Histopathological assessment of tissue samples after prolonged formalin fixation has been described previously, but currently there is only limited knowledge regarding the feasibility of molecular pathology on such tissue. In this pilot study, we tested routine molecular pathology methods (DNA isolation, DNA pyrosequencing/next-generation sequencing, DNA methylation analysis, RT-PCR, clonality analysis and fluorescence in situ hybridization) on tissue samples from 11 tumor entities as well as non-neoplastic brain tissue from 43 body donors during the gross anatomy course at Ulm University (winter semester 2019/20 and 2020/21). The mean post mortem interval until fixation was 2.5 ± 1.6 days (range, 1-6 days). Fixation was performed with aqueous formaldehyde solution (formalin, 1.5-2%). The mean storage time of body donors was 12.8 ± 5.6 months (range, 7-25 months). While most diagnostic methods were successful, samples showed significant variability in DNA quality and evaluability. DNA pyrosequencing as well as next-generation sequencing was successful in all investigated samples. Methylation analyses were partially not successful in some extend due to limited intact DNA yield for these analyses. Taken together, the use of prolonged formalin-fixed tissue samples from body donors offers new avenues in research and education, as these samples could be used for morpho-molecular studies and the establishment of biobanks, especially for tissue types that cannot be preserved and studied in vivo. Pathological ward rounds, sample collection, and histopathological and molecular workup have been integrated in the gross anatomy course in Ulm as an integral part of the curriculum, linking anatomy and pathology and providing medical students early insight into the broad field of (molecular) pathology.

OBJECTIVE: Lung cancer, primarily non-small cell lung cancer (NSCLC), is the leading cause of cancer deaths globally. In Greece in 2020, 8,960 new cases were reported. NSCLC\'s 5-year survival rates range from 54% (stage I) to less than 2% (stage IV); however, innovative therapies like immune check points inhibitors (ICIs) and targeted treatments have notably enhanced outcomes. The aim of this study was to assess the 1st and 2nd line treatment patterns with the introduction of new treatment modalities. Additionally, we evaluated biomarker testing approaches in NSCLC.
METHODS: LACHESIS was a retrospective multinational study, collecting and analyzing data from adult patients from Russia, Bulgaria, and Greece with metastatic NSCLC either newly diagnosed or relapsed from earlier stages, who had the option to undergo biomarker testing (genetic alterations/programmed death-ligand 1 protein expression levels, PD-L1), and who received 1st line treatment for squamous (SQ) or non-squamous (N-SQ) NSCLC. Subsequent lines of therapy were also reported.
RESULTS: The Greek site registered retrospective data from 250 NSCLC patients, of whom 206 were newly diagnosed (ND) metastatic NSCLC patients and 44 were patients relapsed from earlier stages. Seventy-two had SQ NSCLC and 169 had N-SQ NSCLC. For these patients, treatment patterns including immunotherapy±chemotherapy combinations were recorded. Biomarker testing patterns, including genetic alterations and PD-L1 expression levels were also documented.
CONCLUSIONS: LACHESIS provides treatment patterns and biomarker testing data. Greek patients were treated according to international guidelines, with immunotherapy as a viable option, particularly for PD-L1 levels over 50%. Biomarker testing, crucial for non-squamous (N-SQ) cases, should yield timely results for driver mutations, prioritizing patient benefits.

BACKGROUND: Melioidosis is a neglected but often fatal tropical disease. The disease has broad clinical manifestations, which makes diagnosis challenging and time consuming. To improve diagnosis, we aimed to evaluate the performance of the CRISPR-Cas12a system (CRISPR-BP34) to detect Burkholderia pseudomallei DNA across clinical specimens from patients suspected to have melioidosis.
METHODS: We conducted a prospective, observational cohort study of adult patients (aged ≥18 years) with melioidosis at Sunpasitthiprasong Hospital, a tertiary care hospital in Thailand. Participants were eligible for inclusion if they had culture-confirmed B pseudomallei infection from any clinical samples. Data were collected from patient clinical records and follow-up telephone calls. Routine clinical samples (blood, urine, respiratory secretion, pus, and other body fluids) were collected for culture. We documented time taken for diagnosis, and mortality at day 28 of follow-up. We also performed CRISPR-BP34 detection on clinical specimens collected from 330 patients with suspected melioidosis and compared its performance with the current gold-standard culture-based method. Discordant results were validated by three independent qualitative PCR tests. This study is registered with the Thai Clinical Trial Registry, TCTR20190322003.
RESULTS: Between Oct 1, 2019, and Dec 31, 2022, 876 patients with culture-confirmed melioidosis were admitted or referred to Sunpasitthiprasong Hospital, 433 of whom were alive at diagnosis and were enrolled in this study. Median time from sample collection to diagnosis by culture was 4·0 days (IQR 3·0-5·0) among all patients with known survival status at day 28, which resulted in delayed treatment. 199 (23%) of 876 patients died before diagnosis and 114 (26%) of 433 patients in follow-up were treated, but died within 28 days of admission. To test the CRISPR-BP34 assay, we enrolled and collected clinical samples from 114 patients with melioidosis and 216 patients without melioidosis between May 26 and Dec 31, 2022. Application of CRISPR-BP34 reduced the median sample-to-diagnosis time to 1·1 days (IQR 0·7-1·5) for blood samples, 2·3 h (IQR 2·3-2·4) for urine, and 3·3 h (3·1-3·4) for respiratory secretion, pus, and other body fluids. The overall sensitivity of CRISPR-BP34 was 93·0% (106 of 114 samples [95% CI 86·6-96·9]) compared with 66·7% (76 of 114 samples [57·2-75·2]) for culture. The overall specificity of CRISPR-BP34 was 96·8% (209 of 216 samples [95% CI 93·4-98·7]), compared with 100% (216 of 216 samples [98·3-100·0]) for culture.
CONCLUSIONS: The sensitivity, specificity, speed, and window of clinical intervention offered by CRISPR-BP34 support its prospective use as a point-of-care diagnostic tool for melioidosis. Future development should be focused on scalability and cost reduction.
BACKGROUND: Chiang Mai University Thailand and Wellcome Trust UK.

