Exosomal mRNA Cargo are biomarkers of tumor and immune cell populations in pediatric osteosarcoma.

Osteosarcoma is the commonest malignant bone tumor of children and adolescents and is characterized by a high risk of recurrence despite multimodal therapy, especially in metastatic disease. This suggests the presence of clinically undetected cancer cells that persist, leading to cancer recurrence. We sought to evaluate the utility of peripheral blood exosomes as a more sensitive yet minimally invasive blood test that could aid in evaluating treatment response and surveillance for potential disease recurrence. We extracted exosomes from the blood of pediatric osteosarcoma patients at diagnosis (n=7) and after neoadjuvant chemotherapy (n=5 subset), as well as from age-matched cancer-free controls (n=3). We also obtained matched tumor biopsy samples (n=7) from the cases. Exosome isolation was verified by CD9 immunoblot and characterized on electron microscopy. Profiles of 780 cancer-related transcripts were analysed in mRNA from exosomes of osteosarcoma patients at diagnosis and control patients, matched post-chemotherapy samples, and matched primary tumor samples. Peripheral blood exosomes of osteosarcoma patients at diagnosis were significantly smaller than those of controls and overexpressed extracellular matrix protein gene THBS1 and B cell markers MS4A1 and TCL1A. Immunohistochemical staining of corresponding tumor samples verified the expression of THBS1 on tumor cells and osteoid matrix, and its persistence in a treatment-refractory patient, as well as the B cell origin of the latter. These hold potential as liquid biopsy biomarkers of disease burden and host immune response in osteosarcoma. Our findings suggest that exosomes may provide novel and clinically-important insights into the pathophysiology of cancers such as osteosarcoma.

Leveraging Off-Target Reads in Panel Sequencing for Homologous Recombination Repair Deficiency Screening in Tumor.

Targeted tumor only sequencing has become a standard practice in cancer diagnostics. This study aims to develop an approach for robust copy number variant calling in tumor samples using only off-target region (OTR) reads. We also established a clinical use case for homologous recombination deficiency (HRD) score estimation (HRDest) using the sum of telomeric-allelic imbalance and large-scale state transition scores without the need for loss of heterozygosity information. A strong correlation was found between HRD score and the sum of telomeric-allelic imbalance + large-scale state transition in The Cancer Genome Atlas cohort (ρ = 0.99, P < 2.2 × 10-16) and in a clinical in-house cohort of 34 tumors (ρ = 0.9, P = 5.1 × 10-13) comparing whole-exome sequencing and targeted sequencing data. HRDest scores from 1086 clinical cases were compared with The Cancer Genome Atlas data set. There were no significant differences in HRD score distribution within the analyzed tumor types. As a control, commercially available HRD standards were also sequenced, and the HRDest scores obtained from the OTR reads were well within the HRD reference range provided by the manufacturer. In conclusion, OTR reads of tumor-only panel sequencing can be used to determine genome-wide copy number variant profiles and to approximate HRD scores.

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.

Combinatorial Hypofractionated Radiotherapy and Pembrolizumab in Anaplastic Thyroid Cancer.

Objectives Anaplastic thyroid cancer (ATC) is an aggressive disease associated with poor outcomes and resistance to therapies. Our study aim was to evaluate the activity of a combinatorial regimen of sandwich sequencing of pembrolizumab immunotherapy and hypofractionated radiotherapy (RT). Methods In this case series, patients with ATC received hypofractionated RT (QUAD-shot) and intravenous pembrolizumab 200mg every 3-4 weeks. Pembrolizumab was continued until disease progression or up till 24 months. Concurrent Lenvatinib treatment was allowed. Primary endpoint was best overall response (BOR) and progression-free survival (PFS). Additionally, we performed immune profiling of circulating T cells in a responder to investigate the immune response to our combinatorial treatment. Results At median follow-up of 32.6 months (IQR: 26.4-38.8), of a cohort of 5 patients, BOR was 80%; with 2 complete responses (CR) and 2 partial responses (PR). Patients who achieved CR remained disease-free at last follow-up. Median PFS was 7.6 months (IQR: 6.2-NR), and 1-year PFS and overall survival rate was 40% (95% CI: 13.7-100) for both. Treatment was well-tolerated, with mostly grade 1-2 adverse events. Immune profiling of one partial responder revealed an increase in activated CD4 and CD8 T cells post-QUAD-shot RT, which was further enhanced during the maintenance phase of pembrolizumab. Conclusions Herein, we reported a case series of 5 patients with ATC, with 2 long-term survivors who were treated with surgical debulking followed by QUAD-shot RT and pembrolizumab, possibly due to synergy of local and systemic treatments in activating anti-tumour immunogenic cytotoxicity. This regimen warrants further investigation in a larger cohort of patients.

