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INTRODUCTION: This study aims to describe whether the clinical behaviour of nonfunctioning pituitary neuroendocrine tumours/nonfunctioning pituitary adenomas (NF-PitNETs/NFPAs) is affected by pituitary cell lineage differentiation, focusing on patients with silent corticotroph tumours (SCTs) and silent gonadotroph tumours (SGTs).

METHODS: Patients (N = 101) who underwent primary surgery for NF-PitNETs/NFPAs from August 2014 to March 2020 at Uppsala University Hospital were included. Data on sex, age, MRI, pituitary function and immunohistochemical analysis of anterior pituitary hormone and transcription factor expression, were explored.

RESULTS: Seventy-three patients had SGTs, and 18 had SCTs. Binary logistic regression revealed that having SCT versus SGT (OR 6.41 (CI: 1.20-34.42), p = 0.03), being older at the time of surgery (OR 1.07 (CI: 1.02-1.12), p = 0.01), and having a larger preoperative tumour volume (OR 1.17 (CI: 1.04-1.32), p = 0.01) were associated with an increased likelihood of postoperative pituitary failure. Patients with preoperative pituitary failure were older at the time of surgery (p = 0.01) and more often had preoperative elevation of prolactin levels (p = 0.01) than patients without preoperative pituitary failure. SCT patients were younger at the time of surgery than SGT patients (p = 0.003), but no significant difference in preoperative tumour volume was detected.

CONCLUSION: The results indicate that cell lineage differentiation in NF-PitNETs/NFPAs influences clinical behaviour. Patients with SCTs were younger at the time of surgery, and harbouring a SCT was associated with an increased likelihood of having postoperative pituitary failure. These findings emphasise the importance of routine immunohistochemical analyses of anterior pituitary hormone and transcription factor expression to identify silent corticotroph tumours.

The vast majority of tumours in the sellar region are pituitary neuroendocrine tumours, also called pituitary adenomas. Sellar gangliocytomas (GCs), benign tumours that originate from neuronal ganglionic cells, account for less than 1% of sellar tumours. Even rarer are mixed gangliocytoma-pituitary neuroendocrine tumours (GC-PitNET). These tumours are often associated with hormone hypersecretion, most commonly resulting in acromegaly. The histogenesis of mixed GC-PitNET is currently unclear. In this paper, we present comprehensive clinical, immunohistochemical, targeting enrichment next generation DNA sequencing, and genome-wide methylation data from four patients with mixed GC-PitNETs, three with acromegaly and one with Cushing's disease. Transcriptomic data are also included for two of the patients. Our findings indicate that mixed GC-PitNETs have different clinical course, with the acromegaly patients showing greater resistance to pharmacological therapy, as well as different protein expression and molecular features compared to respective pure PitNETs. The transcriptomic data on two patients with somatotroph GC-PitNET show involvement of mitochondrial and ribosomal genes, suggesting a distinct gene expression pattern, in comparison with pure somatotroph tumours. Furthermore, the expression pattern of selected stem cell markers, mainly SOX9, supports a common origin of the neuroendocrine and ganglionic tumour components, suggesting the involvement of stem cells in tumorigenesis.

As integral parts of the transcription super elongation complex, the ENL and AF9 proteins are two master regulators of gene expression involved in the development and homeostasis of various tissues. They are encoded by MLLT1 and MLLT3, two genes that frequently undergo oncogenic mutations or chromosomal translocations in cancers. In addition, we discovered an internal promoter driving the production of a YEATS (named after the five proteins first shown to contain this domain: Yaf9, ENL, AF9, Taf14, and Sas5)-domain-devoid AF9 isoform massively expressed in healthy hematopoietic stem cells (HSCs), as well as in the blasts of patients with extremely poor-prognosis acute myeloid leukemia (AML). Here, we show that such internal promoter is also active in a subset of digestive tissues, including the small intestine, colon and stomach. We also reveal the existence of a similar internal promoter in the MLLT1 locus that is inactive in the hematopoietic lineage and AML but functional in other healthy tissues, including the pituitary gland, brain, thyroid, uterus and lung. Furthermore, the YEATS-domain-devoid MLLT1 isoform may exhibit tumor-type-specific biomarker values, whereas full-length MLLT1 does not. It is highly expressed in the gonadotroph subtype of pituitary tumors, and its expression in non-small cell lung cancer inversely correlates with the epidermal growth factor mutation and a transcriptomic signature associated with a good prognosis. Thus, both MLLT1 and MLLT3 have tissue- and tumor-type-specific internal promoters driving expression of similar YEATS domain-devoid isoforms. This suggests that the functions of both MLLT1 and MLLT3 in tissue homeostasis and cancers, as well as their potentials as biomarkers should be re-evaluated taking into account expression of these YEATS-domain-devoid ENL and AF9 transcription factors.

Background

Synonymous mutations (SMs) change the mRNA nucleotide sequences without altering the corresponding amino acid sequence and are usually overlooked due to their perceived lack of influence on protein function. However, emerging reports suggest that SMs play a significant role in disease development and progression.

