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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 WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Background In esophageal cancer (EC), there is a paucity of knowledge regarding the interplay between the tumor immune microenvironment and response to neoadjuvant treatment and, therefore, which factors may influence outcomes. Thus, our goal was to investigate the changes in the immune microenvironment with neoadjuvant treatment in EC by assessing the expression of immune related genes and their association with prognosis. Methods We examined the transcriptome of paired pre- and post-neoadjuvant treated EC specimens. Based on these findings, we validated the presence of tumor-infiltrating neutrophils using CD15+ immunohistochemistry in a discovery cohort of patients with residual pathologic disease. We developed a nomogram as a predictor of progression-free survival (PFS) incorporating the variables CD15+ cell count, tumor regression grade, and tumor grade. Results After neoadjuvant treatment, there was an increase in genes related to myeloid cell differentiation and a poor prognosis associated with high neutrophil (CD15+) counts. Our nomogram incorporating CD15+ cell count was predictive of PFS with a C-index of 0.80 (95% confidence interval [CI] 0.68–0.9) and a concordance probability estimate (CPE) of 0.77 (95% CI 0.69–0.86), which indicates high prognostic ability. The C-index and CPE of the validation cohort were 0.81 (95% CI 0.69–0.91) and 0.78 (95% CI 0.7–0.86), respectively. Conclusions Our nomogram incorporating CD15+ cell count can potentially be used to identify patients at high risk of recurrent disease and thus stratify patients who will benefit most from adjuvant treatment.

Patients who receive chimeric antigen receptor (CAR)–T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8 + memory T cell progenitors that can become either functional stem-like T (T STEM ) cells or dysfunctional T progenitor exhausted (T PEX ) cells. To that end, we demonstrated that T STEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate T STEM -like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, T STEM -like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4 + T cells during T STEM -like CAR-T cell production. Adoptive transfer of T STEM -like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of T STEM -like CAR-T cells and an increased memory T cell pool. Last, T STEM -like CAR-T cells and anti–programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8 + CAR + T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated T STEM -like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.

Cancer of unknown primary (CUP) constitutes a group of metastatic cancers in which standardized clinical investigations fail to identify a tissue of origin (TOO). Gene-expression profiling (GEP) has been used to resolve TOO, and DNA sequencing to identify potential targeted treatments; however, these methods have not been widely applied together in CUP patients. To assess the diagnostic utility of DNA and RNA tests for TOO classification, we applied GEP classification and/or gene-panel DNA sequencing in 215 CUP patients. Based on a retrospective review of pathology reports and clinical data, 77% of the cohort were deemed True-CUPs (T-CUP). Among the remaining cases, a latent primary diagnosis (10%) (LP-CUP) or TOO was highly suspected based on combined clinicopathological data (13%) (histology-resolved CUP, HR-CUP). High-medium confidence GEP classifications were made for 80% of LP/HR-CUPs, and these classifications were consistent with a pathologist-assigned diagnosis in 94% of cases, while only 56% of T-CUPs had high-medium confidence predictions. The frequency of somatic mutations in cancer genes was similar to 2,785 CUPs from AACR GENIE Project. DNA features, GEP classification, and oncovirus detection assisted making a TOO diagnosis in 37% of T-CUPs. Gene mutations and mutational signatures of diagnostic utility were found in 31% T-CUPs. GEP classification was useful in 13% of cases and viral detection in 4%. Among resolved T-CUPs, lung and biliary were the most frequently identified cancer types, while kidney cancer represented another minor subset. Multivariate survival analysis showed that unresolved T-CUPs had poorer overall survival than LP/HR-CUPs (Hazard ratio=1.9, 95% CI 1.1 − 3.2, p=0.016), while the risk of death was similar in genomically-resolved T-CUPs and LP/HR-CUPs. In conclusion, combining DNA and RNA profiling with clinicopathological data supported a putative TOO diagnosis in over a third of T-CUPs. DNA sequencing benefited T-CUP tumors with atypical transcriptional patterns that hindered reliable GEP classification.

