy-number gains on 7p and 7q.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(d) Tumor 30, stable genome.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(e) Tumor 32, stable genome.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(f) Tumor 34, stable genome.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(g) Tumor 51, stable genome.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eTPIT-lineage, h-i:\u003c/p\u003e \u003cp\u003e(h) Tumor 52 with near-haploid genome with LOH on multiple chromosomes due to whole chromosome loss and expected endoreduplication. (i) Tumor 56 with near-haploid genome with LOH on multiple chromosomes due to whole chromosome loss and detected endoreduplication.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eOncocytic phenotype\u003c/h2\u003e \u003cp\u003eAmong the 43 tumors, ten were classified as oncocytic, 23 showed oncocytic metaplasia-like features, and 10 lacked oncocytic features. Neither correlation nor regression analyses demonstrated association between oncocytic morphology and mitochondrial density as assessed by VDAC1 expression. Lineage-specific analyses likewise revealed no significant relationship between VDAC1 staining intensity and oncocytic features.\u003c/p\u003e \u003cp\u003eThe presence of mtDNA mutations was also not associated with oncocytic morphology or VDAC1-defined mitochondrial density. Given the absence of correlation between oncocytic appearance and mitochondrial mass, representative cases are shown to illustrate this discrepancy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Both, non-oncocytic and tumors with oncocytic features showed variable VDAC1 intensity. These examples highlight that oncocytic morphology is an unreliable surrogate for mitochondrial density, and vice versa. Additionally, no significant association was observed between genome stability (absence of chromosomal alterations) and oncocytic features or mitochondrial density.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e(e) Tumor 41 with oncocytic features, VDAC1 intensity ranging from 175 to 200 (shown). No mtDNA mutation detected.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003ePreviously, we explored chromosomal alteration patterns across all lineages of pituitary neuroendocrine tumors (PitNETs), also known as pituitary adenomas[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Gonadotroph tumors (SF1-lineage) \u0026ndash; typically occurring in older patients and presenting as larger, invasive tumors \u0026ndash; showed almost no chromosomal alterations and were characterized by a stable genome or only chromosomal imbalances due to copy-number gains, suggesting biological characteristics distinct from other PitNET lineages. Although several PitNETs displayed oncocytic features, no clear relationship with chromosomal alteration patterns could be established, in contrast to oncocytic thyroid lesions [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKurelac at al. previously investigated pituitary adenomas in the context of oxidative phosphorylation (OXPHOS) system, reporting stable genome or copy-number gains in tumors with oncocytic phenotype (formerly termed pituitary oncocytoma/oncocytic adenoma), together with frequent disruptive mtDNA mutations affecting complex I [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Following the revision of PitNET nomenclature [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] by introduction of transcription factors, the entity of pituitary oncocytic adenoma was abandoned, and reassessment of these cases allows only limited extrapolation to current lineage definitions. These earlier findings motivated us to investigate OXPHOS system alterations in a molecularly defined and contemporary subtyped PitNET cohort.\u003c/p\u003e \u003cp\u003eWe found that PitNETs commonly show increased mitochondrial density, as reflected by elevated VDAC1 expression, compared to normal adenohypophyseal tissue. However, this increase was not accompanied by uniform up- or downregulation of OXPHOS system components. Instead, increased SDHA (subunit of CII) and ATP5F1A (subunit of CV) expression was positively associated with mitochondrial mass, whereas NDUFB8 (subunit of CI) and MT-CO1 (subunit of CIV) correlated inversely. This pattern suggests that mitochondrial expansion in PitNETs may reflect compensatory metabolic adaptation, with induction of complexes II and V in the context of impaired or depleted complexes I and IV.