Clinical and genetic characteristics of BAP1-mutated non-uveal and uveal melanoma.

Background: Screening for gene mutations has become routine clinical practice across numerous tumor entities, including melanoma. BAP1 gene mutations have been identified in various tumor types and acknowledged as a critical event in metastatic uveal melanoma, but their role in non-uveal melanoma remains inadequately characterized. Methods: A retrospective analysis of all melanomas sequenced in our department from 2014-2022 (n=2650) was conducted to identify BAP1 mutated samples. Assessment of clinical and genetic characteristics was performed as well as correlations with treatment outcome. Results: BAP1 mutations were identified in 129 cases and distributed across the entire gene without any apparent hot spots. Inactivating BAP1 mutations were more prevalent in uveal (55%) compared to non-uveal (17%) melanomas. Non-uveal BAP1 mutated melanomas frequently exhibited UV-signature mutations and had a significantly higher mutation load than uveal melanomas. GNAQ and GNA11 mutations were common in uveal melanomas, while MAP-Kinase mutations were frequent in non-uveal melanomas with NF1, BRAF V600 and NRAS Q61 mutations occurring in decreasing frequency, consistent with a strong UV association. Survival outcomes did not differ among non-uveal melanoma patients based on whether they received targeted or immune checkpoint therapy, or if their tumors harbored inactivating BAP1 mutations. Conclusion: In contrast to uveal melanomas, where BAP1 mutations serve as a significant prognostic indicator of an unfavorable outcome, BAP1 mutations in non-uveal melanomas are primarily considered passenger mutations and do not appear to be relevant from a prognostic or therapeutic perspective.

Liquid Biopsies as Non-Invasive Tools for Mutation Profiling in Multiple Myeloma: Application Potential, Challenges, and Opportunities.

Over the last decades, the survival of multiple myeloma (MM) patients has considerably improved. However, despite the availability of new treatments, most patients still relapse and become therapy-resistant at some point in the disease evolution. The mutation profile has an impact on MM patients' outcome, while typically evolving over time. Because of the patchy bone marrow (BM) infiltration pattern, the analysis of a single bone marrow sample can lead to an underestimation of the known genetic heterogeneity in MM. As a result, interest is shifting towards blood-derived liquid biopsies, which allow for a more comprehensive and non-invasive genetic interrogation without the discomfort of repeated BM aspirations. In this review, we compare the application potential for mutation profiling in MM of circulating-tumor-cell-derived DNA, cell-free DNA and extracellular-vesicle-derived DNA, while also addressing the challenges associated with their use.

Molecular Pathology of Breast Tumors: Diagnostic and Actionable Genetic Alterations.

Breast cancer is a heterogenous disease with various histologic subtypes, molecular profiles, behaviors, and response to therapy. After the histologic assessment and diagnosis of an invasive breast carcinoma, the use of biomarkers, multigene expression assays and mutation profiling may be used. With improved molecular assays, the identification of somatic genetic alterations in key oncogenes and tumor suppressor genes are playing an increasingly important role in many areas of breast cancer care. This review summarizes the most clinically significant somatic alterations in breast tumors and how this information is used to facilitate diagnosis, provide potential treatment options, and identify mechanisms of resistance.

Targeted mutational profiling of Epstein Barr virus-positive mucocutaneous ulcer: Implications for differential diagnosis with EBV-positive diffuse large B-cell lymphoma.

Epstein Barr Virus-positive mucocutaneous ulcer (EBVMCU) can be difficult to distinguish from EBV-positive diffuse large B cell lymphoma (DLBCL). We used targeted next-generation sequencing (NGS) to explore genetic alterations in EBVMCU to aid in this diagnostic challenge. Ten cases of EBVMCU were evaluated by a targeted NGS panel of 164 genes. Targeted NGS identified 18 variants in 15 genes in eight cases of EBVMCU. Loss of function TET2 variants were most frequently identified (3 of 10 cases, 30 %). One TET2 variant occurred at low variant allele frequency (VAF) of 3 %, which may be suggestive of clonal hematopoiesis of indeterminate potential. One case harbored a loss of function DNMT3A variant at low VAF. Two cases demonstrated missense variants in the IRF8 gene. Both variants occurred at a VAF close to 50 % and with an estimated high burden of disease (75 %). Two cases of mucosal gastrointestinal involvement had no reportable variants. Mutational profiling of EBVMCU identified TET2 loss of function variants at an elevated frequency in our cohort; however, the findings are not specific and its clinical significance cannot be completely elucidated. Further studies are needed to confirm the findings in an independent and larger cohort of EBVMCU, to determine the cell of origin of the variants, and to further assess their significance in the pathogenesis of this disorder.

