Imaging extrachromosomal DNA (ecDNA) in cancer.

Extrachromosomal DNA (ecDNA) are circular regions of DNA that are found in many cancers. They are an important means of oncogene amplification, and correlate with treatment resistance and poor prognosis. Consequently, there is great interest in exploring and targeting ecDNA vulnerabilities as potential new therapeutic targets for cancer treatment. However, the biological significance of ecDNA and their associated regulatory control remains unclear. Light microscopy has been a central tool in the identification and characterisation of ecDNA. In this review we describe the different cellular models available to study ecDNA, and the imaging tools used to characterise ecDNA and their regulation. The insights gained from quantitative imaging are discussed in comparison with genome sequencing and computational approaches. We suggest that there is a crucial need for ongoing innovation using imaging if we are to achieve a full understanding of the dynamic regulation and organisation of ecDNA and their role in tumourigenesis.

The frequency and clinical outcome of mono-hit and multi-hit TP53 aberrations in newly diagnosed multiple myeloma.

We investigated the frequency and outcome of mono-hit and multi-hit TP53 aberrations [biallelic or ≥1 TP53 mutations (TP53mut) or TP53mut with variant allele frequency (VAF) ≥55%] in an Indian cohort of newly diagnosed multiple myeloma (NDMM) patients. We employed fluorescence insitu hybridisation (FISH; n=457) and targeted next-generation sequencing (NGS; n=244) on plasma cell-enriched samples. We also studied the impact of TP53mut in cases with and without TP53 deletions (TP53del). In our cohort with a median age of 60 years, TP53del and TP53mut were seen in 12.9% (n=59/457; 14-95% cells) and 10.2% (n=25/244; 30 variants; VAF 3.4-98.2%; median 38.2%) respectively. Mono-hit and multi-hit-TP53 aberrations were observed in 10.2% and 7.8%, respectively. Compared to TP53-wild-type (TP53wt), mono-hit and multi-hit TP53 aberrations were associated with significantly poorer progression-free survival (PFS) (22.6 vs 12.1 vs 9.5 months; p=0.004) and overall survival (OS) [not reached (NR) vs 13.1 vs 15.6 months respectively; p=0.024]. However, multi-hit TP53 did not significantly differ in OS/PFS compared to mono-hit cases. Compared to TP53wt, PFS and OS were significantly poorer in patients with TP53mut only (9.5 vs 22.6 months and 12.1 months vs NR, respectively; p=0.020/0.004). TP53mut retained its significance even in the presence of any Revised International Staging System (HR 2.1; 95% CI 1.1-3.8; p=0.015) for OS. The detection of additional cases with TP53 aberrations, as well as poor survival associated with the presence of mutation alone, supports TP53mut testing in NDMM at least in patients without TP53del and other high-risk cytogenetic abnormalities.

Comparison of an amplicon-based large panel next generation sequencing (NGS) assay with conventional testing methods for MET and HER2 amplification in lung and breast cancers.

The frequency of MET and HER2 amplification being detected by next generation sequencing (NGS) is increasing due to NGS being increasingly adopted for molecular profiling of cancers. However, the accuracy of NGS in detecting these gene amplifications remains uncertain due to conflicting reports in the scientific literature. We studied the accuracy of an amplicon-based large panel NGS assay in detecting MET and HER2 amplification in lung and breast cancers, respectively, by comparing it against conventional testing methods. Amongst 48 lung cancers, four of five cancers that were MET amplified on fluorescence in situ hybridisation (FISH) were classified as amplified on NGS while 42 of the remaining 43 non-amplified cancers were classified as non-amplified on NGS, giving a sensitivity of 80%, specificity of 97.7% and overall concordance of 95.8%. Of the 46 breast cancers tested, only six of the nine cancers that were HER2-positive on immunohistochemistry (IHC)/FISH were HER2-positive on NGS, while all the remaining HER2-negative cases were negative on NGS, giving a sensitivity of 66.7%, specificity of 100% and overall concordance of 93.5%. All the false-negative cases had low level gene amplification (MET:CEP7 or HER2:CEP17 FISH ratio of <3). The low sensitivity for HER2 amplification may be confounded by the small sample size and disproportionate number of cases with low level amplification. In summary, the NGS assay has good concordance with conventional testing methods but may be less sensitive in detecting low level gene amplification.

