Implementation of an ISO 15189 accredited next generation sequencing service for cell-free total nucleic acid (cfTNA) analysis to facilitate driver mutation reporting in blood: the experience of a clinical diagnostic laboratory.

AIMS: Next generation sequencing (NGS) on tumour tissue is integral to the delivery of personalised medicine and targeted therapy. NGS on liquid biopsy, a much less invasive technology, is an emerging clinical tool that has rapidly expanded clinical utility. Gene mutations in cell-free total nucleic acids (cfTNA) circulating in the blood are representative of whole tumour biology and can reveal different mutations from different tumour sites, thus addressing tumour heterogeneity challenges. METHODS: The novel Ion Torrent Genexus NGS system with automated sample preparation, onboard library preparation, templating, sequencing, data analysis and Oncomine Reporter software was used. cfTNA extracted from plasma was verified with the targeted pan-cancer (~50 genes) Oncomine Precision Assay (OPA). Assessment criteria included analytical sensitivity, specificity, limits of detection (LOD), accuracy, repeatability, reproducibility and the establishment of performance metrics. RESULTS: An ISO 15189 accredited, minimally invasive cfTNA NGS diagnostic service has been implemented. High sensitivity (>83%) and specificity between plasma and tissue were observed. A sequencing LOD of 1.2% was achieved when the depth of coverage was >22 000×. A reduction (>68%) in turnaround time (TAT) of liquid biopsy results was achieved: 5 days TAT for in-house analysis from sample receipt to a final report issued to oncologists as compared with >15 days from reference laboratories. CONCLUSION: Tumour-derived somatic variants can now be reliably assessed from plasma to provide minimally invasive tumour profiling. Successful implementation of this accredited service resulted in:Appropriate molecular profiling of patients where tumour tissue is unavailable or inaccessible.Rapid TAT of plasma NGS results.

RB1: governor of the cell cycle in health and disease-a primer for the practising pathologist.

RB1 stands as the pioneering discovery in tumour-suppressor genes, marking a pivotal breakthrough in comprehending cancer development. This overview delves into the role of RB1 in both health and disease, exploring its association with the tumourigenesis of various cancers and a distinct subset of soft-tissue neoplasms. Additionally, we discuss the application of immunohistochemistry and fluorescence in situ hybridisation to detect RB1 alterations.

Combined analysis of albumin in situ hybridisation and C reactive protein immunohistochemistry for the diagnosis of intrahepatic cholangiocarcinoma: towards a molecular classification paradigm.

AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a diagnosis of exclusion that can pose a challenge to the pathologist despite thorough clinical workup. Although several immunohistochemical markers have been proposed for iCCA, none of them reached clinical practice. We here assessed the combined usage of two promising diagnostic approaches, albumin in situ hybridisation (Alb-ISH) and C reactive protein (CRP) immunohistochemistry, for distinguishing iCCA from other adenocarcinoma primaries. METHODS: We conducted Alb-ISH and CRP immunohistochemistry in a large European iCCA cohort (n=153) and compared the results with a spectrum of other glandular adenocarcinomas of different origin (n=885). In addition, we correlated expression patterns with clinicopathological information and mutation data. RESULTS: Alb-ISH was highly specific for iCCA (specificity 98.8%) with almost complete negativity in perihilar CCA and only rare positives among other adenocarcinomas (sensitivity 69.5%). CRP identified the vast majority of iCCA cases (sensitivity 84.1%) at a lower specificity of 86.4%. Strikingly, the combination of CRP and Alb-ISH boosted the diagnostic sensitivity to 88.0% while retaining a considerable specificity of 86.1%. Alb-ISH significantly correlated with CRP expression, specific tumour morphologies and small or large duct iCCA subtypes. Neither Alb-ISH nor CRP was associated with iCCA patient survival. 16 of 17 recurrent mutations in either IDH1, IDH2 and FGFR2 affected Alb-ISH positive cases, while the only KRAS mutation corresponded to an Alb-ISH negative case. CONCLUSIONS: In conclusion, we propose a sequential diagnostic approach for iCCA, integrating CRP immunohistochemistry and Alb-ISH. This may improve the accuracy of CCA classification and pave the way towards a molecular-guided CCA classification.

Novel and unusual USP6 fusion partners in aneurysmal bone cyst and their role in pathogenesis and histopathological evaluation of this disease.