Characterization of Variant RNAs Encapsidated during Bromovirus Infection by High-Throughput Sequencing.

Previously, we described the RNA recombinants accumulating in tissues infected with the bromoviruses BMV (Brome mosaic virus) and CCMV (Cowpea chlorotic mottle virus). In this work, we characterize the recombinants encapsidated inside the purified virion particles of BMV and CCMV. By using a tool called the Viral Recombination Mapper (ViReMa) that detects recombination junctions, we analyzed a high number of high-throughput sequencing (HTS) short RNA sequence reads. Over 28% of BMV or CCMV RNA reads did not perfectly map to the viral genomes. ViReMa identified 1.40% and 1.83% of these unmapped reads as the RNA recombinants, respectively, in BMV and CCMV. Intra-segmental crosses were more frequent than the inter-segmental ones. Most intra-segmental junctions carried short insertions/deletions (indels) and caused frameshift mutations. The mutation hotspots clustered mainly within the open reading frames. Substitutions of various lengths were also identified, whereas a small fraction of crosses occurred between viral and their host RNAs. Our data reveal that the virions can package detectable amounts of multivariate recombinant RNAs, contributing to the flexible nature of the viral genomes.

Relapses in early-stage follicular lymphoma frequently develop via a divergent evolution from their clonally related precursor cells.

Follicular lymphoma (FL) develops through a stepwise acquisition of cooperative genetic changes with t(14;18)(q32;q21)/IGH::BCL2 occurring early at the pre-B stage of B-cell development. Patients with FL typically show an indolent clinical course, remitting and relapsing with the eventual development of resistance to treatments. Interestingly, the majority of transformed FL do not progress directly from FL but originate from their clonally related lymphoma precursor (CLP) cells. To examine whether such divergent tumour evolution also underpins the relapses in patients with early-stage FL, we investigated by targeted next-generation sequencing 13 cases (stage I = 9, stage II = 4), who showed complete remission (mean: 5 years; range: 1-11.5 years) following local radiotherapy but subsequently relapsed (≥2 in 5). A clonal relationship between the diagnostic FL and relapses was confirmed in 11 cases. In six cases, common and distinct variants were seen between the paired diagnostic and relapsed lymphomas, indicating their divergent evolution from a CLP. In two cases, different B-cell clones were involved in the diagnostic and relapsed lymphomas, including one case involving two different BCL2 translocations. In the remaining five cases, the relapsed lymphoma developed via a linear progression (n = 4) or a mixed evolutionary path (n = 1). These findings may bear important implications in the routine diagnosis and management of relapsed FL. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Clock-like Mutation Signature May Be Prognostic for Worse Survival Than Signatures of UV Damage in Cutaneous Melanoma.

Novel treatment modalities comprising immune checkpoint inhibitors and targeted therapies have revolutionized treatment of metastatic melanoma. Still, some patients suffer from rapid progression and decease within months after a diagnosis of stage IV melanoma. We aimed to assess whether genomic alterations may predict survival after the development of stage IV disease, irrespective of received therapy. We analyzed tumor samples of 79 patients with stage IV melanoma using a custom next-generation gene-sequencing panel, MelArray, designed to detect alterations in 190 melanoma-relevant genes. We classified the patients: first, as short survivors (survival ≤6 months after stage IV disease, n = 22) and long survivors (survival >6 months, n = 57); second, by using a cut-off of one year; and third, by comparing the longest surviving 20 patients to the shortest surviving 20. Among analyzed genes, no individual gene alterations, or combinations of alterations, could be dichotomously associated with survival. However, the cohort's mutational profiles closely matched three known mutational signatures curated by the Catalog of Somatic Mutations in Cancer (COSMIC): UV signature COSMIC_7 (cosine-similarity 0.932), clock-like signature COSMIC_5 (cosine-similarity 0.829), and COSMIC_30 (cosine-similarity 0.726). Patients with UV signature had longer survival compared to patients with clock-like and COSMIC 30 (p < 0.0001). Subgroup dichotomization at 6 months showed that 75% of patients with UV signature survived longer than 6 months, and about 75% of patients with clock-like signature survived less than 6 months after development of stage IV disease. In our cohort, clock-like COSMIC_5 mutational signature predicted poor survival while a UV signature COSMIC_7 predicted longer survival. The prognostic value of mutational signatures should be evaluated in prospective studies.