Most NUTM1-rearranged neoplasms (NRNs) have fusions between NUTM1 and BRD (bromodomain-containing) family members and are termed NUT carcinomas (NCs) because they show some squamous differentiation. However, some NRNs are associated with fusions between NUTM1 and members of the MAD (MAX dimerization) gene family of MYC antagonists. Here we describe a small round cell malignancy from the gastro-esophageal junction with a previously unreported fusion between NUTM1 and the MAD family member MXI1. In contrast to NCs, the MXI1-NUTM1 tumor did not show squamous differentiation and did not express MYC, TP63 or SOX2, genes known to be targets of BRD-NUTM1 proteins and critical for NC oncogenesis. Transcriptome analysis showed paradoxical enrichment of MYC target genes in the MXI1-NUTM1 tumor despite the lack of MYC expression. When expressed in vitro MXI1-NUTM1 partially phenocopied MYC, enhancing cell proliferation and cooperating with oncogenic HRAS to produce anchorage-independent cell growth. These data provide evidence that MAD family members, which are normally repressors of MYC activity, can be converted into MYC-like mimics by fusion to NUTM1. The pathological features and novel oncogenic mechanism of the MXI1-NUTM1 tumor show that identification of NUTM1 fusion partners can be important for accurate diagnostic classification of some NRN subtypes, and potentially may guide therapeutic options.

Acute myeloid leukemias (AML) are comprised of multiple cell types with distinct capabilities to propagate the disease and resist therapy. Approximately 20% of AML patients carry gain- of-function mutations in IDH1 or IDH2 that result in over-production of the onco-metabolite 2-HG. Although IDH inhibitors can induce complete morphological remission, almost all patients eventually relapse. Analysis of clinical samples suggests that a population of IDH mutant cells is able to persist during treatment eventually acquiring 2-HG independence and drug resistance. Herein we characterized the molecular and cellular responses to the clinical IDH1 inhibitor AG-120 at high resolution using a novel multi-allelic mouse model of IDH1 mutant AML. We demonstrate that AG-120 exerts cell type-dependent effects on leukemic cells promoting delayed disease regression. Although IDH1 inhibition alone was not able to fully eradicate the disease, we uncovered that it increases cycling of rare leukemic stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitized IDH1 mutant AML to azacitidine with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non- genetic heterogeneity on treatment response and provide mechanistic rationale for a drug combination that is being tested in clinical trials.STATEMENT OF SIGNIFICANCEInhibition of mutant IDH1 in AML is insufficient to eliminate the disease but promotes proliferation of quiescent leukemic stem cells. Our data provide a mechanistic explanation for the observed synergy between IDH inhibitors and azacitidine and suggest that IDH inhibitors may also synergize with other drugs that preferentially target actively dividing cells.

Intraductal papillomas (IDP) are challenging breast findings because of their variable risk of progression to malignancy. The molecular events driving IDP development and genomic features of malignant progression are poorly understood. In this study, genome-wide CNA and/or targeted mutation analysis was performed on 44 cases of IDP, of which 20 cases had coexisting ductal carcinoma in situ (DCIS), papillary DCIS or invasive ductal carcinoma (IDC). CNA were rare in pure IDP, but 69% carried an activating PIK3CA mutation. Among the synchronous IDP cases, 55% (11/20) were clonally related to the synchronous DCIS and/or IDC, only one of which had papillary histology. In contrast to pure IDP, PIK3CA mutations were absent from clonal cases. CNAs in any of chromosomes 1, 16 or 11 were significantly enriched in clonal IDP lesions compared to pure and non-clonal IDP. The observation that 55% of IDP are clonal to DCIS/IDC indicates that IDP can be a direct precursor for breast carcinoma, not limited to the papillary type. The absence of PIK3CA mutations and presence of CNAs in IDP could be used clinically to identify patients at high risk of progression to carcinoma.