AIMS: The purpose of this study is to report novel and unusual USP6 fusion partners in aneurysmal bone cysts (ABCs). These findings may be useful in routine diagnostics as well as in studying the biology of USP6-related disorders. METHODS: A cohort of seven patients diagnosed with ABC examined between 2014 and 2023 at Motol University Hospital in Prague was included into this retrospective non-randomised study. All cases were analysed using histopathological evaluation, immunohistochemistry and Anchored multiplex RNA methods. Demographic characteristics and clinical data were also analysed. RESULTS: We identified two novel (ZFX and IP6K2), three unusual (MEF2A, EIF1 and COL1A2) and two common (CDH11) fusion partners with USP6 gene among all seven cases of ABC. CONCLUSIONS: Cases in our study were diagnosed as ABCs due to characteristic clinical and morphological presentation. However, not all cases are as self-evident, and molecular testing is necessary. The identification of these gene alterations can be useful in distinction between true ABC and ABC-like changes among many benign and malignant bone tumours.

Novel scoring system provides high separation of diploidy and triploidy to aid partial hydatidiform mole diagnosis: an adaption of HER2 D-DISH for ploidy analysis.

AIMS: Diagnosis of hydatidiform mole or molar pregnancy based on morphology alone can be challenging, particularly in early gestation, necessitating the use of ancillary techniques for accurate diagnosis. We sought to adapt the VENTANA HER2 dual-colour dual-hapten in-situ hybridisation (D-DISH) assay by using the internal chromosome 17 enumeration probe to determine ploidy status. METHODS: We selected 25 products of conception, consisting of molar and non-molar cases, to validate the HER2 D-DISH assay. These cases had prior morphological assessment by a perinatal pathologist and ploidy analysis using molecular cytogenetics. Three independent observers, blinded to the original histopathological and genetic diagnosis, scored 10 representative areas on each slide. Interobserver variability was assessed by comparing the total scores of each observer using analysis of variance (ANOVA) and the kappa statistic. RESULTS: Our ploidy scoring system accurately determined the correct number of diploid and triploid conceptuses, demonstrating complete concordance with pre-existing ploidy status and the initial diagnosis. Interobserver agreement between three independent scorers was robust: ANOVA (p=0.36) and kappa statistic (0.812, p<0.001). We achieved clear separation of average nuclear signals for diploid and triploid conceptuses, which was statistically significant (p<0.05). Employing our innovative scoring system, known as the 'rule of 5', we established ploidy decision thresholds for all 25 cases. CONCLUSIONS: Our modified HER2 D-DISH ploidy assay simplifies the process of ploidy determination and improves the accuracy of morphological diagnosis of molar pregnancy. The HER2 D-DISH assay was selected for ploidy analysis due to the widespread availability of in-situ hybridisation in pathology laboratories.

Appraisal of hydatidiform mole incidence and registration rates in Ireland following the establishment of a National Gestational Trophoblastic Disease Registry.

AIMS: This study aimed to re-evaluate the incidence of hydatidiform mole (HM) and determine gestational trophoblastic disease (GTD) registration rates in Ireland following the establishment of the National GTD Registry in 2017. METHODS: We performed a 3-year retrospective audit of HM cases (January 2017 to December 2019) reported in our centre. In 2019, we surveyed Irish pathology laboratories to determine the number of HMs diagnosed nationally and compared this data to that recorded in the National GTD Registry. Additionally, we compared both local and national HM incidence rates to those reported internationally. RESULTS: In the 3-year local audit, we identified 87 HMs among 1856 products of conception (POCs) providing a local HM incidence rate of 3.92 per 1000 births. The 1-year pathology survey recorded 170 HMs in 6008 POCs, yielding a national incidence rate of 2.86 per 1000 births. Importantly, the local HM incidence rate exceeded the national incidence rate by 37% and the local partial HM incidence (1 in 296 births) was 64% higher than the nationally incidence rate (1 in 484 births). Notably, 42% of the HM and atypical POCs diagnosed nationally were not reported to the National GTD Registry. CONCLUSIONS: Our study reveals increased HM incidence rates both locally and nationally compared with previous Irish studies. The higher local PHM incidence may reflect more limited access to ploidy analysis in other pathology laboratories nationally. Significantly, almost half of the women with diagnosed or suspected HM were not registered with the National GTD Centre.

PHOX2B: a diagnostic cornerstone in neurocristopathies and neuroblastomas.