Somatic mutation profiling, tumor-infiltrating leukocytes, tertiary lymphoid structures and PD-L1 protein expression in HER2-amplified colorectal cancer.

The status of human epidermal growth factor receptor 2 (HER2) for the prognosis in colorectal cancer (CRC) is controversial, and the characteristics of the somatic mutation spectrum, tumor-infiltrating leukocytes, tertiary lymphoid structures and PD-L1 protein are unknown in HER2-amplified colorectal cancer (HACC). In order to explore these characteristics along with their correlation with clinicopathological factors and prognosis in HACC. Samples of 812 CRC patients was collected. After immunohistochemistry (IHC), 59 of 812 were found to be HER2-positive, then 26 of 59 samples were further determined to be HER2 amplification by fluorescence in situ hybridization (FISH). Somatic mutation profiling of HACC was analysed using whole exome sequencing (WES). Multiplex fluorescence immunohistochemistry (mIHC) was used for tumor-infiltrating leukocytes and tertiary lymphoid structures (TLSs), while PD-L1 protein was detected by IHC. Our results indicate that the detection rates of HER2 positivity by IHC and FISH were 7.3% and 3.2% respectively, and HER2 amplification is correlated with distant tumour metastasis. The somatic mutation profiling revealed no differences between HACC and HER2-negative CRC. However, TP 53 strongly correlated with poor prognosis in HACC. Furthermore, tumor-infiltrating T cells and TLSs in the tumor immune microenvironment, as well as PD-L1 expression, were higher in HACC than in HER2-negative controls. However, none of them were associated with the prognosis of HACC. In all, HER2 amplification is correlated with distant metastasis and TP53 gene mutation may be a potential protective mechanism of HACC.

Breast Cancer Pathology in the Era of Genomics.

The era of genomic medicine provides an opportunity for pathologists to offer greater detail about the molecular underpinnings of a patient's cancer and thereby more targeted therapeutic options. In this review article, the role of genomics in breast cancer pathology is discussed, as it pertains to risk management, classification of special tumor types, predictive and prognostic testing, identification of actionable therapeutic targets, and monitoring for disease progression or development of treatment resistance.

A comparison of DNA sequencing and gene expression profiling to assist tissue of origin diagnosis in cancer of unknown primary.

Cancer of unknown primary (CUP) is a syndrome defined by clinical absence of a primary cancer after standardised investigations. Gene expression profiling (GEP) and DNA sequencing have been used to predict primary tissue of origin (TOO) in CUP and find molecularly guided treatments; however, a detailed comparison of the diagnostic yield from these two tests has not been described. Here, we compared the diagnostic utility of RNA and DNA tests in 215 CUP patients (82% received both tests) in a prospective Australian study. Based on retrospective assessment of clinicopathological data, 77% (166/215) of CUPs had insufficient evidence to support TOO diagnosis (clinicopathology unresolved). The remainder had either a latent primary diagnosis (10%) or clinicopathological evidence to support a likely TOO diagnosis (13%) (clinicopathology resolved). We applied a microarray (CUPGuide) or custom NanoString 18-class GEP test to 191 CUPs with an accuracy of 91.5% in known metastatic cancers for high-medium confidence predictions. Classification performance was similar in clinicopathology-resolved CUPs - 80% had high-medium predictions and 94% were concordant with pathology. Notably, only 56% of the clinicopathology-unresolved CUPs had high-medium confidence GEP predictions. Diagnostic DNA features were interrogated in 201 CUP tumours guided by the cancer type specificity of mutations observed across 22 cancer types from the AACR Project GENIE database (77,058 tumours) as well as mutational signatures (e.g. smoking). Among the clinicopathology-unresolved CUPs, mutations and mutational signatures provided additional diagnostic evidence in 31% of cases. GEP classification was useful in only 13% of cases and oncoviral detection in 4%. Among CUPs where genomics informed TOO, lung and biliary cancers were the most frequently identified types, while kidney tumours were another identifiable subset. In conclusion, DNA and RNA profiling supported an unconfirmed TOO diagnosis in one-third of CUPs otherwise unresolved by clinicopathology assessment alone. DNA mutation profiling was the more diagnostically informative assay. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Liquid Biopsy-Derived DNA Sources as Tools for Comprehensive Mutation Profiling in Multiple Myeloma: A Comparative Study.