Chromosomal defects in multiple myeloma.

Multiple myeloma is a plasma cell neoplasm driven by primary (e.g. hyperdiploidy; IGH translocations) and secondary (e.g. 1q21 gains/amplifications; del(17p); MYC translocations) chromosomal events. These are important to detect as they influence prognosis, therapeutic response and disease survival. Currently, cytogenetic testing is most commonly performed by interphase fluorescence in situ hybridisation (FISH) on aspirated bone marrow samples. A number of variations to FISH methodology are available, including prior plasma cell enrichment and incorporation of immunophenotypic plasma cell identification. Other molecular methods are increasingly being utilised to provide a genome-wide view at high resolution (e.g. single nucleotide polymorphism (SNP) microarray analysis) and these can detect abnormalities in most cases. Despite their wide application at diagnostic assessment, both FISH and SNP-array have relatively low sensitivity, limiting their use for identification of prognostically significant low-level sub-clones or for disease monitoring. Next-generation sequencing is increasingly being used to detect mutations and new FISH techniques such as by flow cytometry are in development and may address some of the current test limitations. Here we review the primary and secondary cytogenetic aberrations in myeloma and discuss the range of techniques available for their assessment.

CDKN2A/B deletion in IDH-mutant astrocytomas: An evaluation by Fluorescence in-situ hybridization.

INTRODUCTION: CDKN2A/B homozygous deletion is one of the defining features of grade 4 in IDH-mutant astrocytic tumours. AIM: To evaluate CDKN2A/B-deletion in IDH-mutant astrocytic tumours and its clinicopathological impact. MATERIALS AND METHODS: CDKN2A/B-deletion was evaluated by Fluorescence in-situ hybridisation (FISH) and interpreted by two recently accepted methods. RESULTS: Eighty-three out of 94 cases (histologically-grade 2: 3, grade 3: 46, grade 4: 34) were interpretable on FISH. Concordant CDKN2A/B-deletion was observed in 71% (27/38) of lower-grade tumours (n = 49) and 90% (27/30) of histological grade 4 tumours (n = 34). Both the interpretation methods showed good agreement (Kappa = 0.75). CDKN2A/B-deletion showed an inverse correlation for < 10% MIB-1 labeling index (p = 0.01) while that by method-2 showed a significant correlation for grade 4 (p = 0.02). No significant correlation was observed for any other clinicopathological parameters. Twenty-four patients showed progression/recurrence (including deaths), and no significant difference in frequency of CDKN2A/B deletion was observed among cases with disease progression across different histological grades. CONCLUSIONS: CDKN2A/B-deletion was observed across all the histological grades of IDH-mutant astrocytic tumours, expectedly more in the higher grade. FISH, as a method, can be used for the detection of CDKN2A/B homozygous deletion, when there is concordant interpretation.

Translational practice of fluorescence in situ hybridisation to identify neuroblastic tumours with TERT rearrangements.

Recently, telomerase reverse transcriptase (TERT) gene rearrangements have been identified in neuroblastoma (NB), the typical pathological type of neuroblastic tumours (NTs); however, the prevalence of TERT rearrangements in other types of NT remains unknown. This study aimed to develop a practical method for detecting TERT defects and to evaluate the clinical relevance of TERT rearrangements as a biomarker for NT prognosis. A TERT break-apart probe for fluorescence in situ hybridisation (FISH) was designed, optimised, and applied to assess the genomic status of TERT in Chinese children with NTs at the Beijing Children's Hospital from 2016 to 2019. Clinical, histological, and genetic characteristics of TERT-rearranged NTs were further addressed. Genomic TERT rearrangements could be effectively detected by FISH and were mutually exclusive with MYCN amplification. TERT rearrangements were identified in 6.0% (38/633) of NTs overall, but 12.4% (31/250) in high-risk patients. TERT rearrangements identified a subtype of aggressive NTs with the characteristics of Stage 3/4, high-risk category, over 18 months old, and presenting all histological subtypes of NB and ganglioneuroblastoma nodular. Moreover, TERT rearrangements were significantly associated with elevated TERT expression levels and decreased survival chances. Multivariable analysis confirmed that it was an independent prognostic marker for NTs. FISH is an easily applicable method for evaluating TERT defects, which define a subgroup of NTs with unfavourable prognosis. TERT rearrangements would contribute to characterising NT molecular signatures in clinical practice.