Paired-like homeobox 2B (PHOX2B) is a gene essential in the development of the autonomic nervous system. PHOX2B mutations are associated with neurocristopathies-Hirschsprung disease (HSCR) and congenital central hypoventilation syndrome (CCHS)-and peripheral neuroblastic tumours. PHOXB2 plays an important role in the diagnostics of these conditions.Genotyping of a PHOX2B pathogenic variant is required to establish a diagnosis of CCHS. In HSCR patients, PHOX2B immunohistochemical staining has proven to be a valuable tool in identifying this disease. Furthermore, PHOXB2 is a predisposition gene for neuroblastoma, in which PHOX2B immunohistochemical staining can be used as a highly sensitive and specific diagnostic marker. The utility of PHOX2B immunohistochemistry in pheochromocytoma and paraganglioma has also been studied but yields conflicting results.In this review, an overview is given of PHOX2B, its associated diseases and the usefulness of PHOX2B immunohistochemistry as a diagnostic tool.

Rapid microdissection of tissue sections via laser ablation.

We demonstrate a method for tissue microdissection using scanning laser ablation that is approximately two orders of magnitude faster than conventional laser capture microdissection. Our novel approach uses scanning laser optics and a slide coating under the tissue that can be excited by the laser to selectively eject regions of tissue for further processing. Tissue was dissected at 0.117 s/mm2 without reduction in yield, sequencing insert size or base quality compared with undissected tissue. From eight cases, 58-416 mm2 of tissue was obtained from one to four slides in 7-48 seconds total dissection time per case. These samples underwent exome sequencing and we found the variant allelic fraction increased in regions enriched for tumour as expected. This suggests that our ablation technique may be useful as a tool in both clinical and research labs.

Increased PI3K pathway activity is associated with recurrent breast cancer in patients with low and intermediate 21-gene recurrence score.

AIMS: We investigated key signalling pathways' activity and mutational status of early-stage breast carcinomas with low and intermediate 21-gene recurrence score (RS) to identify molecular features that may predict recurrence. METHODS: This is a retrospective case-control study of 18 patients with recurrent breast carcinoma with low and intermediate 21-gene RS (<25) and control group of 15 non-recurrent breast cancer patients. DNA and mRNA were extracted from tumour tissue. mRNA expression of genes involved in oestrogen receptor (ER), androgen receptor (AR), PI3K and MAPK signalling pathways was measured by real-time quantitative reverse transcription-qPCR (OncoSIGNal G4 test, InnoSIGN). Tumour mutational landscape was assessed by targeted DNA sequencing (Oncomine Precision Assay). RESULTS: There were no statistical differences between the groups' demographic and clinicopathological characteristics. PI3K pathway showed significantly higher activity in cases compared with controls (p=0.0014). Receiver operating characteristic curve analysis showed an area under the curve of 0.79 for PI3K pathway activity in the prediction of recurrent disease in low and intermediate 21-gene RS breast cancer. There was no difference in ER, AR and MAPK pathway activity. PIK3CA alterations were the most common driver mutations, but no difference was found between the groups (p=0.46) and no association with PI3K pathway activity (p=0.86). Higher Ki67 gene expression was associated with recurrences (p=0.042) CONCLUSION: Increased PI3K pathway activity, independent of PIK3CA mutations, may play a role in the recurrence of early-stage breast cancer with low and intermediate 21-gene RS. Pathway analysis can help to identify high-risk patients in this setting.

Genomic discordances and heterogeneous mutational burden, PD-L1 expression and immune infiltrates of non-small cell lung cancer metastasis.

AIMS: To investigate the genomic discordances and heterogeneous mutational burden, PD-L1 expression and immune cell (IC) infiltrates of non-small cell lung cancer (NSCLC) metastasis. METHODS: Surgical samples from 41 cases of NSCLC with metastatic tumours (MTs) and paired primary tumours (PTs) were collected. PD-L1 expression and ICs were quantified using image-based immunohistochemistry profiling. Whole exome sequencing was employed to explore discrepancies in genomic characteristics, tumour mutational burden (TMB) and tumour neoantigen burden (TNB) in 28 cases. RESULTS: Non-synonymous mutations in MTs were slightly more than in PTs, with only 42.34% of mutations shared between paired PTs and MTs. The heterogeneity of TMB showed no significant difference (p=0.785) between MTs and PTs, while TNB significantly increased in MTs (p=0.013). MTs generally exhibited a higher density of PD-L1+ cells and a higher tumour proportion score with a lower density of IC infiltrates. Subgroup analysis considering clinicopathological factors revealed that the heterogeneity of immune biomarkers was closely associated with the histology of lung adenocarcinoma, metastatic sites of extrapulmonary, time intervals and treatment history. Prognosis analysis indicated that a high density of CD8+ T cells was a low-risk factor, whereas a high density of PD-L1+ cells in MTs was a high-risk factor for cancer-related death in metastatic NSCLC. CONCLUSIONS: The mutational burden, PD-L1 expression and IC infiltrates undergo changes during NSCLC metastasis, which may impact the immunotherapeutic benefits in patients with NSCLC with metastatic progression and should be monitored according to clinical scenarios.