OBJECTIVE: 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.
CONCLUSIONS: 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.

OBJECTIVE: Due to the lack of large clinical cohorts in the Chinese populations with colorectal cancer (CRC) and gastric cancer (GC), there is no consensus among the preferred panel for microsatellite instability (MSI)-PCR testing. This study aims to evaluate a more appropriate panel.
METHODS: We tested the MSI status of 2572 patients with CRC and GC using the NCI panel and 2 mononucleotide panels (5 and 6 mononucleotide panels). Immunohistochemistry (IHC) was employed to perform mismatch repair protein testing in 1976 samples.
RESULTS: We collected 2572 patients with CRC and GC. The National Cancer Institute (NCI) panel failed to detect 13 cases. Of the 2559 cases that received results from all three panels, 2544 showed consistent results. In the remaining 15 cases, 9 showed discrepancies between MSI-H and MSI-L, and 6 showed discrepancies between MSI-L and microsatellite stability (MSS). The misdiagnosis rate of MSI-L was significantly lower in two mononucleotide panels than in the NCI panel (12.5% vs 87.5%, p=0.010) in CRC. In patients with GC, only the NCI panel detected three MSI-L cases, while the results of the two mononucleotide panels were one MSI-H and two MSS. Based on their IHC results, the MSI-L misdiagnosis rate of the NCI panel was 33.3%. Furthermore, compared with two mononucleotide panels, the NCI panel had a much lower rate of all loci instability in CRC (90.8% and 90.3% vs 25.2%) and GC (89.5% and 89.5% vs 12.0%).
CONCLUSIONS: In Chinese patients with CRC and GC, the five and six mononucleotide panels have advantages for detecting MSI over the NCI panel.

BACKGROUND: Molecular diagnostic tests may improve antibiotic prescribing by enabling earlier tailoring of antimicrobial therapy. However, clinicians\' trust and acceptance of these tests will determine their application in practice.
OBJECTIVE: To examine ICU prescribers\' views on the application of molecular diagnostics in patients with suspected hospital-acquired and ventilator-associated pneumonia (HAP/VAP).
METHODS: Sixty-three ICU clinicians from five UK hospitals completed a cross-sectional questionnaire between May 2020 and July 2020 assessing attitudes towards using molecular diagnostics to inform initial agent choice and to help stop broad-spectrum antibiotics early.
RESULTS: Attitudes towards using molecular diagnostics to inform initial treatment choices and to stop broad-spectrum antibiotics early were nuanced. Most (83%) were positive about molecular diagnostics, agreeing that using results to inform broad-spectrum antibiotic prescribing is good practice. However, many (58%) believed sick patients are often too unstable to risk stopping broad-spectrum antibiotics based on a negative result.
CONCLUSIONS: Positive attitudes towards the application of molecular diagnostics to improve antibiotic stewardship were juxtapositioned against the perceived need to initiate and maintain broad-spectrum antibiotics to protect unstable patients.