Routine breast cancer screening has increased detection of pre-invasive lesions such as ductal carcinoma in situ (DCIS). Currently, DCIS cases are mostly treated with wide local excision combined with adjuvant radiotherapy and/or hormonal therapy, even though only 25% of DCIS treated with surgery alone recur with 50% of recurrences being invasive. At present, prediction of recurrence risk is based on histopathological features and patient characteristics but reliable identification of individual recurrence risk is still challenging. Certain genetic aberrations (i.e. copy number variation, somatic mutations) are associated with breast cancer progression. While most recurrences are assumed to be genetically related to the primary tumour (“clonal recurrence”), the prevalence of ipsilateral independent primary events (“non-clonal recurrence”) after treatment for DCIS is uncertain. If this is a substantial proportion of cases, their presence would compromise attempts to find tumour intrinsic biomarkers of recurrence. To enable the discovery of better recurrence biomarkers, this project initially aims to identify cases with primary DCIS and later clonal ipsilateral recurrence. Sections from formalin-fixed paraffin-embedded blocks from primary DCIS cases and their subsequent recurrences were obtained from Nottingham City Hospital (UK). Two pathologists reviewed representative sections to identify areas for microdissection. Tumour epithelial cells were needle-microdissected and DNA was isolated. Sequencing libraries were prepared using a) a custom capture panel of 110 breast cancer-related genes or b) NEBNext Ultra II for low-coverage whole-genome sequencing (LC-WGS) depending on DNA quality and quantity. Sequencing was then performed on Illumina NextSeq 500 system with an average coverage of a) 400x and b) 1.3x, respectively. Data was processed by an in-house bioinformatics pipeline to detect and filter for high confidence somatic variants. Copy number profiles were obtained using CopywriteR (panel off-target reads), PureCN (panel on-target reads) and Control-FREEC (LC-WGS). Of 33 patients initially ascertained on the basis of a recurrent breast event, 30 had an ipsilateral recurrent tumour after 4.7 years on average, and 24 primary/recurrence pairs had sufficient data available to determine clonality. Eighteen pairs (75%) were classified as clonal by the following criteria: occurring in similar locations, showing a similar or higher tumour grade, and sharing ≥ 3 copy number breakpoints or likely somatic variants in the genes included in the panel. Six pairs (25%) did not meet this criteria and were classified as non-clonal. Preliminary data analysis shows that invasive recurrences (n = 12) were detected on average 3 years later than DCIS recurrences (n = 6), despite constant surveillance. The most commonly observed somatic mutations occurred in the TP53 and PIK3CA genes. Genetic variants in TP53 and PIK3CA were equally common in primary tumours with clonal and non-clonal recurrence. Frequent copy number alterations involved losses on 16q as well as gains on 1q, 8q and 17q, whereat the 8q gain was less profound. The presence of 25% non-clonal recurrences shows that differentiating between clonal and non-clonal recurrences might be crucial for further recurrence risk biomarker discovery. Thus, this project will proceed with comparing genetic variation in primary DCIS with clonal recurrence to DCIS cases without later recurrence. These samples are currently being obtained and analysed. Citation Format: Dorothea Lesche, Hugo Saunders, Sakshi Mahale, Magnus Zenthoven, Siobhan Huges, Tanjina Kader, Niko Thio, Islam M Miligy, Jia-Min Pang, Andrew R Green, Emad A Rakha, Ian G Campbell, Stephen B Fox, Kylie Gorringe. Evaluation of genomic changes in ductal carcinoma in situ as potential biomarkers of recurrence risk [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-21.

Objective Mucinous ovarian carcinoma (MOC) is an uncommon ovarian cancer histotype that responds poorly to conventional chemotherapy regimens. Although long overall survival outcomes can occur with early detection and optimal surgical resection, recurrent and advanced disease are associated with extremely poor survival. There are no current guidelines specifically for the systemic management of recurrent MOC. We analyzed data from a large cohort of women with MOC to evaluate the potential for clinical utility from a range of systemic agents. Methods We analyzed gene copy number (n = 191) and DNA sequencing data (n = 184) from primary MOC to evaluate signatures of mismatch repair deficiency and homologous recombination deficiency, and other genetic events. Immunohistochemistry data were collated for ER, CK7, CK20, CDX2, HER2, PAX8 and p16 (n = 117–166). Results Molecular aberrations noted in MOC that suggest a match with current targeted therapies include amplification of ERBB2 (26.7%) and BRAF mutation (9%). Observed genetic events that suggest potential efficacy for agents currently in clinical trials include: KRAS/NRAS mutations (66%), TP53 missense mutation (49%), RNF43 mutation (11%), ARID1A mutation (10%), and PIK3CA/PTEN mutation (9%). Therapies exploiting homologous recombination deficiency (HRD) may not be effective in MOC, as only 1/191 had a high HRD score. Mismatch repair deficiency was similarly rare (1/184). Conclusions Although genetically diverse, MOC has several potential therapeutic targets. Importantly, the lack of response to platinum-based therapy observed clinically corresponds to the lack of a genomic signature associated with HRD, and MOC are thus also unlikely to respond to PARP inhibition.

BackgroundCancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers where a primary tissue of origin (TOO) is uncertain. Most patients with CUP have limited treatment options and poor survival outcomes. Immune checkpoint inhibitors (ICIs) can be efficacious in some patients with CUP, but the optimal predictive biomarkers are unknown. We therefore assessed immune and genomic biomarkers as well as predicted TOO in patients with CUP, including a subset treated with ICIs.MethodsPatients with CUP were subject to gene-expression profiling (GEP) and DNA panel sequencing. Immune and stromal-related gene expression was explored by NanoString, including genes associated with immunotherapy response (IR) in other solid malignancies. ICI responsive cancer types were assigned based on Food and Drug Administration-approved indications, and either detection of a latent primary tumor or the TOO was suspected based on genomics informed pathology review. Tumor mutation burden (TMB) and gene mutations were also assessed.ResultsA total of 219 patients with CUP were included, 215 assessed for TOO in a previous study, with the majority (163) receiving both RNA and DNA tests. Of GEP profiled cases, 33% (59/175) had a high IR gene-expression score. Of the DNA sequenced cases, 16% (32/203) had high TMB (>10 mutations/Mb), including two with mismatch repair deficiency. Low correlation was observed between TMB and an IR score (R=0.26, pConclusionsA significant fraction of CUP tumors had genomic features previously associated with ICI response. High IR score was the most sensitive predictive feature of ICI response, warranting evaluation in a larger patient series.

Significant advances have been made in our understanding of the tumor immune microenvironment (TIM) and tumor infiltrating lymphocytes (TILs). Nevertheless, there is little understanding of the changes in the TIM in response to neoadjuvant chemoradiotherapy (CRT). Thus, our aim was to investigate the changes in the TIM with neoadjuvant CRT in EC by assessing the immune cell infiltrate, the expression of immune related genes, and their association with treatment response and prognosis. Methods To decipher the effects of neoadjuvant CRT on the TIM, we obtained 58 paired pre-treatment and post neoadjuvant CRT treated EC specimens. TILs and tumor infiltrating neutrophils (TIN) were quantified in pre-treatment biopsies and surgical resection specimens following neoadjuvant CRT. To evaluate the immune transcriptomics, RNA was extracted from these specimens and gene expression was assessed using the Nanostring Platform based on the PanCancer Immune Profiling Panel. Immunohistochemistry (IHC) was performed to validate findings from the immune transcriptomics. Results TIL counts were not prognostic for disease specific survival (DSS). We observed higher expression of immune-suppressive inflammatory chemokines (TGFß-1 and IL-16) in post-neoadjuvant treated samples compared to pre-treatment biopsies. In samples collected after neoadjuvant CRT, low expression of genes related to anti-tumor T cell cytotoxic activity1 was significantly associated with disease recurrence. In patients with residual disease, multivariate analysis revealed a high neutrophil count, but not TIL count, was significantly associated with inferior DSS (HR 3.8 [1.3– 10.8]; p = 0.01). Conclusion In EC, the tumor microenvironment after neoadjuvant CRT remains largely immune-suppressive. We discovered that the presence of TINs in patients with residual disease post neoadjuvant CRT is an independent adverse prognostic factor. Collectively, our results suggest that an inflammatory pro-tumoral microenvironment associated with TINs may contribute to treatment resistance and progressive disease in EC.