Chromosomal radiosensitivity in oncological and non-oncological patients with rheumatoid arthritis and connective tissue diseases.

BACKGROUND: The risk of developing late radiotoxicity after radiotherapy in patients with high chromosomal radiosensitivity after radiotherapy could potentially be higher compared to the risk in patients with average radiosensitivity. In case of extremely high radiosensitivity, dose reduction may be appropriate. Some rheumatic diseases (RhD), including connective tissue diseases (CTDs) appear to be associated with higher radiosensitivity. The question arises as to whether patients with rheumatoid arthritis (RA) also generally have a higher radiosensitivity and whether certain parameters could indicate clues to high radiosensitivity in RA patients which would then need to be further assessed before radiotherapy. METHODS: Radiosensitivity was determined in 136 oncological patients with RhD, 44 of whom were RA patients, and additionally in 34 non-oncological RA patients by three-colour fluorescence in situ hybridization (FiSH), in which lymphocyte chromosomes isolated from peripheral blood are analysed for their chromosomal aberrations of an unirradiated and an with 2 Gy irradiated blood sample. The chromosomal radiosensitivity was determined by the average number of breaks per metaphase. In addition, correlations between certain RA- or RhD-relevant disease parameters or clinical features such as the disease activity score 28 and radiosensitivity were assessed. RESULTS: Some oncological patients with RhD, especially those with connective tissue diseases have significantly higher radiosensitivity compared with oncology patients without RhD. In contrast, the mean radiosensitivity of the oncological patients with RA and other RhD and the non-oncological RA did not differ. 14 of the 44 examined oncological RA-patients (31.8%) had a high radiosensitivity which is defined as ≥ 0.5 breaks per metaphase. No correlation of laboratory parameters with radiosensitivity could be established. CONCLUSIONS: It would be recommended to perform radiosensitivity testing in patients with connective tissue diseases in general. We did not find a higher radiosensitivity in RA patients. In the group of RA patients with an oncological disease, a higher percentage of patients showed higher radiosensitivity, although the average radiosensitivity was not high.

Molecular Detection of the Harmful Raphidophyte Chattonella subsalsa Biecheler by Whole-Cell Fluorescence in-situ Hybridisation Assay.

Species of the genus Chattonella (Raphidophyceae) are a group of marine protists that are commonly found in coastal waters. Some are known as harmful microalgae that form noxious blooms and cause massive fish mortality in finfish aquaculture. In Malaysia, blooms of Chattonella have been recorded since the 1980s in the Johor Strait. In this study, two strains of Chattonella were established from the strait, and morphological examination revealed characteristics resembling Chattonella subsalsa. The molecular characterization further confirmed the species' identity as C. subsalsa. To precisely detect the cells of C. subsalsa in the environment, a whole-cell fluorescence in-situ hybridisation (FISH) assay was developed. The species-specific oligonucleotide probes were designed in silico based on the nucleotide sequences of the large subunit (LSU) and internal transcribed spacer 2 (ITS2) of the ribosomal DNA (rDNA). The best candidate signature regions in the LSU-rRNA and ITS2-rDNA were selected based on hybridisation efficiency and probe parameters. The probes were synthesised as biotinylated probes and tested by tyramide signal amplification with FISH (FISH-TSA). The results showed the specificity of the probes toward the target cells. FISH-TSA has been proven to be a potential tool in the detection of harmful algae in the environment and could be applied to the harmful algal monitoring program.