Genomic heterogeneity at baseline is associated with T790M resistance mutations in EGFR-mutated lung cancer treated with the first-/second-generation tyrosine kinase inhibitors.

This study analyzed whether extended molecular profiling can predict the development of epidermal growth factor receptor (EGFR) gene T790M mutation, which is the most frequent resistance alteration in non-small cell lung cancer (NSCLC) after treatment with the first-/second-generation (1G/2G) EGFR inhibitors (tyrosine kinase inhibitors [TKIs]), but only weakly associated with clinical characteristics. Whole exome sequencing (WES) was performed on pretreatment tumor tissue with matched normal samples from NSCLC patients with (n = 25, detected in tissue or blood rebiopsies) or without (n = 14, negative tissue rebiopsies only) subsequent EGFR p.T790M mutation after treatment with 1G/2G EGFR TKI. Several complex genetic biomarkers were assessed using bioinformatic methods. After treatment with first-line afatinib (44%) or erlotinib/gefitinib (56%), median progression-free survival and overall survival were 12.1 and 33.7 months, respectively. Clinical and tumor genetic characteristics, including age (median, 66 years), sex (74% female), smoking (69% never/light smokers), EGFR mutation type (72% exon 19 deletions), and TP53 mutations (41%) were not significantly associated with T790M mutation (p > 0.05). By contrast, complex biomarkers including tumor mutational burden, the clock-like mutation signature SBS1 + 5, tumor ploidy, and markers of subclonality including mutant-allele tumor heterogeneity, subclonal copy number changes, and median tumor-adjusted variant allele frequency were significantly higher at baseline in tumors with subsequent T790M mutation (all p < 0.05). Each marker alone could predict subsequent development of T790M with an area under the curve (AUC) of 0.72-0.77, but the small number of cases did not allow confirmation of better performance for biomarker combinations in leave-one-out cross-validated logistic regression (AUC 0.69, 95% confidence interval: 0.50-0.87). Extended molecular profiling with WES at initial diagnosis reveals several complex biomarkers associated with subsequent development of T790M resistance mutation in NSCLC patients receiving first-/second-generation TKIs as the first-line therapy. Larger prospective studies will be necessary to define a forecasting model.

The Prognostic Value of CD39 as a Marker of Tumor-Specific T Cells in Triple-Negative Breast Cancer in Asian Women.

Triple-negative breast cancer (TNBC) has a poor prognosis with limited therapeutic options available for affected patients. Efforts are ongoing to identify surrogate markers for tumor-specific CD8+ T cells that can predict the response to immune checkpoint inhibitor (ICI) therapies, such as programmed cell death protein 1 or programmed cell death ligand-1 blockade. We have previously identified tumor-specific CD39+CD8+ T cells in non-small cell lung cancer that might help predict patient responses to programmed cell death protein 1 or programmed cell death ligand-1 blockade. Based on this finding, we conducted a comparative interrogation of TNBC in an Asian cohort to evaluate the potential of CD39 as a surrogate marker of tumor-specific CD8+ T cells. Using ICI-treated TNBC mouse models (n = 24), flow cytometric analyses of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes revealed that >99% of tumor-specific CD8+ T cells also expressed CD39. To investigate the relationship between CD39+CD8+ T-cell density and CD39 expression with disease prognosis, we performed multiplex immunohistochemistry staining on treatment-naive human TNBC tissues (n = 315). We saw that the proportion of CD39+CD8+ T cells in human TNBC tumors correlated with improved overall survival, as did the densities of other CD39+ immune cell infiltrates, such as CD39+CD68+ macrophages. Finally, increased CD39 expression on CD8+ T cells was also found to predict the response to ICI therapy (pembrolizumab) in a separate cohort of 11 TNBC patients. These findings support the potential of CD39+CD8+ T-cell density as a prognostic factor in Asian TNBC patients.

Multicentric pilot study to standardize clinical whole exome sequencing (WES) for cancer patients.