\u003c/p\u003e \u003cp\u003eComplex I deficiency and mtDNA mutations were observed predominantly in SF1-lineage tumors, followed by TPIT-lineage tumors, whereas no mtDNA mutations were detected in PIT1-lineage \u0026ndash; likely reflecting selection bias. Kurelac et al. reported disruptive CI mtDNA mutation (m.11832G\u0026thinsp;\u0026gt;\u0026thinsp;A) in an oncocytic tumor with TSH/PRL-expression (likely PIT1-lineage under current classification) accompanied by copy-number losses along chromosome 13 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Another oncocytic tumor without hormone expression harboured a different disruptive complex I mtDNA mutation (m.11873insC), also co-occurring with copy-number losses along chromosome 13. A third oncocytic growth hormone-expressing tumor (again, likely PIT1-lineage) showed copy-number losses across chromosomes 10, 13, and 16, harboured \u003cem\u003eGNAS\u003c/em\u003e mutation (c.2530C\u0026thinsp;\u0026gt;\u0026thinsp;T; p.Arg844Cys), and lacked mtDNA mutations. Together, these cases illustrate that the relationship between oncocytic morphology, genome stability, and mitochondrial mutations is not straightforward.\u003c/p\u003e \u003cp\u003eOur findings extend these observations by identifying disruptive complex I mtDNA mutations in two TPIT-lineage tumors, which show highly unstable near-haploid genome extensive loss of heterozygosity of multiple chromosomes due to whole chromosome loss. This observation underscores the biological diversity of PitNETs and indicates that mitochondrial dysfunction is not restricted to genomically stable tumors. Despite the frequent presence of oncocytic features across all lineages, neither mitochondrial mass nor mtDNA mutation status correlated with oncocytic morphology, and we therefore could not confirm a previously assumed association between mtDNA mutations and the oncocytic phenotype [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOXPHOS system abnormalities showed a strikingly non-uniform pattern. While complex I deficiency \u0026mdash; often associated with mtDNA mutations \u0026mdash; was the most frequent alteration, deficiencies of complexes II\u0026ndash;V were uncommon and typically occurred as part of combined multi-complex defects. Reduced MT-CO1 (subunit CIV) expression was rare and consistently accompanied complex I deficiency. Upregulation of SDHA (subunit CII), UQCRC2 (subunit CIII), and especially ATP5F1A (subunit CV) was observed in subsets of tumors across all lineages.\u003c/p\u003e \u003cp\u003eTo our knowledge, this study provides the first detailed description of extensive intratumoral heterogeneity of OXPHOS system complex expression in PitNETs. Heterogeneous immunohistochemical staining \u0026mdash; previously described in papillary thyroid carcinoma and non-neoplastic tissue [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] \u0026mdash; was common in our cohort, particularly in SF1-lineage tumors, and was present in all tumors harbouring mtDNA mutations. Similar heterogeneity and/or reduced expression was also observed in multiple tumors without detectable mtDNA mutations, suggesting a potential contribution of nuclear-encoded OXPHOS-related genes (not assessed). The coexistence of strongly and weakly stained tumor regions supports clonal metabolic divergence and likely reflects variable heteroplasmy. Whether this heterogeneity affects hormone secretion, proliferation, or treatment response in PitNETs remains to be determined.\u003c/p\u003e \u003cp\u003eIn summary, PitNETs demonstrate lineage-specific and highly heterogeneous OXPHOS system patterns. Genomically stable SF1-lineage tumors frequently shown complex I deficiency and mtDNA mutations, while similar alterations may also occur in TPIT-lineage tumors with highly disrupted near-haploid genomes. These finding highlight the biological complexity of PitNETs and suggest that mitochondrial dysfunction represents an additional layer of heterogeneity beyond lineages and chromosomal alteration patterns. Further studies in larger cohorts, including analysis of nuclear-encoded OXPHOS-related genes, are needed to clarify the role of mitochondrial dysfunction in PitNET biology and its potential clinical relevance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: M.M.J., W.E.C., R.G.F., H.M; Methodology: R.G.F., J.A.M., H.M.; Formal analysis and investigation: M.M.J., R.G.F., S.H., L.E., J.A.M., H.M.; Writing - original draft preparation: M.M. J., R.G.F., J.A.M.; Writing - review and editing: all authors; Resources: I.P., L.B., W.F., M.V., N.B.; Supervision: H.M.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData available on request from the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWHO Classification of Tumours Editorial Board Endocrine and neuroendocrine tumours. 