Angiogenesis underlies development, physiology and pathogenesis of cancer, eye and cardiovascular diseases. Inhibiting aberrant angiogenesis using anti-angiogenic therapy (AAT) has been successful in the clinical treatment of cancer and eye diseases. However, resistance to AAT inevitably occurs and its molecular basis remains poorly understood. Here, we uncover molecular modifiers of the blood endothelial cell (EC) response to a widely used AAT bevacizumab by performing a pooled genetic screen using three-dimensional microcarrier-based cell culture and CRISPR–Cas9. Functional inhibition of the epigenetic reader BET family of proteins BRD2/3/4 shows unexpected mitigating effects on EC survival and/or proliferation upon VEGFA blockade. Moreover, transcriptomic and pathway analyses reveal an interaction between epigenetic regulation and anti-angiogenesis, which may affect chromosomal structure and activity in ECs via the cell cycle regulator CDC25B phosphatase. Collectively, our findings provide insight into epigenetic regulation of the EC response to VEGFA blockade and may facilitate development of quality biomarkers and strategies for overcoming resistance to AAT., Through three-dimensional CRISPR screening, He et al. report that functional inhibition of BET family of proteins BRD2/3/4 shows mitigating effects on blood endothelial cell (EC) survival and/or proliferation upon VEGFA blockade. An interaction between epigenetic regulation and anti-angiogenesis, which may affect chromosomal structure and activity in ECs through CDC25B phosphatase, is potentially involved with EC resistance to anti-angiogenic therapy.

Mucinous ovarian carcinoma (MOC) is a unique subtype of ovarian cancer with an uncertain etiology, including whether it genuinely arises at the ovary or is metastatic disease from other organs. In addition, the molecular drivers of invasive progression, high-grade and metastatic disease are poorly defined. We perform genetic analysis of MOC across all histological grades, including benign and borderline mucinous ovarian tumors, and compare these to tumors from other potential extra-ovarian sites of origin. Here we show that MOC is distinct from tumors from other sites and supports a progressive model of evolution from borderline precursors to high-grade invasive MOC. Key drivers of progression identified are TP53 mutation and copy number aberrations, including a notable amplicon on 9p13. High copy number aberration burden is associated with worse prognosis in MOC. Our data conclusively demonstrate that MOC arise from benign and borderline precursors at the ovary and are not extra-ovarian metastases., Whether mucinous ovarian carcinoma (MOC) arises from cells at the ovary or from metastases from other primary sites is an unanswered question. Here, Cheasley et al perform a genetic analysis of the disease, showing that MOC arises at the ovary.

Bi-allelic loss-of-function (LoF) variants in the base excision repair (BER) gene NTHL1 cause a high-risk hereditary multi-tumor syndrome that includes breast cancer, but the contribution of heterozygous variants to hereditary breast cancer is unknown. An analysis of 4985 women with breast cancer, enriched for familial features, and 4786 cancer-free women revealed significant enrichment for NTHL1 LoF variants. Immunohistochemistry confirmed reduced NTHL1 expression in tumors from heterozygous carriers but the NTHL1 bi-allelic loss characteristic mutational signature (SBS 30) was not present. The analysis was extended to 27,421 breast cancer cases and 19,759 controls from 10 international studies revealing 138 cases and 93 controls with a heterozygous LoF variant (OR 1.06, 95% CI: 0.82–1.39) and 316 cases and 179 controls with a missense variant (OR 1.31, 95% CI: 1.09–1.57). Missense variants selected for deleterious features by a number of in silico bioinformatic prediction tools or located within the endonuclease III functional domain showed a stronger association with breast cancer. Somatic sequencing of breast cancers from carriers indicated that the risk associated with NTHL1 appears to operate through haploinsufficiency, consistent with other described low-penetrance breast cancer genes. Data from this very large international multicenter study suggests that heterozygous pathogenic germline coding variants in NTHL1 may be associated with low- to moderate- increased risk of breast cancer.