Spesies genus Chattonella (Raphidophyceae) ialah sekumpulan protista marin yang biasa ditemui di perairan laut pantai. Sesetengahnya dikenali sebagai mikroalga berbahaya yang membentuk ledakan alga berbahaya dan menyebabkan kematian ikan secara besar-besaran dalam akuakultur ikan sirip. Di Malaysia, ledakan alga Chattonella telah direkodkan sejak tahun 1980-an di Selat Johor. Dalam kajian ini, dua strain Chattonella telah didirikan dari selat, dan pemeriksaan morfologi mendedahkan ciri-ciri yang menyerupai Chattonella subsalsa. Pencirian molekul seterusnya mengesahkan identiti spesies sebagai C. subsalsa. Untuk mengesan dengan tepat sel-sel C. subsalsa di dalam persekitaran, ujian penghibridan in-situ berpendarfluor (FISH) ke atas sel keseluruhan telah dibangunkan. Prob oligonukleotida spesies telah direka secara spesifik secara siliko berdasarkan jujukan nukleotida subunit besar (LSU) dan spacer transkripsi dalaman 2 (ITS2) gen DNA ribosom (rDNA). Calon terbaik kawasan tanda dalam LSU-rRNA dan ITS2-rDNA telah dipilih berdasarkan kecekapan penghibridan dan parameter prob. Prob telah disintesis sebagai prob biotinilasi dan diuji dengan penguatan isyarat tyramide dengan FISH (FISH-TSA). Keputusan menunjukkan kekhususan prob ke atas sel sasaran. FISH-TSA telah terbukti sebagai alat yang berpotensi dalam pengesanan alga berbahaya di alam sekitar dan boleh digunakan untuk program pemantauan alga berbahaya.

Improvement of diagnosis in children with Burkitt lymphoma in Kenya: feasibility study for the implementation of fluorescence in situ hybridisation testing for MYC and the MYC/IGH translocation.

Background: Indiana University (IU) initiated fluorescence in situ hybridisation (FISH) methodology for Burkitt Lymphoma (BL) to advance the accuracy and speed of diagnosis in the AMPATH Reference Laboratory at Moi Teaching and Referral Hospital (MTRH) in Eldoret, Kenya. Standard diagnostic testing for BL at MTRH includes morphology of the biopsy specimen or aspirate and limited immunohistochemistry panels. Methods: Tumour specimens from 19 children enrolled from 2016 to 2018 in a prospective study to improve the diagnosis and staging of children with suspected BL were evaluated. Touch preps from biopsy specimens or smears from fine needle aspiration were collected, stained with Giemsa and/or H&E and reviewed by pathologists to render a provisional diagnosis. Unstained slides were stored and later processed for FISH. Duplicate slides were split between two laboratories for analysis. Flow cytometry results were available for all specimens. Results from the newly established FISH laboratory in Eldoret, Kenya were cross-validated in Indianapolis, Indiana. Results: Concordance studies found 18 of 19 (95%) of specimens studied yielded analysable FISH results for one or both probe sets (MYC and MYC/IGH) in both locations. There was 94% (17/18) concordance of results between the two FISH laboratories. FISH results were 100% concordant for the 16 specimens with a histopathological diagnosis of BL and two of three non-BL cases (one case no result in IU FISH lab). FISH was similarly concordant with flow cytometry for specimens with positive flow results with the exception of a nasopharyngeal tumour with positive flow results for CD10 and CD20 but was negative by FISH. The modal turn-around time for FISH testing on retrospective study specimens performed in Kenya ranged between 24 and 72 hours. Conclusion: FISH testing was established, and a pilot study performed, to assess the feasibility of FISH as a diagnostic tool for the determination of BL in a Kenyan paediatric population. This study supports FISH in limited resource settings to improve the accuracy and speed of diagnosis of BL in Africa.

Signet ring cell-like diffuse large B-cell lymphoma involving the breast: a case report.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) with signet ring cell components is extremely rare. Here, we present a case of DLBCL with signet ring cell components involving the breast, which can be easily confused with invasive lobular carcinoma of the breast or metastatic signet ring cell carcinoma of gastrointestinal origin. CASE PRESENTATION: A 66-year-old woman presented with a painless mass in her left breast. Enhanced magnetic resonance imaging (MRI) of the breast revealed a 42 × 29 × 28 mm mass in the left breast. Histological examination revealed a diffuse or scattered arrangement of round cells mixed with signet ring-like cells. Immunohistochemically, the neoplastic cells were positive for PAX-5, CD79a, CD20, Bcl-6, and MUM-1 but and negative for cytokeratin, ER, PR, E-cadherin, and P120. The Ki-67 proliferation index was approximately 70%. Fluorescence in situ hybridisation (FISH) demonstrated non-rearrangement of Bcl-2, Bcl-6, and c-MYC genes. Immunohistochemistry and FISH examination confirmed the diagnosis of DLBCL. Subsequently, immunofluorescence showed both IgM and IgG deposits in the signet ring-like lymphocytes. After confirming the diagnosis, the patient received four courses of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy in a specialist hospital and achieved partial remission; however, she unfortunately died of secondary pneumocystis pneumonia infection 3 months later. CONCLUSION: Malignant lymphoma with signet ring cell morphology is quite uncommon, and this variant can be a diagnostic pitfall. We emphasise that pathologists should consider lymphoma in the differential diagnosis of malignant breast tumours.