Methods

Whole exome sequencing, RNA-sequencing, and droplet digital PCR were performed to identify the SMs from the malignant glioma patients. MutaRNA was used to predict the effect of SMs on RNA structure in silico. SHAPE-MaP was performed to probe and assess the effect of SMs on RNA structure in-cellulo.

Results

Here, we report that a Cancer-Associated SM in TP53 codon valine 203 (CASM203) results in the induction of the alternative translation initiated p53 protein isoform, p47. In-cell high-throughput RNA structural mapping showed that CASM203 mimics the Protein Kinase RNA-Like ER Kinase (PERK)-mediated p53 mRNA secondary structure that induces p47 expression of during the unfolded protein response (UPR).

Conclusions

Overall, the single gain-of-function SM mimics the UPR-mediated p53 stress response, by generating RNA secondary structures akin to the PERK-mediated p53 mRNA structural switch. This illustrates the link between RNA structures and cellular biology and underscores the importance of SMs in cancer biology and their potential to further refine genetic diagnostics.

The vast majority of pituitary neuroendocrine tumors (PitNETs) are benign and slow growing with a low relapse rate over many years after surgical resection. However, about 40% are locally invasive and may not be surgically cured, and about one percentage demonstrate an aggressive clinical behavior. Exceptionally, these aggressive tumors may metastasize outside the sellar region to the central nervous system and/or systemically. The 2017 (4th Edition) WHO Classification of Pituitary Tumors abandoned the terminology "atypical adenoma" for tumors previously considered to have potential for a more aggressive behavior since its prognostic value was not established. The 2022 (5th Edition) WHO Classification of the Pituitary Tumors emphasizes the concept that morphological features distinguish indolent tumors from locally aggressive ones, however, the proposed histological subtypes are not consistent with the real life clinical characteristics of patients with aggressive tumors/carcinomas. So far, no single clinical, radiological or histological parameter can determine the risk of growth or malignant progression. Novel promising molecular prognostic markers, such as mutations in ATRX, TP53, SF3B1, and epigenetic DNA modifications, will need to be verified in larger tumor cohorts. In this review, we provide a critical analysis of the WHO guidelines for prognostic stratification and diagnosis of aggressive and metastatic PitNETs. In addition, we discuss the new WHO recommendations for changing ICD-O and ICD-11 codes for PitNET tumor behavior from a neoplasm either "benign" or "unspecified, borderline, or uncertain behavior" to "malignant" neoplasm regardless of the clinical presentation, histopathological subtype, and tumor location. We encourage multidisciplinary initiatives for integrated clinical, histological and molecular classification, which would enable early recognition of these challenging tumors and initiation of more appropriate and aggressive treatments, ultimately improving the outcome.

Aggressive pituitary neuroendocrine tumors (PitNETs)/adenomas are characterized by progressive growth despite surgery and all standard medical therapies and radiotherapy. A subset will metastasize to the brain and/or distant locations and are termed metastatic PitNETs (pituitary carcinomas). Studies of potential prognostic markers have been limited due to the rarity of these tumors. A few recurrent somatic mutations have been identified, and epigenetic alterations and chromosomal rearrangements have not been explored in larger cohorts of aggressive and metastatic PitNETs. In this study, we performed genome-wide methylation analysis, including copy-number variation (CNV) calculations, on tumor tissue specimens from a large international cohort of 64 patients with aggressive (48) and metastatic (16) pituitary tumors. Twelve patients with non-invasive pituitary tumors (Knosp 0–2) exhibiting an indolent course over a 5 year follow-up served as controls. In an unsupervised hierarchical cluster analysis, aggressive/metastatic PitNETs clustered separately from benign pituitary tumors, and, when only specimens from the first surgery were analyzed, three separate clusters were identified: aggressive, metastatic, and benign PitNETs. Numerous CNV events affecting chromosomal arms and whole chromosomes were frequent in aggressive and metastatic, whereas benign tumors had normal chromosomal copy numbers with only few alterations. Genome-wide methylation analysis revealed different CNV profiles and a clear separation between aggressive/metastatic and benign pituitary tumors, potentially providing biomarkers for identification of these tumors with a worse prognosis at the time of first surgery. The data may refine follow-up routines and contribute to the timely introduction of adjuvant therapy in patients harboring, or at risk of developing, aggressive or metastatic pituitary tumors.

Background: Cells with stem cell features have been described in pituitary neuroendocrine tumours (PitNETs). Transcription factors SOX2 and SOX9 are stem cell-associated markers while the pituitary progenitor marker PROP1 is involved in anterior pituitary development. We characterised the presence of these markers known to be present in the human pituitary in non-functioning (NF) PitNETs.

Methods: We investigated the pituitary transcription factors SOX2, SOX9 and PROP1 by immunohistochemistry (IHC) (N = 125) and RT-qPCR (N = 78) in a retrospective cohort of clinically NF-PitNETs. The markers were scored based on the percentage of immunolabeled cells. IHC staining scores were compared to reintervention rates for the whole cohort, and to expression of FSH, LH or ER in gonadotroph NF-PitNETs.

