The giant diploid faba genome unlocks variation in a global protein crop.

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emissions and loss of biodiversity1. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value2. Faba bean (Vicia faba L.) has a high yield potential and is well suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has expanded to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, although with substantial copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association analysis to dissect the genetic basis of seed size and hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate the improvement of sustainable protein production across the Mediterranean, subtropical and northern temperate agroecological zones.

Comparison of tumors with HER2 overexpression versus HER2 amplification in HER2-positive breast cancer patients.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2)-positive tumors are defined by protein overexpression (3+) or gene amplification using immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH), respectively. HER2-positive tumors have historically included both IHC(3+) and IHC(2+, equivocal)/FISH(+) tumors and received the same treatment. Differences in biology between these two tumor types, however, are poorly understood. Considering anti-HER2 drugs bind directly to HER2 protein on the cell surface, we hypothesized anti-HER2 therapies would be less effective in IHC(2+)/FISH(+) tumors than in IHC(3+) tumors, leading to differences in patient outcomes. METHODS: A total of 447 patients with HER2-positive invasive carcinoma who underwent curative surgery were retrospectively investigated. HER2 status was assessed in surgical specimens, except in patients who received neo-adjuvant chemotherapy, where biopsy specimens were employed. RESULTS: Age, tumor size, lymph node status and ER status were independent factors relating to disease-free-survival, but no difference was observed between IHC(3+) and IHC(2+)/FISH(+) tumors. Kaplan-Meier analysis found patient outcomes did not differ, even after stratifying into those that did (n = 314), or did not (n = 129), receive chemotherapy with anti-HER2 drugs. In 134 patients who received NAC, pathological complete response rates in IHC(3+) and IHC(2+)/FISH(+) tumors were 45% and 21%, respectively. Survival after developing metastasis was significantly shorter in the IHC(2+)/FISH(+) group. CONCLUSIONS: The prognosis of patients with IHC(2+)/FISH(+) tumors did not differ from IHC(3+) tumors. However, the significance of HER2 protein overexpression in relation to treatment response remains unclear and warrants further investigations.

Multiple genetic paths including massive gene amplification allow Mycobacterium tuberculosis to overcome loss of ESX-3 secretion system substrates.

Mycobacterium tuberculosis (Mtb) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. Mtb strains deleted for the genes encoding substrates of the ESX-3 T7SS, esxG or esxH, require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of esxG or esxH deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of Mtb chromosome evolution involving "accordion-type" amplification.

Detection of MDM2 amplification by shallow whole genome sequencing of cell-free DNA of patients with dedifferentiated liposarcoma.

High-level amplification of MDM2 and other genes in the 12q13-15 locus is a hallmark genetic feature of well-differentiated and dedifferentiated liposarcomas (WDLPS and DDLPS, respectively). Detection of this genomic aberration in plasma cell-free DNA may be a clinically useful assay for non-invasive distinction between these liposarcomas and other retroperitoneal tumors in differential diagnosis, and might be useful for the early detection of disease recurrence. In this study, we performed shallow whole genome sequencing of cell-free DNA extracted from 10 plasma samples from 3 patients with DDLPS and 1 patient with WDLPS. In addition, we studied 31 plasma samples from 11 patients with other types of soft tissue tumors. We detected MDM2 amplification in cell-free DNA of 2 of 3 patients with DDLPS. By applying a genome-wide approach to the analysis of cell-free DNA, we also detected amplification of other genes that are known to be recurrently affected in DDLPS. Based on the analysis of one patient with DDLPS with longitudinal plasma samples available, we show that tracking MDM2 amplification in cell-free DNA may be potentially useful for evaluation of response to treatment. The patient with WDLPS and patients with other soft tissue tumors in differential diagnosis were negative for the MDM2 amplification in cell-free DNA. In summary, we demonstrate the feasibility of detecting amplification of MDM2 and other DDLPS-associated genes in plasma cell-free DNA using technology that is already routinely applied for other clinical indications. Our results may have clinical implications for improved diagnosis and surveillance of patients with retroperitoneal tumors.

Pyrotinib in Patients with HER2-Amplified Advanced Non-Small Cell Lung Cancer: A Prospective, Multicenter, Single-Arm Trial.