Stellate cell expression of SPARC-related modular calcium-binding protein 2 is associated with human non-alcoholic fatty liver disease severity.

Background & Aims: Histological assessment of liver biopsies is the gold standard for diagnosis of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD), despite its well-established limitations. Therefore, non-invasive biomarkers that can offer an integrated view of the liver are needed to improve diagnosis and reduce sampling bias. Hepatic stellate cells (HSCs) are central in the development of hepatic fibrosis, a hallmark of NASH. Secreted HSC-specific proteins may, therefore, reflect disease state in the NASH liver and serve as non-invasive diagnostic biomarkers. Methods: We performed RNA-sequencing on liver biopsies from a histologically characterised cohort of obese patients (n = 30, BMI >35 kg/m2) to identify and evaluate HSC-specific genes encoding secreted proteins. Bioinformatics was used to identify potential biomarkers and their expression at single-cell resolution. We validated our findings using single-molecule fluorescence in situ hybridisation (smFISH) and ELISA to detect mRNA in liver tissue and protein levels in plasma, respectively. Results: Hepatic expression of SPARC-related modular calcium-binding protein 2 (SMOC2) was increased in NASH compared to no-NAFLD (p.adj <0.001). Single-cell RNA-sequencing data indicated that SMOC2 was primarily expressed by HSCs, which was validated using smFISH. Finally, plasma SMOC2 was elevated in NASH compared to no-NAFLD (p <0.001), with a predictive accuracy of AUROC 0.88. Conclusions: Increased SMOC2 in plasma appears to reflect HSC activation, a key cellular event associated with NASH progression, and may serve as a non-invasive biomarker of NASH. Impact and implications: Non-alcoholic fatty liver disease (NAFLD) and its progressive form, non-alcoholic steatohepatitis (NASH), are the most common forms of chronic liver diseases. Currently, liver biopsies are the gold standard for diagnosing NAFLD. Blood-based biomarkers to complement liver biopsies for diagnosis of NAFLD are required. We found that activated hepatic stellate cells, a cell type central to NAFLD pathogenesis, upregulate expression of the secreted protein SPARC-related modular calcium-binding protein 2 (SMOC2). SMOC2 was elevated in blood samples from patients with NASH and may hold promise as a blood-based biomarker for the diagnosis of NAFLD.

Molecular testing in melanoma for the surgical pathologist.

The diagnostic work-up of melanocytic tumours has undergone significant changes in the last years following the exponential growth of molecular assays. For the practising pathologist it is often difficult to sort through the multitude of different tests that are currently available for clinical use. The molecular tests used in melanocytic pathology can be broadly divided into four categories: (1) tests that predict response to systemic therapy in melanoma; (2) tests that predict prognosis in melanoma; (3) tests useful in determining the type or class of melanocytic tumour; and (4) tests useful in the differential diagnosis of naevus versus melanoma (primarily used as an aid in the diagnosis of histologically ambiguous melanocytic lesions). This review will present an updated synopsis of major molecular ancillary tests used in clinical practice.

The Chromosome Number and rDNA Loci Evolution in Onobrychis (Fabaceae).

The evolution of chromosome number and ribosomal DNA (rDNA) loci number and localisation were studied in Onobrychis Mill. Diploid and tetraploid species, as well as two basic chromosome numbers, x = 7 and x = 8, were observed among analysed taxa. The chromosomal distribution of rDNA loci was presented here for the first time using fluorescence in situ hybridisation (FISH) with 5S and 35S rDNA probes. Onobrychis species showed a high polymorphism in the number and localisation of rDNA loci among diploids, whereas the rDNA loci pattern was very similar in polyploids. Phylogenetic relationships among the species, inferred from nrITS sequences, were used as a framework to reconstruct the patterns of basic chromosome number and rDNA loci evolution. Analysis of the evolution of the basic chromosome numbers allowed the inference of x = 8 as the ancestral number and the descending dysploidy and polyploidisation as the major mechanisms of the chromosome number evolution. Analyses of chromosomal patterns of rRNA gene loci in a phylogenetic context resulted in the reconstruction of one locus of 5S rDNA and one locus of 35S rDNA in the interstitial chromosomal position as the ancestral state in this genus.