The analysis of bone marrow (BM) samples in multiple myeloma (MM) patients can lead to the underestimation of the genetic heterogeneity within the tumor. Blood-derived liquid biopsies may provide a more comprehensive approach to genetic characterization. However, no thorough comparison between the currently available circulating biomarkers as tools for mutation profiling in MM has been published yet and the use of extracellular vesicle-derived DNA for this purpose in MM has not yet been investigated. Therefore, we collected BM aspirates and blood samples in 30 patients with active MM to isolate five different DNA types, i.e., cfDNA, EV-DNA, BM-DNA and DNA isolated from peripheral blood mononucleated cells (PBMNCs-DNA) and circulating tumor cells (CTC-DNA). DNA was analyzed for genetic variants with targeted gene sequencing using a 165-gene panel. After data filtering, 87 somatic and 39 germline variants were detected among the 149 DNA samples used for sequencing. cfDNA showed the highest concordance with the mutation profile observed in BM-DNA and outperformed EV-DNA, CTC-DNA and PBMNCs-DNA. Of note, 16% of all the somatic variants were only detectable in circulating biomarkers. Based on our analysis, cfDNA is the preferable circulating biomarker for genetic characterization in MM and its combined use with BM-DNA allows for comprehensive mutation profiling in MM.

Genetic and Methylation Analysis of CTNNB1 in Benign and Malignant Melanocytic Lesions.

Melanocytic neoplasms have been genetically characterized in detail during the last decade. Recurrent CTNNB1 exon 3 mutations have been recognized in the distinct group of melanocytic tumors showing deep penetrating nevus-like morphology. In addition, they have been identified in 1-2% of advanced melanoma. Performing a detailed genetic analysis of difficult-to-classify nevi and melanomas with CTNNB1 mutations, we found that benign tumors (nevi) show characteristic morphological, genetic and epigenetic traits, which distinguish them from other nevi and melanoma. Malignant CTNNB1-mutant tumors (melanomas) demonstrated a different genetic profile, instead grouping clearly with other non-CTNNB1 melanomas in methylation assays. To further evaluate the role of CTNNB1 mutations in melanoma, we assessed a large cohort of clinically sequenced melanomas, identifying 38 tumors with CTNNB1 exon 3 mutations, including recurrent S45 (n = 13, 34%), G34 (n = 5, 13%), and S27 (n = 5, 13%) mutations. Locations and histological subtype of CTNNB1-mutated melanoma varied; none were reported as showing deep penetrating nevus-like morphology. The most frequent concurrent activating mutations were BRAF V600 (n = 21, 55%) and NRAS Q61 (n = 13, 34%). In our cohort, four of seven (58%) and one of nine (11%) patients treated with targeted therapy (BRAF and MEK Inhibitors) or immune-checkpoint therapy, respectively, showed disease control (partial response or stable disease). In summary, CTNNB1 mutations are associated with a unique melanocytic tumor type in benign tumors (nevi), which can be applied in a diagnostic setting. In advanced disease, no clear characteristics distinguishing CTNNB1-mutant from other melanomas were observed; however, studies of larger, optimally prospective, cohorts are warranted.

Defining Optimal Conditions for Tumor Extracellular Vesicle DNA Extraction for Mutation Profiling.