Objective: To clarify the clinicopathological, especially molecular, features of early-onset gastric cancer with the aim of informing analysis of treatment strategies. Methods: In this retrospective case-control study, we examined data from a dedicated gastric cancer database in Zhongshan Hospital affiliated to Fudan University. The original cohort comprised 2506 patients with gastric cancer who had undergone gastrectomy in Zhongshan Hospital Fudan University from July 2020 to October 2021, including 198 with early-onset gastric cancer (aged ≤45 years) and 2,308 with non-early gastric cancer. We used a simple random sampling method to select 396 of the 2,308 patients aged >45 years (ratio of 1:2) as the control group and then compared molecular diagnostic data and clinicopathological features of the two groups. Results: The median age was 39 years in the early-onset gastric cancer group, while 66 years in the control group. The clinicopathological features of early-onset gastric cancer included female predominance (59.1% [117/198] vs. 27.8% [110/396], χ2=54.816, P<0.001), less comorbidity (32.3% [64/198] vs. 57.1% [226/396], χ2=32.355, P<0.001), poorer differentiation (93.9% [186/198] vs. 74.5% [295/396], χ2=30.777, P<0.001) and higher proportion of diffuse type (40.4% [80/198] vs. 15.9% [63/396], χ2=69.639, P<0.001), distant metastasis (7.1% [14/198] vs. 2.8% [11/396], χ2=6.034, P=0.014). Regarding treatment, distal gastrectomy was more commonly performed than proximal gastrectomy (55.1% [109/198] vs. 47.0% [186/396], 1.5% [3/198] vs. 8.3% [33/396], χ2=11.644, P=0.003). Family history of gastric cancer, TNM stage, tumor size, lymph node dissection, nerve invasion, nodes harboring metastases, range of lymph node dissection, digestive tract reconstruction procedure, implementation of laparoscopic surgery, combined resection, and preoperative treatment did not differ significantly between the two groups (all P>0.05). Molecular diagnosis showed there was a smaller percentage of mismatch repair deficiency in the early-onset gastric cancer than in the control group (1.0% [2/198] vs. 10.1% [40/396], χ2=16.301, P<0.001), and a higher rate of positivity for Claudin 18.2 (77.8% [154/198] vs. 53.0% [210/396], χ2=5.442,P<0.001). HER-2 and Epstein-Barr virus positivity rates did not differ significantly between the two groups. Conclusion: Early-onset gastric cancer is a distinct type of gastric cancer with a high degree of malignancy, and treatment targeting Claudin 18.2 may be effective.
目的： 探讨早发型胃癌的临床病理特点，尤其是分子诊断特征，并分析其治疗策略。 方法： 采用回顾性病例对照研究方法。基于复旦大学附属中山医院胃癌专病数据库，收集2020年7月至2021年10月期间，于复旦大学附属中山医院普通外科行胃切除术的共2 506例胃癌患者的临床资料。其中，年龄≤45岁者198例，定义为早发型胃癌（早发型胃癌组）；另从2 308例非早发型胃癌患者按1∶2比例，采用简单随机抽样方法、选择396例患者作为对照（年龄>45岁，非早发型胃癌组）。对两组临床病理特征、分子诊断特征以及治疗策略进行比较。 结果： 早发型胃癌组中位年龄39岁，非早发型胃癌组则为66岁。相较非早发型胃癌组，早发型胃癌组以女性为主［59.1%（117/198）比27.8%（110/396），χ2=54.816，P<0.001］，合并症较少［32.3%（64/198）比57.1%（226/396），χ2=32.355，P<0.001］，低分化或未分化比例更高［93.9%（186/198）比74.5%（295/396），χ2=30.777，P<0.001］，Lauren分型弥漫型比例高［40.4%（80/198）比15.9%（63/396），χ2=69.639，P<0.001］，远处转移比例更高［7.1%（14/198）比2.8%（11/396），χ2=6.034，P=0.014］；在治疗上，远端胃切除比例高、近端胃切除比例低［分别为55.1%（109/198）比47.0%（186/396），1.5%（3/198）比8.3%（33/396），χ2=11.644，P=0.003］。两组在胃癌家族史、TNM分期、肿瘤大小、淋巴结清扫数、神经侵犯、癌结节以及淋巴结清扫范围、消化道重建方式、是否腹腔镜手术、是否行合并切除和术前治疗等方面比较，差异无统计学意义（均P>0.05）。分子诊断情况的比较，相比非早发型胃癌组，早发型胃癌错配修复基因缺陷比例更低［1.0%（2/198）比10.1%（40/396），χ2=16.301，P<0.001］，Claudin18.2阳性率较高［77.8%（154/198）比53.0%（210/396），χ2=5.442，P<0.001］。两组HER-2阳性比例和EBV+阳性比例差异无统计学意义（均P>0.05）。 结论： 早发型胃癌是一种恶性程度较高的特殊类型胃癌，Claudin18.2可能是其有效治疗靶点。.