PURPOSE: In this study, we aimed to evaluate the efficacy and safety of pyrotinib, a pan-HER inhibitor, in patients with HER2-amplified non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: In this prospective, multicenter, single-arm trial (ChiCTR1800020262), patients with advanced NSCLC with HER2 amplification, as determined by next-generation sequencing, were enrolled and administered pyrotinib orally at 400 mg per day. The primary endpoint was 6-month progression-free survival (PFS) rate. Other endpoints included objective response rate (ORR), disease control rate (DCR), PFS, overall survival (OS), and safety. RESULTS: The enrolled cohort included 27 patients with HER2 amplification. The 6-month PFS rate was 51.9% [95% confidence interval (CI), 34.0-69.3]. The median PFS (mPFS) was 6.3 months (95% CI, 3.0-9.6 months), and median OS was 12.5 months (95% CI, 8.2-16.8 months). Pyrotinib elicited a confirmed ORR of 22.2% (95% CI, 10.6%-40.8%). Patients administered pyrotinib as first-line treatment achieved an mPFS of 12.4 months. Moreover, 30.8% of the patients who had progressed on EGFR tyrosine kinase inhibitor (TKI) responded to pyrotinib. Patients with brain metastases had an ORR of 40%. Treatment-related adverse events (TRAE) occurred in all patients (grade 3, 22.2%), but no grade 4 or higher TRAEs were documented. Diarrhea was the most frequent TRAE (all, 92.6%; grade 3, 7.4%). Loss of HER2 amplification was detected upon disease progression. CONCLUSIONS: Pyrotinib provided antitumor efficacy with a manageable safety profile in HER2-amplified patients with NSCLC.

Recurrent Superenhancer of the Oncogene POU5F1B in Colorectal Cancers.

Superenhancer usages in single cancer form such as colorectal cancer (CRC) may provide novel efficient targeting candidates. It is unclear whether CRC contains recurrent superenhancers that confer a predisposition to malignancy. We investigated the superenhancer profile of CRC cell line HCT116 and compared it to that of a healthy sigmoid colon. We found that HCT116 had lost most of the normal colon superenhancer activities but gained a new set of tumor-favoring superenhancers that facilitate tumor proliferation, growth signalling, and hypoxia resistance. Inhibiting the superenhancers by JQ-1 treatment had significantly decreased the colony formation capability of HCT116. Then, by comparing the superenhancer genes and robust CRC upregulated genes, we identified a superenhancer associated with a common CRC upregulated oncogene, POU5f1B. POU5f1B overexpression is related to the worse outcome in CRCs. Via performing ChIP-PCR in 35 clinical samples and investigating CRC anti-H3K27ac ChiP-seq public dataset consisting of 36 samples, we further identified that the superenhancer of oncogene POU5F1B is recurrently activated in CRCs, taking 62 and 72 per cent, respectively. Moreover, JQ-1 treatment successfully inhibited the POU5F1B expression in 5 out of 6 POU5F1B superenhancer-positive samples. Therefore, we concluded that the superenhancer activation of POU5F1B contributes partially to its high expression in CRCs, in addition to the well-known gene amplification aetiology.

Expression of a Cutinase of Moniliophthora roreri with Polyester and PET-Plastic Residues Degradation Activity.

Cutinases are enzymes produced by phytopathogenic fungi like Moniliophthora roreri. The three genome-located cutinase genes of M. roreri were amplified from cDNA of fungi growing in different induction culture media for cutinase production. The mrcut1 gene was expressed in the presence of a cacao cuticle, while the mrcut2 and mrcut3 genes were expressed when an apple cuticle was used as the inducer. The sequences of all genes were obtained and analyzed by bioinformatics tools to determine the presence of signal peptides, introns, glycosylation, and regulatory sequences. Also, the theoretical molecular weight and pI were obtained and experimentally confirmed. Finally, cutinase 1 from M. roreri (MRCUT1) was selected for heterologous expression in Escherichia coli. Successful overexpression of MRCUT1 was observed with the highest enzyme activity of 34,036 U/mg under the assay conditions at 40°C and pH 8. Furthermore, the degradation of different synthetic polyesters was evaluated; after 21 days, 59% of polyethylene succinate (PES), 43% of polycaprolactone (PCL), and 31% of polyethylene terephthalate (PET) from plastic residues were degraded. IMPORTANCE Plastic pollution is exponentially increasing; even the G20 has recognized an urgent need to implement actions to reduce it. In recent years, searching for enzymes that can degrade plastics, especially those based on polyesters such as PET, has been increasing as they can be a green alternative to the actual plastic degradation process. A promising option in recent years refers to biological tools such as enzymes involved in stages of partial and even total degradation of some plastics. In this context, the MRCUT1 enzyme can degrade polyesters contained in plastic residues in a short time. Besides, there is limited knowledge about the biochemical properties of cutinases from M. roreri. Commonly, fungal enzymes are expressed as inclusion bodies in E. coli with reduced activity. Interestingly, the successful expression of one cutinase of M. roreri in E. coli with enhanced activity is described.