A growing number of druggable targets and national initiatives for precision oncology necessitate broad genomic profiling for many cancer patients. Whole exome sequencing (WES) offers unbiased analysis of the entire coding sequence, segmentation-based detection of copy number alterations (CNAs), and accurate determination of complex biomarkers including tumor mutational burden (TMB), homologous recombination repair deficiency (HRD), and microsatellite instability (MSI). To assess the inter-institution variability of clinical WES, we performed a comparative pilot study between German Centers of Personalized Medicine (ZPMs) from five participating institutions. Tumor and matched normal DNA from 30 patients were analyzed using custom sequencing protocols and bioinformatic pipelines. Calling of somatic variants was highly concordant with a positive percentage agreement (PPA) between 91 and 95% and a positive predictive value (PPV) between 82 and 95% compared with a three-institution consensus and full agreement for 16 of 17 druggable targets. Explanations for deviations included low VAF or coverage, differing annotations, and different filter protocols. CNAs showed overall agreement in 76% for the genomic sequence with high wet-lab variability. Complex biomarkers correlated strongly between institutions (HRD: 0.79-1, TMB: 0.97-0.99) and all institutions agreed on microsatellite instability. This study will contribute to the development of quality control frameworks for comprehensive genomic profiling and sheds light onto parameters that require stringent standardization.

Analysis of rare fusions in NSCLC: Genomic architecture and clinical implications.

OBJECTIVES: Molecular diagnosis for targeted therapies has been improved significantly in non-small-cell lung cancer (NSCLC) patients in recent years. Here we report on the prevalence of rare fusions in NSCLC and dissect their genomic architecture and potential clinical implications. MATERIALS AND METHODS: Overall, n = 5554 NSCLC patients underwent next-generation sequencing (NGS) for combined detection of oncogenic mutations and fusions either at primary diagnosis (n = 5246) or after therapy resistance (n = 308). Panels of different sizes were employed with closed amplicon-based, or open assays, i.e. anchored multiplex PCR (AMP) and hybrid capture-based, for detection of translocations, including "rare" fusions, defined as those beyond ALK, ROS1, RET and <0.5 % frequency in NSCLC. RESULTS: Rare fusions involving EGFR, MET, HER2, BRAF and other potentially actionable oncogenes were detected in 0.5% (n = 26) of therapy-naive and 2% (n = 6) TKI-treated tumors. Detection was increased using open assays and/or larger panels, especially those covering >25 genes, by approximately 1-2% (p = 0.001 for both). Patient characteristics (age, gender, smoking, TP53 co-mutations (56%), or mean tumor mutational burden (TMB) (4.8 mut/Mb)) showed no association with presence of rare fusions. Non-functional alterations, i.e. out-of-frame or lacking kinase domains, comprised one-third of detected rare fusions and were significantly associated with simultaneous presence of classical oncogenic drivers, e.g. EGFR or KRAS mutations (p < 0.001), or use of larger panels (frequency of non-functional among the detected rare fusions 57% for 25+ gene- vs. 12% for smaller panels, p < 0.001). As many rare fusions were identified before availability of targeted therapy, mean survival for therapy-naïve patients was 23.8 months, comparable with wild-type tumors. CONCLUSION: Approximately 1-2% of advanced NSCLC harbor rare fusions, which are potentially actionable and may support diagnosis. Routine adoption of broad NGS assays capable to identify exact fusion points and potentially retained protein domains can increase the yield of therapeutically relevant molecular information in advanced NSCLC.

Anastomosing Hemangioma on 68Ga-DOTATATE PET/CT: A Potential Pitfall.

ABSTRACT: Anastomosing hemangioma (AH) is a rare benign vascular lesion that primarily involves the genitourinary tract. Cases have also rarely been reported in other organs. AH is often discovered incidentally and resembles angiosarcoma histologically. On imaging, it may mimic other vascular lesions such as renal cell carcinoma and neuroendocrine tumors. We present a case of a 32-year-old woman with incidentally detected AH involving the kidneys, adrenal glands, liver, and retroperitoneum, initially presumed to be neuroendocrine tumors based on imaging findings on CT and 68Ga-DOTATATE PET scans.

Breast metastasis presenting as swelling with calcification - A case report and review of the literature.

Primary breast carcinomas often present as ill-defined, infiltrative lesions which may contain calcifications, whereas metastatic cancers from non-mammary sites are often more well-circumscribed, sharply demarcated from the adjacent breast tissue and are usually not associated with calcifications, although there are exceptions. We report an atypical case of a lady with lung adenocarcinoma with pleural involvement, who presented with diffuse breast swelling with calcifications on imaging from metastatic lung adenocarcinoma, the first of its kind in the literature. We postulate that the pathophysiology of this was due to lymphatic spread of the tumour from the pleura resulting in retrograde lymphovascular congestion of the breast, resulting in swelling and dystrophic calcification.