(1) Background: Extracellular vesicles (EVs) have emerged as crucial players in the communication between cells in both physiological and pathological scenarios. The functions of EVs are strongly determined by their molecular content, which includes all bioactive molecules, such as proteins, lipids, RNA, and, as more recently described, double-stranded DNA. It has been shown that in oncological settings DNA associated with EVs (EV-DNA) is representative of the genome of parental cells and that it reflects the mutational status of the tumor, gaining much attention as a promising source of biomarker mutant DNA. However, one of the challenges in studies of EV-DNA is the lack of standardization of protocols for the DNA extraction from EVs, as well as ways to assess quality control, which hinders its future implementation in clinics. (2) Methods: We performed a comprehensive comparison of commonly used approaches for EV-DNA extraction by assessing DNA quantity, quality, and suitability for downstream analyses. (3) Results: We here established strategic points to consider for EV-DNA preparation for mutational analyses, including qPCR and NGS. (4) Conclusions: We put in place a workflow that can be applied for the detection of clinically relevant mutations in the EV-DNA of cancer patients.

Genetic and methylation profiles distinguish benign, malignant and spitzoid melanocytic tumors.

Accurate classification of melanocytic tumors is important for prognostic evaluation, treatment and follow-up protocols of patients. The majority of melanocytic proliferations can be classified solely based on clinical and pathological criteria, however in select cases a definitive diagnostic assessment remains challenging and additional diagnostic biomarkers would be advantageous. We analyzed melanomas, nevi, Spitz nevi and atypical spitzoid tumors using parallel sequencing (exons of 611 genes and 507 gene translocation analysis) and methylation arrays (850k Illumina EPIC). By combining detailed genetic and epigenetic analysis with reference-based and reference-free DNA methylome deconvolution we compared Spitz nevi to nevi and melanoma and assessed the potential for these methods in classifying challenging spitzoid tumors. Results were correlated with clinical and histologic features. Spitz nevi were found to cluster independently of nevi and melanoma and demonstrated a different mutation profile. Multiple copy number alterations and TERT promoter mutations were identified only in melanomas. Genome-wide methylation in Spitz nevi was comparable to benign nevi while the Leukocytes UnMethylation for Purity (LUMP) algorithm in Spitz nevi was comparable to melanoma. Histologically difficult to classify Spitz tumor cases were assessed which, based on methylation arrays, clustered between Spitz nevi and melanoma and in terms of genetic profile or copy number variations demonstrated worrisome features suggesting a malignant neoplasm. Comprehensive sequencing and methylation analysis verify Spitz nevi as an independent melanocytic entity distinct from both nevi and melanoma. Combined genetic and methylation assays can offer additional insights in diagnosing difficult to classify Spitzoid tumors.

Multiple primary malignances managed with surgical excision: a case report with next generation sequencing analysis.

BACKGROUND: Multiple primary malignancies (MPM) are defined as tumors originating in the same individual without any correlation between them. In addition to morphological and immunohistochemical analyses, sensitive DNA sequencing methods such as next generation sequencing (NGS) may help to discriminate the common or different genetic alterations driving each malignancy, to better diagnose these uncommon cases. METHODS AND RESULTS: Here we report the case of a man who developed a poorly differentiated gastric adenocarcinoma invading the pancreas followed, two years later, by a colorectal cancer involving also the kidney and the diaphragm. Despite the advanced stage of both diseases, adjuvant chemotherapy was successful. While the second tumor was initially interpreted as a relapse of his stomach cancer, NGS-based mutation profiling of the two carcinomas revealed two distinct malignances, independently developing in different times and indicative of metachronous MPM. Indeed, sequencing of cancer-associated genes identified somatic mutations only in the first gastric cancer, besides germline variants on three different genes (PDGFRA, APC and TP53). However, analysis of both somatic and germline mutations with bio-informatics prediction tools failed to find a correlation between these variants and the unexpectedly good prognosis of both cancers. CONCLUSIONS: In summary, NGS analysis contributed to defined different molecular profiles for two tumors developed in the span of two years, thus allowing diagnosing the case as MPN. However, NGS was unable to establish a direct correlation between the identified alterations and cancer development.