Results: Most tumours showed no or few cells positive for SOX2, SOX9 and PROP1. More patients with SOX2-negative tumours went through reintervention (40 % vs 19 %, p = 0.03). SOX2, SOX9 and PROP1 staining correlated positively to each other (SOX2 and SOX9 r_s = 0.666, SOX2 and PROP1 r_s = 0.704, SOX9 and PROP1 r_s = 0.570, and p < 0.001 for all). In gonadotroph NF-PitNETs, staining for SOX2 and PROP1 was positively associated to FSHβ staining (p < 0.001 for both). Staining for SOX2, SOX9 and PROP1 was positively associated with gene expression of Estrogen Receptor 1 (ESR1) (p < 0.001, p = 0.004 and p < 0.001) and IHC staining for ERα (p = 0.001, p = 0.03 and p = 0.05, respectively).

Conclusion: SOX2, SOX9 and PROP1 were present at low levels in NF-PitNETs. Absence of SOX2 staining was associated with a higher reintervention rate. The stem cell markers correlated positively with markers of gonadotroph differentiation in gonadotroph NF-PitNETs. SOX2 and SOX9 were frequently coexpressed and showed positivity in intratumoural cells with epithelial features, however without coexpression of pituitary transcription factors.

Although most pituitary neuroendocrine tumors (PitNETs)/pituitary adenomas remain intrasellar, a significant proportion of tumors show parasellar invasive growth and 6% to 8% infiltrate the bone structures, thus affecting the prognosis. There is an unmet need to identify novel markers that can predict the parasellar growth of PitNETs. Furthermore, mechanisms that regulate bone invasiveness of PitNETs and factors related to tumor vascularization are largely unknown.

We used genome-wide mRNA analysis in a cohort of 77 patients with PitNETs of different types to explore the differences in gene expression patterns between invasive and noninvasive tumors with respect to the parasellar growth and regarding the rare phenomenon of bone invasiveness. Additionally, we studied the genes correlated to the contrast enhancement quotient, a novel radiological parameter of tumor vascularization.

Most of the genes differentially expressed related to the parasellar growth were genes involved in tumor invasiveness. Differentially expressed genes associated with bone invasiveness are involved in NF-κB pathway and antitumoral immune response. Lack of clear clustering regarding the parasellar and bone invasiveness may be explained by the influence of the cell lineage-related genes in this heterogeneous cohort of PitNETs.

Our transcriptomics analysis revealed differences in the molecular fingerprints between invasive, including bone invasive, and noninvasive PitNETs, although without clear clustering. The contrast enhancement quotient emerged as a radiological parameter of tumor vascularization, correlating with several angiogenesis-related genes. Several of the top genes related to the PitNET invasiveness and vascularization have potential prognostic and therapeutic application requiring further research.

Aggressive pituitary tumors (APTs) and pituitary carcinomas (PCs) are heterogeneous with regard to clinical presentation, proliferative markers, clinical course, and response to therapy. Half of them show an aggressive course only many years after the first apparently benign presentation. APTs and PCs share several properties, but a Ki67 index greater than or equal to 10% and extensive p53 expression are more prevalent in PCs. Mutations in TP53 and ATRX are the most common genetic alterations; their detection might be of value for early identification of aggressiveness. Treatment requires a multimodal approach including surgery, radiotherapy, and drugs. Temozolomide is the recommended first-line chemotherapy, with response rates of about 40%. Immune checkpoint inhibitors have emerged as second-line treatment in PCs, with currently no evidence for a superior effect of dual therapy compared to monotherapy with PD-1 blockers. Bevacizumab has resulted in partial response (PR) in few patients; tyrosine kinase inhibitors and everolimus have generally not been useful. The effect of peptide receptor radionuclide therapy is limited as well. Management of APT/PC is challenging and should be discussed within an expert team with consideration of clinical and pathological findings, age, and general condition of the patient. Considering that APT/PCs are rare, new therapies should preferably be evaluated in shared standardized protocols. Prognostic and predictive markers to guide treatment decisions are needed and are the scope of ongoing research.

We present the case of a male patient who was ultimately diagnosed with Becker muscular dystrophy (BMD; MIM# 300376) after the onset of muscle weakness in his teens progressively led to significant walking difficulties in his twenties. A genetic diagnosis was pursued but initial investigation revealed no aberrations in the dystrophin gene (DMD), although immunohistochemistry and Western blot analysis suggested the diagnosis of dystrophinopathy. Eventually, after more than 10 years, an RNA analysis captured abnormal splicing where 154 nucleotides from intron 43 were inserted between exon 43 and 44 resulting in a frameshift and a premature stop codon. Normal splicing of the DMD gene was also observed. Additionally, a novel variant c.6291–13537A>G in DMD was confirmed in the genomic DNA of the patient. The predicted function of the variant aligns with the mRNA results. To conclude, we here demonstrate that mRNA analysis can guide the diagnosis of non-coding genetic variants in DMD.