Conditioned CAR-T cells by hypoxia-inducible transcription amplification (HiTA) system significantly enhances systemic safety and retains antitumor efficacy.

BACKGROUND: Hypoxia is a striking feature of most solid tumors and could be used to discriminate tumors from normoxic tissues. Therefore, the design of hypoxia-conditioned Chimeric Antigen Receptor (CAR) T cells is a promising strategy to reduce on-target off-tumor toxicity in adoptive cell therapy. However, existing hypoxia-conditioned CAR-T designs have been only partially successful in enhancing safety profile but accompanied with reduced cytotoxic efficacy. Our goal is to further improve safety profile with retained excellent antitumor efficacy. METHODS: In this study, we designed and constructed a hypoxia-inducible transcription amplification system (HiTA-system) to control the expression of CAR in T (HiTA-CAR-T) cells. CAR expression was determined by Flow cytometry, and the activation and cytotoxicity of HiTA-CAR-T cells in vitro were evaluated in response to antigenic stimulations under hypoxic or normoxic conditions. The safety of HiTA-CAR-T cells was profiled in a mouse model for its on-target toxicity to normal liver and other tissues, and antitumor efficacy in vivo was monitored in murine xenograft models. RESULTS: Our results showed that HiTA-CAR-T cells are highly restricted to hypoxia for their CAR expression, activation and cytotoxicity to tumor cells in vitro. In a mouse model in vivo, HiTA-CAR-T cells targeting Her2 antigen showed undetectable CAR expression in all different normoxic tissues including human Her2-expresing liver, accordingly, no liver and systemic toxicity were observed; In contrast, regular CAR-T cells targeting Her2 displayed significant toxicity on human Her2-expression liver. Importantly, HiTA-CAR-T cells were able to achieve significant tumor suppression in murine xenograft models. CONCLUSION: Our HiTA system showed a remarkable improvement in hypoxia-restricted transgene expression in comparison with currently available systems. HiTA-CAR-T cells presented significant antitumor activities in absence of any significant liver or systemic toxicity in vivo. This approach could be also applied to design CAR-T cell targeting other tumor antigens.

Comparison of the multigene panel test and OncoScan™ for the determination of HER2 amplification in breast cancer.

The diagnostic accuracy of the multigene panel test (MPT) and OncoScan™ in the determination of HER2 amplification in breast tumors remains controversial. In the present study, HER2 copy number was analyzed using both MPT and OncoScan™ in 45 breast tumors and was compared with that in fluorescent in situ hybridization (FISH) analysis. Tumors with low cellularity were examined using tumor cell enrichment and fluorescence­activated cell sorting. Both MPT and OncoScan™ exhibited significant correlations with FISH with respect to the determination of HER2 amplification in breast tumors. However, the correlation coefficient was significantly higher for the comparison of MPT and FISH (r=0.770) compared with that between OncoScan™ and FISH (r=0.564). The accuracy of MPT (93.3%) was slightly higher compared with that in OncoScan™ (84.4%) in determining the HER2 status, which was mostly explained by the higher sensitivity of MPT in tumors with low cellularity (83.3 vs. 33.3%), but not in those with high cellularity (81.8 vs. 72.7%). The specificity was 100% for both tests. The MPT exhibited higher sensitivity in the determination of the amplification of other genes, including MYC, fibroblast growth factor receptor 1 and GATA binding protein 3 in tumors with low cellularity compared with that in tumors with high cellularity. OncoScan™ exhibited low sensitivity without tumor cell enrichment. The results suggested that MPT could be a promising method to determine HER2 status in breast tumors and that it could exhibit improved accuracy compared with that in OncoScan™ in tumors with low cellularity.

Systematic identification of novel cancer genes through analysis of deep shRNA perturbation screens.

Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumor-promoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/ß-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by delaying cell cycle and modulates apoptosis in breast cancer. We demonstrate APSiC is a robust statistical framework for discovery of novel cancer genes through analysis of large-scale perturbation screens. The analysis of DRIVE using APSiC is provided as a web portal and represents a valuable resource for the discovery of novel cancer genes.

CCND1 copy number increase and cyclin D1 expression in acral melanoma: a comparative study of fluorescence in situ hybridization and immunohistochemistry in a Chinese cohort.