Genetic and Clinical Characteristics of ARID1A Mutated Melanoma Reveal High Tumor Mutational Load without Implications on Patient Survival.

(1) Background: Melanoma has the highest mortality of all cutaneous tumors, despite recent treatment advances. Many relevant genetic events have been identified in the last decade, including recurrent ARID1A mutations, which in various tumors have been associated with improved outcomes to immunotherapy. (2) Methods: Retrospective analysis of 116 melanoma samples harboring ARID1A mutations. Assessment of clinical and genetic characteristics was performed as well as correlations with treatment outcome applying Kaplan-Meier (log-rank test), Fisher's exact and Chi-squared tests. (3) Results: The majority of ARID1A mutations were in cutaneous and occult melanoma. ARID1A mutated samples had a higher number of mutations than ARID1A wild-type samples and harbored UV-mutations. A male predominance was observed. Many samples also harbored NF1 mutations. No apparent differences were noted between samples harboring genetically inactivating (frame-shift or nonsense) mutations and samples with other mutations. No differences in survival or response to immunotherapy of patients with ARID1A mutant melanoma were observed. (4) Conclusions: ARID1A mutations primarily occur in cutaneous melanomas with a higher mutation burden. In contrast to findings in other tumors, our data does not support ARID1A mutations being a biomarker of favorable response to immunotherapies in melanoma. Larger prospective studies would still be warranted.

Mutation profiling of circulating tumor DNA identifies distinct mutation patterns in non-Hodgkin lymphoma.

OBJECTIVE: Circulating tumor DNA (ctDNA) is emerging as a versatile biomarker for noninvasive genotyping and response monitoring in specific B-cell lymphomas; however, few studies have been conducted to explore ctDNA-based mutation profiling across non-Hodgkin lymphomas (NHLs) and genomic changes after initiation of chemotherapy. METHODS: A targeted sequencing of 362 genes was performed to detect the mutation profiles in paired blood and tissue samples from 42 NHL patients. Genomic alterations were explored in 11 diffuse large B-cell lymphoma (DLBCL) patients using paired blood samples collected pre- and post-R-CHOP chemotherapy. RESULTS: The frequencies of PIM1, MYD88, MYC, ZNF292, JAK, and MAF mutations were higher in aggressive than in indolent B-cell lymphoma and NK/T subtypes. Tumor mutation burden in blood samples was higher in aggressive than in indolent B-cell lymphomas and higher in patients who progressed than in those who responded to treatments. Our data also revealed significant enhance of concordance index through integrating mutated genes that were significantly associated with prognosis into International Prognostic Index-based prognostic model. Moreover, acquisition of mutations such as PCLO_p.L1220Tfs*3 was associated with resistance to R-CHOP in DLBCL patients. CONCLUSIONS: Our findings illustrated distinct mutation patterns across various NHL subtypes and suggested the association of genomic alterations in ctDNA with treatment outcomes.

Circulating Cell-Free DNA-Based Comprehensive Molecular Analysis of Biliary Tract Cancers Using Next-Generation Sequencing.

Biliary tract cancer (BTC) is a rare malignancy with a long disease course and an overall poor prognosis. Despite multiple chemotherapy agents, there is no defined second-line treatment opportunity for advanced BTCs. In the era of precision oncology, NGS plays an important role in identifying mutations that may predict the molecular pathomechanism and manage the BTC therapy. The peripheral blood liquid biopsy (LB) of cancer patients represents variable amounts of cell-free DNA (cfDNA) released from tumor foci of any anatomical location. Our study aimed to identify somatic mutations and tumor variant burden (TVB) in cell-free and matched tumor DNA. We found a positive correlation between the estimated tumor volume and cfDNA yield (r = 0.9326, p < 0.0001). Comparing tissue and LB results, similar TVB was observed. SNVs were proven in 84% of the cases, while in two cases, only the LB sample was informative for molecular analysis. The most important aberrations in BTCs, such as FGFR2, IDH1, IDH2, KRAS, and TP53, could be detected in matched LB samples. Our prospective study demonstrates a minimally invasive testing approach to identify molecular genetic alterations in cholangiocarcinoma and gallbladder cancers. Clinical applications of cfDNA reflect by capturing the outstanding spatial tumor heterogeneity and guarantee novel aspects for the precision oncology treatment.