BackgroundVaginal melanoma (VM) is a rare cancer and has a poor response to immune checkpoint blockade (ICB). CD8+Tissue Resident Memory (TRM) T cells proliferate in response to ICB and correlate with longer survival in metastatic cutaneous melanoma. However, their capacity to respond to VM and their neoantigens is not known.MethodsUsing longitudinal samples, we explored the evolution of VM mutations by whole-exome sequencing and RNAseq, we also defined the immune context using multiplex immunohistochemistry and nanostring pan cancer immune profile. Then using fresh single cell suspensions of the metastatic samples, we explored VM T cells via mass cytometry and single cell RNAseq and T cell receptor sequencing (TCRseq). Finally, we investigated TRM, pre-TRM and exhausted T cell function against melanoma neo-antigens and melanoma differentiation antigens in vitro.ResultsPrimary VM was non-inflamed and devoid of CD8+ TRM cells. In contrast, both metastases showed proliferating CD8+ TRM were clustered at the tumor margin, with increased numbers in the second ICB-refractory metastasis. The first metastasis showed dense infiltration of CD8+ T cells, the second showed immune exclusion with loss of melanoma cell Major histocompatibility complex (MHC)-I expression associated with downregulation of antigen presentation pathway gene expression. CD8+ TRM from both metastases responded to autologous melanoma cells more robustly than all other CD8+ T cell subsets. In addition, CD8+ TRM shared TCR clones across metastases, suggesting a response to common antigens, which was supported by recognition of the same neoantigen by expanded tumor infiltrating lymphocytes.ConclusionsIn this study, we identified TRM clusters in VM metastases from a patient, but not primary disease. We showed TRM location at the tumor margin, and their superior functional response to autologous tumor cells, predicted neoantigens and melanoma differentiation antigens. These CD8+ TRM exhibited the highest tumor-responsive potential and shared their TCR with tumor-infiltrating effector memory T cells. This suggests VM metastases from this patient retain strong antitumor T cell functional responses; however, this response is suppressed in vivo. The loss of VG MHC-I expression is a common immune escape mechanism which was not addressed by anti-PD-1 monotherapy; rather an additional targeted approach to upregulate MHC-I expression is required.

Background Chimeric antigen receptor (CAR-T) cells are a promising new therapy for patients with cancer. However, in contrast to their success in B cell malignancies, CAR-T cells targeting solid cancers have had limited success so far due to their poor proliferation and poor long-term persistence in vivo. To address this issue, we used naive T cells to generate second-generation CAR-T cells recognizing the tumor antigen Lewis Y (LeY), termed ‘early’ CAR-T cells. Methods Purified naive T cells were activated by CD3/CD28 soluble tetrameric antibody complex, retrovirally transduced (LeY scFv-CD3z-CD28 CAR) and expanded in IL-7/IL-15. The early LeY CAR-T cell function was tested in vitro for cytotoxicity (Cr-release and degranulation), proliferation, and cytokine secretion by CBA, either de novo or following chronic stimulation for 1 month. Finally, early CAR-T cell persistence and anti-tumor efficacy was assessed in the OVCAR3-NSG model, in the presence or absence of anti-PD-1. Results The early-CAR-T cells comprised stem cell memory-like (CD95+, CD62L+, CD45RA+) and central memory phenotype (CD95+, CD62L+, CD45RA-) T cells with increased expression of ICOS, Ki67, TCF7 and CD27 (Figure 1). The early-CAR-T cells retained potent antigen-specific cytotoxicity, and secreted significantly higher levels of cytokines (IFN-?, TNF-a and IL-2) and increased proliferation compared to conventional CAR-T cells. Importantly, early-CAR-T cells had a significantly higher proliferative capacity after long-term chronic stimulation compared to conventional CAR-T cells (figure 2), and CD4+ CAR-T cells were critical for effective early CD8+ CAR-T cell proliferation capacity in vitro (figure 3). Early CAR-T cells had significantly better in vivo tumor control compared to conventional CAR-T cells (Figure 4), this was associated with increased CAR-T cell persistence. Because chronically stimulated early-LeY-CAR-T cells expressed PD-1 (figure 2), and OVCAR-3 cells expressed PD-L1 when co-cultured with LeY-CAR-T cells (figure 5), we combined early LeY-CAR-T cells with anti-PD-1 therapy and observed complete tumour regression in these mice. Interestingly, early LeY-CAR-T cell plus anti-PD-1 treatment also enhanced the percentage of circulating stem-cell memory like CAR-T cells in vivo (figure 5). Conclusions Our early CAR-T cells have better cytokine secretion and proliferation than conventional CAR-T cells. Early CAR-T cells also have superior anti-tumor efficacy in vivo, they have better persistence and maintain the circulating T cell memory pool. Importantly, low dose early-LeY-CAR-T cells combined with anti-PD1-treatment leads to complete clearance of LeY+ solid tumors in vivo. The early CAR-T cell production protocol is directly translatable for improving CAR-T cell efficacy in clinical trials for patients with solid tumors.