Identification of immune cell markers associated with ulcerative colitis histological disease activity in colonic biopsies.

AIMS: Accurate determination of histological activity in ulcerative colitis (UC) is essential given its diagnostic and prognostic importance. Data on the relationship between histology and immune cell markers are limited. We aimed to evaluate the association between histological disease activity and immune cell marker concentration in colonic biopsies from patients with UC. METHODS: Sigmoid colon biopsies from 20 patients with UC were retrospectively assessed using the Robarts Histopathology Index (RHI). Targeted mass spectrometry determined the concentration of 18 immune cell markers (cluster of differentiation (CD) 4, CD8, CD19, CD20, CD40, CD56, CD68, CD103, forkhead box p3 (FOXP3), human leucocyte antigen, DR alpha chain (HLA-DRA), interleukin 10 (IL-10), IL-23 subunit alpha (IL-23A), IL-23 receptor (IL-23R), IL-2 receptor alpha chain (IL-2RA), Ki67, lymphocyte-activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1) and PD ligand 1 (PD-L1)). The association between RHI score and immune cell marker concentration was quantified using Spearman's rank correlation coefficient (ρ) and related 95% CIs. RESULTS: Fourteen of the 18 immune cell marker proteins were detected, with tissue concentration ranging from 0.003 to 11.53 fmol/µg. The overall RHI score was positively correlated with CD19, CD20, CD40, FOXP3, LAG-3, PD-1 and PD-L1 concentration (ρ=0.596-0.799) and negatively correlated with CD56 concentration (ρ=-0.460). There was no significant association between RHI score and CD4, CD8, CD68, CD103, HLA-DRA or Ki67 concentration. CONCLUSIONS: This study provides insight into the correlation between immune cell marker expression and histological disease activity and the possible molecular and immunological determinants underlying microscopic disease activity in UC.

Pathology focused review of morphologic subtypes and molecular variants of urothelial carcinoma with an emphasis on clinical/treatment relevance.

Urothelial carcinoma (UC) has significant morbidity, mortality, and remains the most financially costly carcinoma to manage and treat. This review will cover special morphologic features of UC that may be noted by the pathologist and any subsequent significance in terms of clinical management or treatment considerations as mentioned or recommended in the latest WHO 2022 classification of GU tumors. Many important potentially therapy altering morphologic findings can be consistently identified and reported on routine microscopic examination of hematoxylin and eosin (H&E) stained slides. Furthermore, there has been a rapid advancement of molecular diagnostics and tailored therapies throughout oncology, and we will briefly highlight some of these as they relate to the management of UC. We will actively attempt to limit the discussion of histologic descriptions or pathologic diagnostic criteria of these entities and focus rather on the recognition of their importance/implication for clinicians who must make clinical management decisions based upon these findings. Finally, the importance of open lines of communication with the pathologists who review clinical specimens as well as their practice and reporting methods cannot be overstated.

Clinicopathological characterisation of MTAP alterations in gastrointestinal cancers.

BACKGROUND: Methylthioadenosine phosphorylase (MTAP) is an essential metabolic enzyme in the purine and methionine salvage pathway. In cancer, MTAP gene copy number loss (MTAP loss) confers a selective dependency on the related protein arginine methyltransferase 5. The impact of MTAP alterations in gastrointestinal (GI) cancers remains unknown although hypothetically druggable. Here, we aim to investigate the prevalence, clinicopathological features and prognosis of MTAP loss GI cancers. METHODS: Cases with MTAP alterations were retrieved from The Cancer Genome Atlas (TCGA) and a real-world cohort of GI cancers profiled by next-generation sequencing. If MTAP alterations other than loss were found, immunohistochemistry was performed. Finally, we set a case-control study to assess MTAP loss prognostic impact. RESULTS: Findings across the TCGA dataset (N=1363 patients) and our cohort (N=508) were consistent. Gene loss was the most common MTAP alteration (9.4%), mostly co-occurring with CDKN2A/B loss (97.7%). Biliopancreatic and gastro-oesophageal cancers had the highest prevalence of MTAP loss (20.5% and 12.7%, respectively), being mostly microsatellite stable (99.2%). In colorectal cancer, MTAP loss was rare (1.1%), while most MTAP alterations were mutations (5/7, 71.4%); among the latter, only MTAP-CDKN2B truncation led to protein loss, thus potentially actionable. MTAP loss did not confer worse prognosis. CONCLUSIONS: MTAP alterations are found in 5%-10% of GI cancers, most frequently biliopancreatic and gastro-oesophageal. MTAP loss is the most common alteration, identified almost exclusively in MSS, CDKN2A/B loss, upper-GI cancers. Other MTAP alterations were found in colorectal cancer, but unlikely to cause protein loss and drug susceptibility.