Chromosomal instability (CIN) in HAP1 cell lines revealed by multiplex fluorescence in situ hybridisation (M-FISH).

BACKGROUND: HAP1, a near-haploid human leukemic cancer cell line is often used in combination with CRISPR-Cas9 gene editing technology for genetic screens. HAP1 carries the Philadelphia chromosome (Ph) and an additional ~ 30 Mb fragment of chromosome 15 inserted into chromosome 19. The potential use of an in vitro cell line as a model system in biomedical research studies depends on its ability to maintain genome stability. Being a cancer cell line with a near-haploid genome, HAP1 is prone to genetic instability, which is further compounded by its tendency to diploidise in culture spontaneously. Moreover, CRISPR-Cas9 gene editing coupled with prolonged in-vitro cell culturing has the potential to induce unintended 'off-target' cytogenetic mutations. To gain an insight into chromosomal instability (CIN) and karyotype heterogeneity, 19 HAP1 cell lines were cytogenetically characterised, 17 of which were near-haploids and two double-haploids, using multiplex fluorescence in situ hybridisation (M-FISH), at single cell resolution. We focused on novel numerical (N) and structural (S) CIN and discussed the potential causal factors for the observed instability. For each cell line we examined its ploidy, gene editing status and its length of in-vitro cell culturing. RESULTS: Sixteen of the 19 cell lines had been gene edited with passage numbers ranging from 10 to 35. Diploidisation in 17 near-haploid cell lines ranged from 4 to 35% and percentage of N- and S-CIN in [1n] and [2n] metaphases ranged from 7 to 50% with two cell lines showing no CIN. Percentage of cells with CIN in the two double-haploid cell lines were 96% and 100% respectively. The most common S-CIN observed was deletion followed by translocation of both types, non-reciprocal and Robertsonian. Interestingly, we observed a prevalence of S-CIN associated with chromosome 13 in both near-and double-haploid cell lines, with a high incidence of Robertsonian translocation involving chromosome 13. Furthermore, locus-specific BAC (bacterial artificial chromosome) FISH enabled us to show for the first time that the additional chromosome 15 fragment is inserted into the p-arm rather than the q-arm of chromosome 19 of the HAP1 genome. CONCLUSION: Our study revealed a high incidence of CIN leading to karyotype heterogeneity in majority of the HAP1 cell lines with the number of chromosomal aberrations varying between cell lines. A noteworthy observation was the high frequency of structural chromosomal aberrations associated with chromosome 13. We showed that CRISPR-Cas9 gene editing technology in combination with spontaneous diploidisation and prolonged in-vitro cell culturing is potentially instrumental in inducing further chromosomal rearrangements in the HAP1 cell lines with existing CIN. We highlight the importance of maintaining cell lines at low passage and the need for regular monitoring to prevent implications in downstream applications. Our study also established that the additional fragment of chromosome 15 in the HAP1 genome is inserted into chromosome 19p rather than 19q.

Hyperplastic melanocytes with chromosomal aberrations in surrounding skin of subungual melanoma: fluorescence in situ hybridisation analysis using whole-slide digital imaging.