The Molecular Subtype of Adult Acute Lymphoblastic Leukemia Samples Determines the Engraftment Site and Proliferation Kinetics in Patient-Derived Xenograft Models.

In acute lymphoblastic leukemia (ALL), conventional cell lines do not recapitulate the clonal diversity and microenvironment. Orthotopic patient-derived xenograft models (PDX) overcome these limitations and mimic the clinical situation, but molecular stability and engraftment patterns have not yet been thoroughly assessed. We herein describe and characterize the PDX generation in NSG mice. In vivo tumor cell proliferation, engraftment and location were monitored by flow cytometry and bioluminescence imaging. Leukemic cells were retransplanted for up to four passages, and comparative analyses of engraftment pattern, cellular morphology and genomic hotspot mutations were conducted. Ninety-four percent of all samples were successfully engrafted, and the xenograft velocity was dependent on the molecular subtype, outcome of the patient and transplantation passage. While BCR::ABL1 blasts were located in the spleen, KMT2A-positive cases had higher frequencies in the bone marrow. Molecular changes appeared in most model systems, with low allele frequency variants lost during primary engraftment. After the initial xenografting, however, the PDX models demonstrated high molecular stability. This protocol for reliable ALL engraftment demonstrates variability in the location and molecular signatures during serial transplantation. Thorough characterization of experimentally used PDX systems is indispensable for the correct analysis and valid data interpretation of preclinical PDX studies.

TERT promoter mutations are associated with longer progression-free and overall survival in patients with BRAF-mutant melanoma receiving BRAF and MEK inhibitor therapy.

BACKGROUND: Around 50% of cutaneous melanomas harbour therapeutically targetable BRAF V600 mutations. Reliable clinical biomarkers predicting duration of response to BRAF-targeted therapies are still lacking. Recent in vitro studies demonstrated that BRAF-MEK inhibitor therapy response is associated with tumour TERT promoter mutation status. We assessed this potential association in a clinical setting. METHODS: The study cohort comprised 232 patients with metastatic or unresectable BRAF V600-mutated melanoma receiving combined BRAF/MEK inhibitor treatment, including a single-centre retrospective discovery cohort (N = 120) and a prospectively collected multicenter validation cohort (N = 112). Patients were excluded if they received BRAF or MEK inhibitors in an adjuvant setting, as monotherapy, or in combination with immunotherapy. Kaplan-Meier and univariate/multivariate Cox regression analyses were performed as appropriate. RESULTS: median age at first diagnosis was 54 years (range 16-84 years). The majority of patients were men 147/232 (63.4%). Most tumours harboured TERT promoter mutations (72%, N = 167). A survival advantage was observed in both progression-free survival (PFS) and overall survival (OS) for patients with TERT promoter-mutant versus wild-type tumours in both the discovery cohort (mPFS of 9.6 months [N = 87] vs 5.0 months [N = 33]; hazard ratio [HR] = 0.56 [95% confidence interval {CI} 0.33-0.96] and mOS of 33.6 months vs 15.0 months; HR = 0.47 [95%CI 0.32-0.70]) as well as the validation cohort (mPFS of 7.3 months [N = 80] vs 5.8 months [N = 32]; HR = 0.67 [95%CI 0.41-1.10] and mOS of 51.1 months vs 15.0 months; HR = 0.33 [95%CI 0.18-0.63]). In the pooled cohort of TERT promoter-mutant (N = 167) versus wild-type (N = 65) tumours, respectively, PFS was 8.9 versus 5.5 months, (HR = 0.62; 95%CI 0.45-0.87; P = 0.004), and OS was 33.6 versus 17.0 months, (HR = 0.51; 95%CI 0.35-0.75, P = 0.0001). CONCLUSIONS: In patients with melanoma receiving BRAF/MEK-targeted therapies, TERT promoter mutations are associated with longer survival. If validated in larger studies, TERT promoter mutation status should be included as a predictive biomarker in treatment algorithms for advanced melanoma.