Response to anti-HER2 neoadjuvant chemotherapy in HER2-positive invasive breast cancers with different HER2 FISH patterns.

AIMS: Human epidermal growth factor receptor 2 (HER2)-positive patients with breast cancer may have different HER2/CEP17 ratios and HER2 copy numbers, with inconsistent responses to anti-HER2 neoadjuvant chemotherapy (NACT). Our study aimed to explore the relationship between different HER2 fluorescence in situ hybridisation (FISH) patterns in HER2-positive patients with breast cancer and responses to anti-HER2 NACT. METHODS: 527 patients with HER2-positive invasive breast cancer who received anti-HER2 NACT from 2015 to 2022 were included and divided into three groups by FISH results, namely group A: HER2/CEP17<2.0 and HER2 copy numbers ≥6.0, HER2 immunohistochemistry 2/3+; group B: HER2/CEP17≥2.0 and HER2 copy numbers ≥4.0 and <6.0; group C: HER2/CEP17≥2.0 and HER2 copy numbers ≥6.0. We compared clinicopathological characteristics and pathological complete response (pCR) rates of different groups. RESULTS: According to HER2 FISH results, 12 patients (2.3%, 12/527) were in group A, 40 (7.6%, 40/527) were in group B and 475 (90.1%, 475/527) were in group C. The pCR rate was the lowest in group B (5.0%), while the pCR rates in group A and group C were 33.3% and 44.4%, respectively (p (group A vs. B) =0.021, p (group C vs. B) < 0.001). Both univariate and multivariate analyses revealed that HER2 FISH pattern was correlated with pCR rate (p (group C vs. B) < 0.001, p (group C vs. B) = 0.025). CONCLUSIONS: Patients with HER2/CEP17≥2.0 and HER2 copy numbers ≥4.0 and <6.0 do not benefit to the same extent from current anti-HER2 therapies as FISH-positive patients with other patterns.

Clinicopathological analysis of BRAF and non-BRAF MAPK pathway-altered gliomas in paediatric and adult patients: a single-institution study of 40 patients.

AIMS: Mitogen-activated protein kinase (MAPK) pathway alteration is a major oncogenic driver in paediatric low-grade gliomas (LGG) and some adult gliomas, encompassing BRAF (most common) and non-BRAF alterations. The aim was to determine the frequency, molecular spectrum and clinicopathological features of MAPK-altered gliomas in paediatric and adult patients at our neuropathology site in Kuwait. METHODS: We retrospectively searched the data of molecularly sequenced gliomas between 2018 and 2023 for MAPK alterations, revised the pathology in view of the 2021 WHO classification and evaluated the clinicopathological data for possible correlations. RESULTS: Of 272 gliomas, 40 (15%) harboured a MAPK pathway alteration in 19 paediatric (median 9.6 years; 1.2-17.6) and 21 adult patients (median 37 years; 18.9-89.2), comprising 42% and 9% of paediatric and adult cases, respectively. Pilocytic astrocytoma and glioblastoma were the most frequent diagnoses in children (47%) and adults (43%), respectively. BRAF V600E (n=17, 43%) showed a wide distribution across age groups, locations and pathological diagnoses while KIAA1549::BRAF fusion (n=8, 20%) was spatially and histologically restricted to cerebellar paediatric LGGs. Non-V600E variants and BRAF amplifications accompanied other molecular aberrations in high-grade tumours. Non-BRAF MAPK alterations (n=8) included mutations and gene fusions involving FGFR1, NTRK2, NF1, ROS1 and MYB. Fusions included KANK1::NTRK2, GOPC::ROS1 (both infant hemispheric gliomas), FGFR1::TACC1 (diffuse LGG), MYB::QKI (angiocentric glioma) and BCR::NTRK2 (glioblastoma). Paradoxical H3 K27M/MAPK co-mutations were observed in two LGGs. CONCLUSION: The study provided insights into MAPK-altered gliomas in Kuwait highlighting the differences among paediatric and adult patients and providing a framework for planning therapeutic polices.