Most NUTM1-rearranged neoplasms (NRNs) have fusions between NUTM1 and BRD (bromodomain-containing) family members and are termed NUT carcinomas (NCs) because they show some squamous differentiation. However, some NRNs are associated with fusions between NUTM1 and members of the MAD (MAX dimerization) gene family of MYC antagonists. Here we describe a small round cell malignancy from the gastro-esophageal junction with a previously unreported fusion between NUTM1 and the MAD family member MXI1. In contrast to NCs, the MXI1-NUTM1 tumor did not show squamous differentiation and did not express MYC, TP63 or SOX2, genes known to be targets of BRD-NUTM1 proteins and critical for NC oncogenesis. Transcriptome analysis showed paradoxical enrichment of MYC target genes in the MXI1-NUTM1 tumor despite the lack of MYC expression. When expressed in vitro MXI1-NUTM1 partially phenocopied MYC, enhancing cell proliferation and cooperating with oncogenic HRAS to produce anchorage-independent cell growth. These data provide evidence that MAD family members, which are normally repressors of MYC activity, can be converted into MYC-like mimics by fusion to NUTM1. The pathological features and novel oncogenic mechanism of the MXI1-NUTM1 tumor show that identification of NUTM1 fusion partners can be important for accurate diagnostic classification of some NRN subtypes, and potentially may guide therapeutic options.

The current model for breast cancer progression proposes independent 'low grade (LG)-like' and 'high grade (HG)-like' pathways but lacks a known precursor to HG cancer. We applied low-coverage whole-genome sequencing to atypical ductal hyperplasia (ADH) with and without carcinoma to shed light on breast cancer progression. Fourteen out of twenty isolated ADH cases harboured at least one copy number alteration (CNA), but had fewer aberrations than LG or HG ductal carcinoma in situ (DCIS). ADH carried more HG-like CNA than LG DCIS (e.g. 8q gain). Correspondingly, 64% (7/11) of ADH cases with synchronous HG carcinoma were clonally related, similar to LG carcinoma (67%, 6/9). This study represents a significant shift in our understanding of breast cancer progression, with ADH as a common precursor lesion to the independent 'low grade-like' and 'high grade-like' pathways. These data suggest that ADH can be a precursor of HG breast cancer and that LG and HG carcinomas can evolve from a similar ancestor lesion. We propose that although LG DCIS may be committed to a LG molecular pathway, ADH may remain multipotent, progressing to either LG or HG carcinoma. This multipotent nature suggests that some ADH cases could be more clinically significant than LG DCIS, requiring biomarkers for personalising management. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In recent years the networking infrastructure has improved and as a result there has been a tremendous growth in the number of providers and the services they offer. With the wide choice of available services, many clients are inter- ested in differentiating providers based on Quality of Service (QoS) - performance being one of the most important QoS attributes. In this paper we focus on provider differentiation based on client-perceived performance. The client-perceived performance better represents client experience compared to server-side performance measurement used widely today. We analyze and characterize client-perceived performance, based on Internet measurements. Based on this characterization, we propose a technique - last-period prediction (LPP) - for me- dium-term performance prediction between a client-provider pair, which will be used for provider differentiation. The LPP technique is specially designed to capture the characteristics of client-perceived performance, such as periodic fluctuations during the concerned period. Through experiments on the Internet, we demonstrate that the proposed technique pro- vides better prediction accuracy for medium-term prediction of client-perceived performance compared to some popular time series models such as ARIMA, seasonal ARIMA, expo- nential smoothing, and Holt-Winters. Index Terms—Client-side performance, time series prediction, application-oriented performance