Artificial intelligence modelling in differentiating core biopsies of fibroadenoma from phyllodes tumor.

Breast fibroepithelial lesions (FEL) are biphasic tumors which consist of benign fibroadenomas (FAs) and the rarer phyllodes tumors (PTs). FAs and PTs have overlapping features, but have different clinical management, which makes correct core biopsy diagnosis important. This study used whole-slide images (WSIs) of 187 FA and 100 PT core biopsies, to investigate the potential role of artificial intelligence (AI) in FEL diagnosis. A total of 9228 FA patches and 6443 PT patches was generated from WSIs of the training subset, with each patch being 224 × 224 pixel in size. Our model employed a two-stage architecture comprising a convolutional neural network (CNN) component for feature extraction from the patches, and a recurrent neural network (RNN) component for whole-slide classification using activation values from the global average pooling layer in the CNN model. It achieved an overall slide-level accuracy of 87.5%, with accuracies of 80% and 95% for FA and PT slides respectively. This affirms the potential role of AI in diagnostic discrimination between FA and PT on core biopsies which may be further refined for use in routine practice.

Correlating SS18-SSX immunohistochemistry (IHC) with SS18 fluorescent in situ hybridization (FISH) in synovial sarcomas: a study of 36 cases.

The recently introduced, highly sensitive and specific SS18-SSX immunohistochemistry (IHC) is an attractive alternative to SS18 fluorescence in situ hybridization (FISH) testing in synovial sarcoma (SS). However, little is known about how SS18-SSX IHC correlates with SS18 FISH. We correlated the SS18 FISH results of SS from 36 patients with SS18-SSX IHC. Twenty-six tumours had a classic break-apart FISH pattern (1 fused, 1 red and 1 green signal) and all stained positive for the IHC. Ten had an atypical (non-classic) FISH pattern of which 5 stained positive for the IHC. Four of these (including two with novel atypical SS18 FISH patterns) were confirmed to harbour the SS18-SSX fusion on targeted RNA sequencing, while one had classic features of a biphasic SS. The remaining 5 tumours stained negative for the IHC. One had a TPM3-NTRK1 fusion, and one had no fusion, while the remaining three had insufficient tissue/RNA for sequencing. The sensitivity of the IHC was 91% (after excluding the 2 cases with confirmed absence of SS18-SSX fusion). Twenty histologic mimics of SS also stained negative for the IHC (100% specificity). Our study shows that the SS18-SSX IHC is more specific than SS18 FISH in diagnosing SS, especially in cases with atypical FISH patterns. It correlates well with RNA sequencing result and has the potential to replace SS18 FISH testing. A positive IHC result supports the diagnosis of SS, while a tumour with atypical FISH pattern and negative IHC result should undergo further molecular testing.

Genetic differences between benign phyllodes tumors and fibroadenomas revealed through targeted next generation sequencing.

Breast fibroepithelial lesions are biphasic tumors which comprise the common benign fibroadenomas (FAs) and the rarer phyllodes tumors (PTs). This study analyzed 262 (42%) conventional FAs, 45 (7%) cellular FAs, and 321 (51%) benign PTs contributed by the International Fibroepithelial Consortium, using a previously curated 16 gene panel. Benign PTs were found to possess a higher number of mutations, and higher rates of cancer driver gene alterations than both groups of FAs, in particular MED12, TERT promoter, RARA, FLNA, SETD2, RB1, and EGFR. Cases with MED12 mutations were also more likely to have TERT promoter, RARA, SETD2, and EGFR. There were no significant differences detected between conventional FAs and cellular FAs, except for PIK3CA and MAP3K1. TERT promoter alterations were most optimal in discriminating between FAs and benign PTs. Our study affirms the role of sequencing and key mutations that may assist in refining diagnoses of these lesions.

Papillary neoplasms of the breast-reviewing the spectrum.

Papillary neoplasms of the breast encompass a wide range of tumor types ranging from the benign intraductal papilloma to in situ and invasive papillary carcinomas. In this review, we considered each tumor entity listed under the Papillary Neoplasms category in the latest WHO Classification of Breast Tumors (5th edition), namely intraductal papilloma, papillary ductal carcinoma in situ, encapsulated papillary carcinoma, solid-papillary carcinoma, and invasive papillary carcinoma. We examined their pathological features, current issues pertaining to diagnosis and prognostication, as well as the latest molecular findings. We also briefly addressed adenomyoepithelioma and the newly included tall cell carcinoma with reversed polarity, highlighting areas where they overlap with papillary neoplasms.

