Initiation of molecular testing of endometrial carcinomas in a population-based setting: practical considerations and pitfalls.

Since the publication of The Cancer Genome Atlas (TCGA) molecular Classification of endometrial carcinomas in 2013, multiple studies have demonstrated the prognostic and therapeutic importance of this. However, there is great variability on whether and how this is undertaken in different institutions, and this is often dependent on resources and availability of molecular testing. Points of controversy include whether molecular classification is needed on all endometrial carcinomas and whether pure molecular testing is undertaken or a surrogate such as the ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer) Classifier. Herein we report our experience instigating molecular classification of endometrial carcinomas in Northern Ireland. From 1st March 2023, all endometrial carcinomas diagnosed on biopsy in the four pathology laboratories in Northern Ireland were referred to the central molecular pathology laboratory for genomic analysis using a custom next-generation sequencing (NGS) panel; the NGS panel included the entire coding regions of polymerase epsilon (POLE) and TP53 genes, as well as microsatellite instability (MSI) analysis. All cases also underwent immunohistochemical staining with oestrogen receptor (ER), p53, and the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. The molecular results were available by the time of surgery (if a hysterectomy was performed) allowing integration into the final pathology report where a TCGA molecular type was assigned. Two hundred and sixty-seven endometrial carcinomas underwent molecular testing; in five cases, there was insufficient material for testing, leaving 262 cases. The TCGA groups were POLEmut (19; 7.3%), MMRd (63; 24%), p53abn (62; 23.7%), and no specific molecular profile (NSMP) 118 (45%). Seventeen tumours (6.5%) were "multiple-classifiers": five POLEmut-p53abn, two POLEmut-MMRd, one POLE-MMRd-p53abn (all included in the POLEmut TCGA group), and nine MMRd-p53abn (included in the MMRd group). This represents one of the first population-based studies investigating the prevalence of the different TCGA molecular groups of endometrial carcinomas in an unselected population. Performing molecular testing on biopsies enables management to be tailored to the molecular group and allows integration of the TCGA group into the report of the final resection specimen. We hope our experience will facilitate other laboratories in undertaking TCGA molecular classification.

Machuca-Aguado J ，Catherwood M ，Houghton O ，Taylor J ，Shah R ，Ben-Mussa A ，Gonzalez D ，McCluggage WG ... -  《-》

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

《Jove-Journal of Visualized Experiments》

Validation and clinical performance of a single test, DNA based endometrial cancer molecular classifier.

We have previously shown that DNA based, single test molecular classification by next generation sequencing (NGS) (Proactive Molecular risk classifier for Endometrial cancer (ProMisE) NGS) is highly concordant with the original ProMisE classifier and maintains prognostic value in endometrial cancer. Our aim was to validate ProMisE NGS in an independent cohort and assess the performance of ProMisE NGS in real world clinical practice to address if there were any practical challenges or learning points for implementation. We evaluated DNA extracted from an external research cohort of 211 endometrial cancer cases diagnosed in 2016 from Germany, Switzerland, and Austria, across seven European centers, comparing standard molecular classification (NGS for POLE status, immunohistochemistry for mismatch repair and p53) with ProMisE NGS (NGS for POLE and TP53, microsatellite instability assay) for concordance metrics and Kaplan-Meier survival statistics across molecular subtypes. In parallel, we assessed all patients who had undergone a new NGS based molecular classification test (n=334) comparing molecular subtype assignment with the original ProMisE classifier. A total of 545 endometrial cancers were compared. Prognostic differences in progression free, disease specific, and overall survival between the four molecular subtypes were observed for the NGS classifier, recapitulating the survival curves of original ProMisE. In 28 of 545 (5%) discordant cases (8/211 (4%) in the validation set, 20/334 (6%) in the real world cohort), molecular subtype was able to be definitively assigned in all, based on review of the histopathological features and/or additional immunohistochemistry. DNA based molecular classification identified twice as many 'multiple classifier' endometrial cancers; 37 of 545 (7%) compared with 20 of 545 (4%) with original ProMisE. External validation confirmed that single test, DNA based molecular classification was highly concordant (95%) with original ProMisE classification, with prognostic value maintained, representing an acceptable alternative for clinical practice. Careful consideration of reasons for discordance and knowledge of how to correctly assign multiple classifier endometrial cancers is imperative for implementation.