AIMS: Subungual melanoma (SUM) is increasingly being treated with conservative surgery. Consequently, the evaluation of the resection margins has increased in importance. However, in several cases it is difficult to distinguish the in-situ lesion of SUM from hyperplastic melanocytes in the surrounding skin. We examined whether PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemistry and fluorescence in situ hybridisation (FISH) labelling of CCND1 (11q13), RREB1 (6p25), MYB (6q23), and centromere 6 (CEP6) genes differentiated SUM from hyperplastic melanocytes. METHODS AND RESULTS: We reviewed specimens of 36 SUM cases and compared PRAME immunostains of invasive melanoma, melanoma in situ, and hyperplastic melanocytes. PRAME-positive cases accounted for 90.5% of invasive melanoma, 88.9% of in situ melanoma, and 59.4% of hyperplastic melanocyte specimens. While invasive and in situ melanomas in more than half of the examined cases were diffusely positive, this was found for only 9.4% of hyperplastic melanocyte cases. Four-coloured FISH using whole-slide digital imaging was used to analyse positive detection rates and changes in chromosomal aberrations. The FISH positive detection rate was 100% in invasive melanomas, 94.7% in melanomas in situ, and 66.7% in hyperplastic melanocytes. The number of RREB1 (6p25) signals per cell was significantly amplified following tumour progression. CONCLUSION: Hyperplastic melanocytes in the surrounding skin of SUM, considered morphologically non-neoplastic, showed chromosomal aberrations similar to those in melanoma. Such cells are also thought similar to the field cells of acral melanomas. Thus, whole-slide digital imaging is a technique that allows the evaluation of individual melanocyte lesions by FISH.

ALK-positive advanced non-small cell lung cancer patients with poor performance status: Outcomes in a real-world scenario.

Background: Anaplastic lymphoma kinase (ALK) inhibitors have shown significant efficacy in ALK -rearranged non-small cell lung cancer (NSCLC) patients with good performance status (PS) in multiple randomised studies. However, there is limited data on patients with poor performance status. Patients and methods: We carried out a retrospective analysis of prospectively collected data of patients with ALK-rearranged NSCLC and Eastern Cooperative Oncology Group (ECOG) PS of 2-4 treated at a single academic cancer centre from January 2013 to November 2018. The outcomes, progression-free survival (PFS) and overall survival (OS) were calculated from the date of diagnosis. SPSS version 20 was used for all statistical calculations. Results: Out of the total 441 ALK-positive patients, 97 (21.9%) had ECOG PS 2-4 (poor PS). The median PFS was 9.3 months (95% CI = 6.6-12.0) as compared to 14.9 months (95% CI = 13.4-16.4) for patients with a PS of 0-1 (HR = 1.38, 95% CI = 1.04-1.84, p = 0.027). The corresponding median OS were 17.9 months (95% CI = 12.8-23.1) and 33.5 months (95% CI = 28.6-38.4), respectively (HR = 1.89, 95% CI = 1.36-2.62, p < 0.001). Among poor PS patients, a subgroup of patients with PS 2 had median OS of 20.6 months (95% CI = 10.8-47.3) as compared to 8.6 months for PS 3-4 (95% CI = 7.8-27.8) (HR = 1.79, 95% CI = 1.01-3.20, p = 0.047). The patients treated with upfront ALK inhibitors had better survival as opposed to those treated with chemotherapy. On multivariate analysis, PS 3-4, smoking, stage 4 and not using ALK inhibitors as first-line therapy were associated significantly with poor outcomes. Conclusion: The ALK-rearranged NSCLC patients with poor PS derived significant benefits with ALK inhibitors. The outcomes were significantly poorer as compared to patients with PS 0-1; the subgroup of patients with PS 2 had better outcomes as compared to patients with PS 3-4.

Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences.

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.

The highlights of the 15th international conference of the international mesothelioma interest group - Do molecular concepts challenge the traditional approach to pathological mesothelioma diagnosis?