Liquid Biopsy in Breast Cancer: A Focused Review.

CONTEXT.­: The role of liquid biopsy in cancer management has been gaining increased prominence in the past decade, with well-defined clinical applications now being established in lung cancer. Recently, the US Food and Drug Administration also approved the Therascreen PIK3CA RGQ polymerase chain reaction assay as a companion diagnostic assay to detect PIK3CA mutations in breast cancer for both tissue and liquid biopsies, bringing the role of liquid biopsy in breast cancer management to the fore. Its utility in other aspects of breast cancer, however, is yet to be clearly defined. OBJECTIVE.­: To review the studies that looked at liquid biopsies in breast cancer and examine their potential for clinical application in the areas of early diagnosis, prognostication, monitoring disease response, detecting minimal residual disease, and predicting risk of progression or relapse. We focus mainly on circulating tumor cells and circulating tumor DNA. DATA SOURCES.­: Peer-reviewed articles in PubMed. CONCLUSIONS.­: Liquid biopsies in breast cancers have yielded promising results, especially in the areas of monitoring treatment response and predicting disease progression or relapse. With further study, and hopefully coupled with continued improvements in technologies that isolate tumor-derived materials, liquid biopsies may go on to play a greater role in the breast cancer clinic.

Bizarre giant cells in human angiosarcoma exhibit chemoresistance and contribute to poor survival outcomes.

Giant cells (GC) are a poorly understood subset of tumor cells that have been increasingly recognized as a potential contributor to tumor heterogeneity and treatment resistance. We aimed to characterize the biological and clinical significance of GC in angiosarcoma, an aggressive rare cancer of endothelial origin. Archival angiosarcoma samples were examined for the presence of GC and compared with clinicopathological as well as NanoString gene expression data. GC were examined in angiosarcoma cell lines MOLAS and ISOHAS using conventional and electron microscopy, single cell whole genome profiling, and other assays. In the cell lines, GC represented a rare population of mitotically active, non-senescent CD31+ cells, and shared similar genomic profiles with regular-sized cells, consistent with a malignant endothelial phenotype. GC remained viable and persisted in culture following exposure to paclitaxel and doxorubicin. In patient samples, GC were present in 24 of 58 (41.4%) cases. GC was correlated with poorer responses to chemotherapy (25.0% vs 73.3%, P = 0.0213) and independently contributed to worse overall survival outcomes (hazard ratio 2.20, 95% confidence interval 1.17-4.15, P = 0.0142). NanoString profiling revealed overexpression of genes, including COL11A1, STC1, and ERO1A, accompanied by upregulation of immune-related metabolic stress and metastasis/matrix remodeling pathways in GC-containing tumors. In conclusion, GC may contribute to chemoresistance and poor prognosis in angiosarcoma.

Multiomic analysis and immunoprofiling reveal distinct subtypes of human angiosarcoma.

Angiosarcomas are rare, clinically aggressive tumors with limited treatment options and a dismal prognosis. We analyzed angiosarcomas from 68 patients, integrating information from multiomic sequencing, NanoString immuno-oncology profiling, and multiplex immunohistochemistry and immunofluorescence for tumor-infiltrating immune cells. Through whole-genome sequencing (n = 18), 50% of the cutaneous head and neck angiosarcomas exhibited higher tumor mutation burden (TMB) and UV mutational signatures; others were mutationally quiet and non-UV driven. NanoString profiling revealed 3 distinct patient clusters represented by lack (clusters 1 and 2) or enrichment (cluster 3) of immune-related signaling and immune cells. Neutrophils (CD15+), macrophages (CD68+), cytotoxic T cells (CD8+), Tregs (FOXP3+), and PD-L1+ cells were enriched in cluster 3 relative to clusters 2 and 1. Likewise, tumor inflammation signature (TIS) scores were highest in cluster 3 (7.54 vs. 6.71 vs. 5.75, respectively; P < 0.0001). Head and neck angiosarcomas were predominant in clusters 1 and 3, providing the rationale for checkpoint immunotherapy, especially in the latter subgroup with both high TMB and TIS scores. Cluster 2 was enriched for secondary angiosarcomas and exhibited higher expression of DNMT1, BRD3/4, MYC, HRAS, and PDGFRB, in keeping with the upregulation of epigenetic and oncogenic signaling pathways amenable to targeted therapies. Molecular and immunological dissection of angiosarcomas may provide insights into opportunities for precision medicine.