With the increasing role of molecular genetics in the diagnostics of intracranial tumors, delivering sufficient representative tissue for such analyses is of paramount importance. This study explored the rate of successful diagnosis after frame-based stereotactic biopsies of intracranial lesions.
Consecutive patients undergoing frame-based stereotactic biopsies in 2020 and 2021 were included in this retrospective analysis. Cases were classified into three groups: conclusive, diagnosis with missing molecular genetics (MG) data, and inconclusive neuropathological diagnosis.
Of 145 patients, a conclusive diagnosis was possible in n = 137 cases (94.5%). For 3 cases (2.0%), diagnosis was established with missing MG data. In 5 cases (3.5%), an inconclusive (tumor) diagnosis was met. Diagnoses comprised mainly WHO 4 glioblastomas (n = 73, 56%), CNS lymphomas (n = 23, 16%), inflammatory diseases (n = 14, 10%), and metastases (n = 5, 3%). Methylomics were applied in 49% (n = 44) of tumor cases (panel sequencing in n = 28, 30% of tumors). The average number of specimens used for MG diagnostics was 5, while the average number of specimens provided was 15. In a univariate analysis, insufficient DNA was associated with an inconclusive diagnosis or a diagnosis with missing MG data (p < 0.001). Analyses of planned and implemented trajectories of cases with diagnosis with missing MG data or inconclusive diagnosis (n = 8) revealed that regions of interest were reached in almost all cases (n = 7).
Although stereotactic frame-based biopsies deliver a limited amount of tissue, they bear high histopathological and molecular genetic diagnostic yields. Given the proven surgical precision of the planned biopsy trajectories, optimizing surveyed lesion regions could help improve the rate of conclusive diagnoses.

BACKGROUND: Bacterial pathogens cause substantial diarrhea morbidity and mortality among children living in endemic settings, yet antimicrobial treatment is only recommended for dysentery or suspected cholera.
METHODS: AntiBiotics for Children with severe Diarrhea was a 7-country, placebo-controlled, double-blind efficacy trial of azithromycin in children 2-23 months of age with watery diarrhea accompanied by dehydration or malnutrition. We tested fecal samples for enteric pathogens utilizing quantitative polymerase chain reaction to identify likely and possible bacterial etiologies and employed pathogen-specific cutoffs based on genomic target quantity in previous case-control diarrhea etiology studies to identify likely and possible bacterial etiologies.
RESULTS: Among 6692 children, the leading likely etiologies were rotavirus (21.1%), enterotoxigenic Escherichia coli encoding heat-stable toxin (13.3%), Shigella (12.6%), and Cryptosporidium (9.6%). More than one-quarter (1894 [28.3%]) had a likely and 1153 (17.3%) a possible bacterial etiology. Day 3 diarrhea was less common in those randomized to azithromycin versus placebo among children with a likely bacterial etiology (risk difference [RD]likely, -11.6 [95% confidence interval {CI}, -15.6 to -7.6]) and possible bacterial etiology (RDpossible, -8.7 [95% CI, -13.0 to -4.4]) but not in other children (RDunlikely, -0.3% [95% CI, -2.9% to 2.3%]). A similar association was observed for 90-day hospitalization or death (RDlikely, -3.1 [95% CI, -5.3 to -1.0]; RDpossible, -2.3 [95% CI, -4.5 to -.01]; RDunlikely, -0.6 [95% CI, -1.9 to .6]). The magnitude of risk differences was similar among specific likely bacterial etiologies, including Shigella.
CONCLUSIONS: Acute watery diarrhea confirmed or presumed to be of bacterial etiology may benefit from azithromycin treatment.
BACKGROUND: NCT03130114.

OBJECTIVE: Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPIs) represent a standard of care for the clinical management of high-grade serous ovarian cancer (HGSOC). The recognition of homologous recombination deficiency (HRD) has emerged as a predictive biomarker of response for first-line PARPIs treatment in patients with HGOSC. On the other hand, this test is extremely complex and therefore it is often externalised. Regrettably, the reliability of outsourced HRD testing can be troubled by inconclusive results and high rejection rates. In this methodological study, we assessed the technical feasibility, interassay and interlaboratory reproducibility of in-house HRD testing using three different commercially available next-generation sequencing assays.
METHODS: A total of n=20 epithelial ovarian cancer samples previously analysed with MyChoice CDx were subjected to HRD retesting using three different platforms in three different major pathology laboratories, that is, SOPHiA DDM HRD Solution, HRD focus and Oncomine homologous recombination repair pathway predesigned panel. Concordance was calculated by Cohen\'s (dual) and Fleiss (triple) κ coefficients.
RESULTS: In-house BRCA1/2 molecular testing yielded a concordance rate >90.0% among all participating centres. HRD scores were successfully calculated by each institution with a concordance rate of 76.5%. Concerning the external gold standard test, the overall percentage of agreement ranged from 80.0% to 90.0% with a positive percentage agreement ranging from 75.0% to 80.0% and a negative percentage agreement ranging from 80.0% to 100%.
CONCLUSIONS: In-house testing for HRD can be reliably performed with commercially available next-generation sequencing assays.