Pathology plays an important role in diagnosing mesothelioma since radiological and clinical findings alone cannot distinguish mesothelioma reliably from its many mimics. The long-held gold standard for pathological diagnosis requires a tissue biopsy that, in addition to mesothelial phenotype, demonstrates invasion, but this is challenged by the WHO recognition of mesothelioma in situ (MIS) and concurrent acknowledgement of all mesotheliomas as malignant. Tumor sampling and ancillary techniques are of paramount importance for diagnosis of MIS. Standardisation of these techniques, cut-off points and terminology, and an updated staging system are urgently required. These clinically relevant issues and the impact of new developments were illustrated at the pathology session of 15th meeting of the International Mesothelioma Interest Group. It was reported that combination of losses in p16 nuclear expression, with cut-off ≤ 1%, and cytoplasmic MTAP with cut-off ≥ 30% demonstrated increased specificity (96%) and high sensitivity (86%) for CDKN2A HD detection. Otherwise, the combination of p16 IHC and CDKN2A HD may improve prognosis. The potential usefulness of pleural effusions for early diagnosis was demonstrated in a retrospective study investigating pleural effusions had been diagnosed as benign prior to mesothelioma diagnosis. Alterations of BAP1 (IHC) and CDKN2A (FISH) were detectable 2 or more years prior diagnosis. Moreover, analysis of gene expression profiles in cytology samples by principal component analysis discriminated reactive hyperpasia from epitheliod mesothelioma. Early diagnosis, including cytology diagnosis, is being acyively investigated. Since no treatment recommendations exist for MIS, pathologists recognise the need for international collaborations to fully characterise this rare entity. Clear communication with the clinical teams is required to ensure optimum patient care. The data reported in this meeting are encouraging and open avenues for further work that will allow even earlier diagnosis and better characterisation of mesothelioma progression, based on changes in gene expression, including epigenetic changes.

A comparison of two methods for the detection of circulating tumour cells in patients with oral cavity cancer.

BACKGROUND: Circulating tumour cells (CTCs) detected in patient blood samples are relevant as diagnostic and prognostic markers offering insights into tumour behaviour and guiding treatment of cancer at an individualised level. The aim of this study was to ascertain the feasibility of detecting CTCs in oral squamous cell carcinoma (OSCC) using two different methods so as to determine the optimal method for the study of this cancer. METHODS: Comparison of the numbers of CTCs, circulating tumour micro-emboli (CTMs) and circulating tumour endothelial cells (CTECs), was undertaken in forty clinical samples of oral squamous cell carcinoma (OSCC) determined by filtration (ISET® ) and in situ fluorescent immunostaining (i-FISH, Cytelligen® ) immunostaining and in situ hybridisation. RESULTS: i-FISH detected CTCs in 80% of samples compared with 40% of samples analysed by microfiltration. i-FISH detected CTCs in a further 40% of samples in which microfiltration did not detect CTCs. No CTC clusters were detected by microfiltration while i-FISH detected CTM in 12.5% of samples. i-FISH analysis detected CTECs in 20/40 samples. CONCLUSION: These results highlight significant differences in detection of CTCs, CTM and CTECs between i-FISH and microfiltration when applied to OSCC samples, suggesting that technologies capable of detecting circulating aneuploid cells more accurately detect CTCs. i-FISH also detected CTM and CTEC not detected using ISET® . With proven prognostic relevance in adenocarcinomas, accurate enumeration of CTCs, CTMs and CTECs may be a clinically useful tool in the management of OSCC and may aid in the reduction of false-negative diagnoses.

9p24.1 alterations and programmed cell death 1 ligand 1 expression in early stage unfavourable classical Hodgkin lymphoma: an analysis from the German Hodgkin Study Group NIVAHL trial.

High programmed cell death 1 ligand 1 (PD-L1) protein expression and copy number alterations (CNAs) of the corresponding genomic locus 9p24.1 in Hodgkin- and Reed-Sternberg cells (HRSC) have been shown to be associated with favourable response to anti-PD-1 checkpoint inhibition in relapsed/refractory (r/r) classical Hodgkin lymphoma (cHL). In the present study, we investigated baseline 9p24.1 status as well as PD-L1 and major histocompatibility complex (MHC) class I and II protein expression in 82 biopsies from patients with early stage unfavourable cHL treated with anti-PD-1-based first-line treatment in the German Hodgkin Study Group (GHSG) NIVAHL trial (ClinicalTrials.gov Identifier: NCT03004833). All evaluated specimens showed 9p24.1 CNA in HRSC to some extent, but with high intratumoral heterogeneity and an overall smaller range of alterations than reported in advanced-stage or r/r cHL. All but two cases (97%) showed PD-L1 expression by the tumour cells in variable amounts. While MHC-I was rarely expressed in >50% of HRSC, MHC-II expression in >50% of HRSC was found more frequently. No obvious impact of 9p24.1 CNA or PD-L1 and MHC-I/II expression on early response to the highly effective anti-PD-1-based NIVAHL first-line treatment was observed. Further studies evaluating an expanded panel of potential biomarkers are needed to optimally stratify anti-PD-1 first-line cHL treatment.