BACKGROUND: CCND1 copy number increase is characteristic of acral melanoma and is useful in distinguishing benign and malignant acral melanocytic lesions. Increase of the gene copy number may result in protein overexpression. This raises the possibility that detection of high expression of cyclin D1 by immunohistochemistry (IHC) may be used as a surrogate for direct evaluation of increase in the CCND1 gene copy number. METHODS: We examined increases in CCND1 copy number with fluorescence in situ hybridization (FISH), and examined cyclin D1 protein expression with IHC in 61 acral melanomas. RESULTS: Using FISH, 29 acral melanomas (29/61, 47.5%) showed increase in the CCND1 copy number, including 8 (8/61, 13.1%) which showed low-level increase in the CCND1 copy number and 21 (21/61, 34.4%) with high-level increase in the CCND1 copy number. By analysis of IHC, the median IHC score was 15% (range: 1-80%) in acral melanomas with no CCND1 copy number alteration. In acral melanomas with low-level CCND1 copy number increase, the median IHC score was 25% (range: 3-90%). In acral melanomas with high-level CCND1 copy number increase, the median IHC score was 60% (range: 1-95%). Comparing FISH and IHC, cyclin D1 protein expression level has no corelation with the CCND1 copy number in acral melanomas which have no CCND1 copy number alteration and low-level CCND1 copy number increase (P = 0.108). Cyclin D1 protein expression level correlated positively with CCND1 copy number in acral melanomas with high-level CCND1 copy number increase (P = 0.038). The sensitivity, specificity and positive predictive value of using cyclin D1 IHC to predict CCND1 FISH result was 72.4, 62.5 and 63.6%. Increase in CCND1 copy number was associated with Breslow thickness in invasive acral melanoma. CONCLUSION: High-level increase in the CCND1 copy number can induce high cyclin D1 protein expression in acral melanomas. However low-level increase and normal CCND1 copy number have no obvious correlation with protein expression. Cyclin D1 IHC cannot serve as a surrogate for CCND1 FISH in acral melanomas.

Is there a correlation between HER2 gene amplification level and response to neoadjuvant treatment with trastuzumab and chemotherapy in HER2-positive breast cancer?

There are contradictory data regarding the correlation between HER2 amplification level determined by in situ hybridization and evolution after treatment with anti-HER2 therapies. The aim of this study was to correlate quantitative results of FISH (ratio HER2/CEP17 and number of HER2 signals/nucleus) with pathological response achieved after neoadjuvant treatment with trastuzumab and chemotherapy. For this purpose, we analysed 100 consecutive HER2-positive cases of breast carcinoma treated with neoadjuvant therapy. HER2 amplification determined by FISH was found in 92 of the 100 cases studied. pCR was obtained in 58% of the patients whose tumours presented amplification. In contrast, no pCR was obtained in the 8 patients with non-amplified tumours. A significant direct correlation between HER2 high amplification (HER2/CEP17 ratio > 5 or HER2 signals/nucleus > 10) and pCR was found. In conclusion, HER2 amplification levels are clinically relevant because they provide oncologists with valuable information on the possibilities of achieving pCR after neoadjuvant treatment.

MYCN amplification levels in primary retinoblastoma tumors analyzed by Multiple Ligation-dependent Probe Amplification.

Background: Retinoblastoma (Rb) is a childhood tumor of the developing retina where predisposition is caused by RB1 pathogenic variants. MYCN amplification (MYCNA) has been implicated in around 2% of sporadic unilateral Rb tumors with no detectable RB1 variants. We audited data from tumors collected between 1993 and 2019 to determine if this is the case for patients treated at Barts Health NHS Trust, and how often it occurred alongside RB1 variants. Materials and methods: Screening for MYCNA was carried out by Multiple Ligation Probe Analysis of tumor and blood samples collected for RB1 genetic screening. The cohort consisted of 149 tumors, of which 114 had matched blood samples. Results: 10/149 (6.7%) tumors were positive for MYCNA in a population containing a disproportionate number of cases negative for RB1 pathogenic variants. Of 65 unbiased tumors collected from 2014 to 2019, 2 (3.1%) had MYCNA. All MYCNA samples were from sporadic, unilateral patients and 3/10 (30%) had RB1 pathogenic variants. MYCNA was not detected in any blood sample. No MYCNA tumor had 6p gain which is usually a common alteration in Rbs. Conclusions:MYCNA occurs in a small fraction of Rbs and can occur in the presence of pathogenic RB1 variants. However, where it occurs alongside RB1 alterations, the age of onset appears to be later. MYCNA has yet to be seen as a heritable change. In sporadic cases with early diagnosis, Rbs with no RB1 pathogenic variant identified should be tested for MYCNA. Conversely, tumors with MYCNA should still be screened for RB1 pathogenic variants.