Improving practice in PD-L1 testing of non-small cell lung cancer in the UK: current problems and potential solutions.

AIMS: Programmed cell death ligand 1 (PD-L1) expression, used universally to predict response of non-small cell lung cancer (NSCLC) to immune-modulating drugs, is a fragile biomarker due to biological heterogeneity and challenges in interpretation. The aim of this study was to assess current PD-L1 testing practices in the UK, which may help to define strategies to improve its reliability and consistency. METHODS: A questionnaire covering NSCLC PD-L1 testing practice was devised and members of the Association of Pulmonary Pathologists were invited to complete this online. RESULTS: Of 44 pathologists identified as involved in PD-L1 testing, 32 (73%) responded. There was good consistency in practice and approach, but there was wide variability in the distribution of PD-L1 scoring. Although the proportions of scores falling into the three groups (negative, low and high) defined by the 1% and 50% 'cut-offs' (38%, 33% and 27%, respectively) reflect the general experience, the range within each group was wide at 23-70%, 10-60% and 15-36%, respectively. CONCLUSIONS: There is inconsistency in the crucial endpoint of PD-L1 testing of NSCLC, the expression score that guides management. Addressing this requires formal networking of individuals and laboratories to devise a strategy for its reduction.

Primary salivary duct carcinoma of the lung: clinicopathological features, diagnosis and practical challenges.

AIMS: To investigate the clinicopathological features, molecular characteristics and diagnostic criteria of primary salivary duct carcinoma of the lung (LSDC). METHODS: We analysed the clinicopathological and molecular features of five cases of LSDC retrieved from the archives of Shanghai Pulmonary Hospital from 2020 to 2022, and reviewed the relevant literature. RESULTS: All patients were men, with an average age of 66 years (age range: 49-79 years), and all lesions were central masses with a mean maximum diameter of 42.6 mm (range: 16-70 mm). Morphologically, LSDC comprised of intraductal and invasive components. Both the intraductal and invasive components of LSDC can exhibit papillary, micropapillary, cribriform, tubule structures and solid proliferation. The intraductal component can exhibit Roman bridge structures, which were usually accompanied by central comedo-like necrosis. Immunohistochemically, LSDCs consistently expressed cytokeratin (CK)7 (5 of 5) and showed variable positivity of androgen receptor (AR) (5 of 5) focally or diffusely; additionally, the tumour cells expressed human epidermal growth factor receptor 2 (HER2) (3+, n=3; 2+, n=2), GATA-binding protein 3 (3 of 5), and gross cystic disease fluid protein-15 (1 of 5), and all of which were negative for thyroid transcription factor-1, napsin A, p40, CK5/6 and p63. The residual basal/myoepithelial cells surrounding the in situ carcinoma expressed p40, CK5/6 and p63. TP53 mutation and HER2 gene amplification (3 of 5) were the most frequent genetic alterations in LSDC. All patients who underwent surgical lobectomies were alive without recurrence or metastasis. CONCLUSIONS: LSDC is a highly rare malignant tumour. The distinctive architecture of in situ carcinoma and tumour cells expressing AR can provide diagnostic indications for LSDC.

Gene of the month: DDIT3.

DNA damage-inducible transcript 3 (DDIT3) gene, mapped to the human chromosome 12q13.3, encodes a protein that belongs to the CCAAT/enhancer-binding protein family of transcription factors. DDIT3 is involved in the proliferative control that responds to endoplasmic reticulum stress in normal conditions, dimerising other transcription factors with basic leucine zipper (bZIP) structural motifs. DDIT3 plays a significant role during cell differentiation, especially adipogenesis, arresting the maturation of adipoblasts. In disease, FUS/EWSR1::DDIT3 fusion is the pathogenic event that drives the development of myxoid liposarcoma. The amplification of DDIT3 in other adipocytic neoplasms mediates the presence of adipoblast-like elements. Another fusion, GLI1::DDIT3, has rarely been documented in other tumours. This paper reviews the structure and function of DDIT3, its role in disease-particularly cancer-and its use and pitfalls in diagnostic testing, including immunohistochemistry as a tissue-based marker.