Jamieson A ，Grube M ，Kommoss F ，Lum A ，Leung S ，Chiu D ，Henderson G ，Heitz F ，Heublein S ，Zeimet AG ，Hasenburg A ，Diebold J ，Walter C ，Staebler A ，Reynolds J ，Lapuk A ，McConechy MK ，Huntsman DG ，Gilks B ，Kommoss S ，McAlpine JN ... -  《-》

Performance of molecular classification in predicting oncologic outcomes of fertility-sparing treatment for atypical endometrial hyperplasia and endometrial cancer.

Endometrial cancers can be classified into 4 molecular sub-groups: (1) POLE mutated (POLEmut), (2) mismatch repair deficiency/microsatellite-instable (MMRd/MSI-H), (3) TP53-mutant or p53 abnormal (p53abn), and (4) no specific mutational profile (NSMP). Although molecular classification is increasingly applied in oncology, its role in guiding fertility-sparing treatments for endometrial cancer remains unclear. This study examines the prognostic role of molecular classification in fertility-sparing treatment and its potential to guide treatment decisions. We conducted a systematic review and meta-analysis of studies applying molecular classifiers in patients with endometrial cancer or atypical hyperplasia who underwent fertility-sparing treatment (International Prospective Register of Systematic Reviews, identification CRD42024555559). A literature search was performed across Scopus, PubMed/MEDLINE, ScienceDirect, and the Cochrane Library (2013-February 2024). Studies included full-text English articles with pre-operative assessments (histology, magnetic resonance imaging, or ultrasound) and molecular classification through next-generation sequencing or Proactive Molecular Risk Classifier for Endometrial Cancer. Both randomized controlled trials and observational studies were considered. Outcomes included complete response, partial response, stable disease, progression, and recurrence, with pooled analyses performed. Eight retrospective cohort studies comprising 363 patients met the inclusion criteria. Next-generation sequencing was used in 5 studies. The distribution of molecular sub-groups was POLEmut (5.8%), p53abn (3.3%), MMRd/MSI-H (12.1%), and NSMP (78.8%). Complete response and recurrence rates were POLEmut (66.6% and 14.3%), p53abn (50% and 33%), MMRd/MSI-H (48.8% and 42.8%), and NSMP (78.4% and 18.4%). Significant differences in complete response (p <.001) and recurrence rates (p = .005) were found across sub-groups. Pairwise analysis revealed lower complete response and higher recurrence rates for MMRd/MSI-H (p <.001, p = .01) and lower response for p53abn (p = .03) than for NSMP. POLEmut did not show superior success to other groups. Molecular classification indicates prognostic value in fertility-sparing treatment for endometrial cancer. NSMP had the highest response rates, whereas MMRd/MSI-H and p53abn were associated with poorer outcomes.

Ferrari FA ，Uccella S ，Franchi M ，Scambia G ，Fanfani F ，Fagotti A ，Pavone M ，Raspagliesi F ，Bogani G ... -  《-》

Can a Liquid Biopsy Detect Circulating Tumor DNA With Low-passage Whole-genome Sequencing in Patients With a Sarcoma? A Pilot Evaluation.