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Naturwissenschaften 77(5):221\u0026ndash;225. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/bf01138485\u003c/span\u003e\u003cspan address=\"10.1007/bf01138485\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pituitary","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pitu","sideBox":"Learn more about [Pituitary]()","snPcode":"11102","submissionUrl":"https://submission.nature.com/new-submission/11102/3","title":"Pituitary","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"oxidative phosphorylation, pituitary adenoma, PitNET","lastPublishedDoi":"10.21203/rs.3.rs-8475859/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8475859/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003ePituitary neuroendocrine tumors (PitNETs), also known as pituitary adenomas, exhibit marked lineage-specific heterogeneity. The underlying molecular biology of certain tumor types, particularly gonadotroph tumors (SF1-lineage) \u0026mdash; which typically exhibit stable genomes \u0026mdash; remains poorly understood. This study aimed to define oxidative phosphorylation (OXPHOS) system patterns across PitNET lineages.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eImmunohistochemistry was performed in 43 previously molecularly and histologically classified PitNETs on tumor and normal adenohypophyseal tissue for VDAC1 (porin) to assess mitochondrial density and OXPHOS subunits of complexes I\u0026ndash;V. Quantified staining intensity scores were used for statistical analyses, and mtDNA sequencing was successful in 21 tumors.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMitochondrial density was significantly increased in PitNETs compared with normal tissue. OXPHOS alterations were non-uniform: complex I deficiency was the most frequent abnormality, often associated with disruptive mtDNA mutations, particularly in genomically stable gonadotroph tumors. Two corticotroph tumors with near-haploid genomes also harboured disruptive complex I mutations. Alterations in other complexes were less common and typically occurred in combination. Staining heterogeneity was frequent (24/43 tumors), including focal expression loss, especially in SF1-lineage and all mtDNA-mutated tumors, but also present in tumors without mtDNA mutations.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ePitNETs display lineage-specific and highly heterogeneous OXPHOS phenotypes. Complex I deficiency and mtDNA mutations occur not only in genomically stable gonadotroph tumors but also in highly disrupted corticotroph tumors with a near-haploid genome. Further studies including sequencing of nuclear-encoded OXPHOS-related genes are required to clarify the contribution of OXPHOS and mitochondrial pathways to PitNET biology and potential clinical applications.\u003c/p\u003e","manuscriptTitle":"Oxidative Phosphorylation Patterns in Pituitary Adenoma/Neuroendocrine Tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-06 04:04:58","doi":"10.21203/rs.3.rs-8475859/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-19T16:14:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-18T09:52:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63037244736484269576415598258932747280","date":"2025-12-31T08:35:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-30T15:21:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-30T08:28:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-30T08:27:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pituitary","date":"2025-12-29T20:53:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pituitary","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pitu","sideBox":"Learn more about [Pituitary]()","snPcode":"11102","submissionUrl":"https://submission.nature.com/new-submission/11102/3","title":"Pituitary","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d0a771a4-d90b-4288-ab62-ffa4b6d9abce","owner":[],"postedDate":"January 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:01:05+00:00","versionOfRecord":{"articleIdentity":"rs-8475859","link":"https://doi.org/10.1007/s11102-026-01658-w","journal":{"identity":"pituitary","isVorOnly":false,"title":"Pituitary"},"publishedOn":"2026-03-11 15:57:57","publishedOnDateReadable":"March 11th, 2026"},"versionCreatedAt":"2026-01-06 04:04:58","video":"","vorDoi":"10.1007/s11102-026-01658-w","vorDoiUrl":"https://doi.org/10.1007/s11102-026-01658-w","workflowStages":[]},"version":"v1","identity":"rs-8475859","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8475859","identity":"rs-8475859","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}