A liquid biopsy is a test that evaluates the status of a disease by analyzing a sample of bodily fluid, most commonly blood. In recent years, there has been progress in the development and clinical application of liquid biopsy methods to identify blood-based, tumor-specific biomarkers for many cancer types. However, the implementation of these technologies to aid in the treatment of patients who have a sarcoma remains behind other fields of cancer medicine. For this study, we chose to evaluate a sarcoma liquid biopsy based on circulating tumor DNA (ctDNA). All human beings have normal cell-free DNA (cfDNA) circulating in the blood. In contrast with cfDNA, ctDNA is genetic material present in the blood stream that is derived from a tumor. ctDNA carries the unique genomic fingerprint of the tumor with changes that are not present in normal circulating cfDNA. A successful ctDNA liquid biopsy must be able to target these tumor-specific genetic alterations. For instance, epidermal growth factor receptor (EGFR) mutations are common in lung cancers, and ctDNA liquid biopsies are currently in clinical use to evaluate the status of disease in patients who have a lung cancer by detecting EGFR mutations in the blood. As opposed to many carcinomas, sarcomas do not have common recurrent mutations that could serve as the foundation to a ctDNA liquid biopsy. However, many sarcomas have structural changes to their chromosomes, including gains and losses of portions or entire chromosomes, known as copy number alterations (CNAs), that could serve as a target for a ctDNA liquid biopsy. Murine double minute 2 (MDM2) amplification in select lipomatous tumors or parosteal osteosarcoma is an example of a CNA due to the presence of extra copies of a segment of the long arm of chromosome 12. Since a majority of sarcomas demonstrate a complex karyotype with numerous CNAs, a blood-based liquid biopsy strategy that searches for these CNAs may be able to detect the presence of sarcoma ctDNA. Whole-genome sequencing (WGS) is a next-generation sequencing technique that evaluates the entire genome. The depth of coverage of WGS refers to how detailed the sequencing is, like higher versus lower power on a microscope. WGS can be performed with high-depth sequencing (that is, > 60×), which can detect individual point mutations, or low-depth sequencing (that is, 0.1× to 5×), referred to as low-passage whole-genome sequencing (LP-WGS), which may not detect individual mutations but can detect structural chromosomal changes including gains and losses (that is, CNAs). While similar strategies have shown favorable early results for specific sarcoma subtypes, LP-WGS has not been evaluated for applicability to the broader population of patients who have a sarcoma. Does an LP-WGS liquid biopsy evaluating for CNAs detect ctDNA in plasma samples from patients who have sarcomas representing a variety of histologic subtypes? This was a retrospective study conducted at a community-based, tertiary referral center. Nine paired (plasma and formalin-fixed paraffin-embedded [FFPE] tissue) and four unpaired (plasma) specimens from patients who had a sarcoma were obtained from a commercial biospecimen bank. Three control specimens from individuals who did not have cancer were also obtained. The paired and unpaired specimens from patients who had a sarcoma represented a variety of sarcoma histologic subtypes. cfDNA was extracted, amplified, and quantified. Libraries were prepared, and LP-WGS was performed using a NextSeq 500 next-generation sequencing machine at a low depth of sequencing coverage (∼1×). The ichorCNA bioinformatics algorithm, which was designed to detect CNAs from low-depth genomic sequencing data, was used to analyze the data. In contrast with the gold standard for diagnosis in the form of histopathologic analysis of a tissue sample, this test does not discriminate between sarcoma subtypes but detects the presence of tumor-derived CNAs within the ctDNA in the blood that should not be present in a patient who does not have cancer. The liquid biopsy was positive for the detection of cancer if the ichorCNA algorithm detected the presence of ctDNA. The algorithm was also used to quantitatively estimate the percent ctDNA within the cfDNA. The concentration of ctDNA was then calculated from the percent ctDNA relative to the total concentration of cfDNA. The CNAs of the paired FFPE tissue and plasma samples were graphically visualized using aCNViewer software. This LP-WGS liquid biopsy detected ctDNA in 9 of 13 of the plasma specimens from patients with a sarcoma. The other four samples from patients with a sarcoma and all serum specimens from patients without cancer had no detectable ctDNA. Of those 9 patients with positive liquid biopsy results, the percent ctDNA ranged from 6% to 11%, and calculated ctDNA quantities were 0.04 to 5.6 ng/mL, which are levels to be expected when ctDNA is detectable. In this small pilot study, we were able to detect sarcoma ctDNA with an LP-WGS liquid biopsy searching for CNAs in the plasma of most patients who had a sarcoma representing a variety of histologic subtypes. These results suggest that an LP-WGS liquid biopsy evaluating for CNAs to identify ctDNA may be more broadly applicable to the population of patients who have a sarcoma than previously reported in studies focusing on specific subtypes. Large prospective clinical trials that gather samples at multiple time points during the process of diagnosis, treatment, and surveillance will be needed to further assess whether this technique can be clinically useful. At our institution, we are in the process of developing a large prospective clinical trial for this purpose.

Anderson CJ ，Yang H ，Parsons J ，Ahrens WA ，Jagosky MH ，Hsu JH ，Patt JC ，Kneisl JS ，Steuerwald NM ... -  《-》