Prognostic value of FGFR1 expression and amplification in patients with HNSCC: A systematic review and meta-analysis.

Fibroblast growth factor receptor 1 (FGFR1) has recently been identified as a promising novel therapeutic target and prognostic marker in different types of cancer. In the present study, a meta-analysis was performed to clarify the correlation between FGFR1 and the survival outcomes of head and neck squamous cell carcinoma (HNSCC) patients. PubMed, Embase, and Web of Science were systematically searched for relevant studies in order to explore the prognostic significance of FGFR1 in HNSCC. Hazards ratios (HR) and 95% confidence intervals (CI) were collected to estimate the correlation between overexpression and amplification of FGFR1 and survival outcomes of HNSCC patients. Nine studies including 2708 patients with HNSCC were finally selected for the meta-analysis. The results indicated that FGFR1 predicted poor overall survival (OS) (HR, 1.97; 95% CI, 1.49-2.61, P<0.001) in HNSCC patients. Futhermore, FGFR1 was related to poor OS in human papillomavirus (HPV) negative HNSCC not in HPV positive HNSCC patients. Subgroup analysis stratified by molecular abnormalities, such as overexpression or amplification showed the similar results. The present study demonstrated that HNSCC patients with FGFR1 overexpression and amplification were more likely to exhibit poorer survival.

Evidence for frequent concurrent DCUN1D1, FGFR1, BCL9 gene copy number amplification in squamous cell lung cancer.

Non-small cell lung cancer (NSCLC) targeted therapies are mostly based on activating mutations and rearrangements which are rare events in Lung Squamous Cell Carcinomas (LUSC). Recently advances in immunotherapy have improved the therapeutic repository for LUSC, but there is still an urgent need for novel targets and biomarkers. We examined 73 cases of LUSC for relative copy number amplification of DCUN1D1, BCL9, FGFR1 and ERBB2 genes and searched for correlations with molecular alterations and clinicopathological characteristics. In our cohort BCL9 gene was amplified in 57.5 % of the cases, followed by DCUN1D1 in 37 %, FGFR1 in 19 % whereas none of the cases were amplified in ERBB2 gene. The majority of the samples exhibited amplification in at least one gene while half of them displayed concurrent amplification of two/three genes. Interestingly, 93 % of the FGFR1 amplified cases were also found co amplified with DCUN1D1 and/or BCL9 genes. Linear correlations were found between BCL9 and DCUN1D1 as well as BCL9 and FGFR1 gene amplification. BCL9 and DCUN1D1 genes' amplification was correlated with poorly differentiated tumors (p = 0.035 and p = 0.056 respectively), implying their possible role in tumor aggressiveness. This is the first study, to the best of our knowledge that examines the correlation of DCUN1D1 and BCL9 genes relative copy number amplification with molecular alterations and clinicopathologic characteristics of squamous cell lung cancer tissue samples. Our findings show concurrent amplification of genes in different chromosomes, with possible involvement in tumor aggressiveness. These results support the complexity of LUSC tumorigenesis and imply the necessity of multiple biomarkers / targets for a more effective therapeutic result in LUSC.

Clinical characteristics and gene mutation analysis of clear cell tumor of the lung.

There were rare clinical reports on clear cell tumor of the lung (CCTL). The clinical characteristics and underlying genetic mutation status of CCTL are poorly understood. From 2012 to 2017, patients pathologically diagnosed with CCTL in our hospital were investigated and analyzed based on clinical manifestations, pathological characteristics, prognosis and full gene mutation status through next generation sequencing (NGS) technology. During a 6-year period, four eligible patients were diagnosed with CCTL through surgical resection and were included in this study. All patients showed solitary nodules or lumps located in the left lung. The average maximum diameter of lesions was 2.5 ± 1.1 cm. Computed tomography (CT) imaging characteristics of these nodules/lumps demonstrated the features of benign tumors. The hematoxylin-eosin (HE) morphology and immunohistochemistry were consistent with the histopathological features of benign CCTL. Subsequent NGS analysis showed frame shift mutations of F2421/E2419, K1466E mutation, and p. 1450_1456 deletion mutation in mTOR gene in two of four patient samples and amplifications of MCL1 were observed in three of four samples. CCTL is a rare type of primary pulmonary mesenchymal tumor with good prognosis. Preliminary diagnosis on CT is usually sclerosing pneumocytoma. It is still unclear whether the occurrence and development of the disease are related to specific gene mutation. In this study, the genomic findings of frame shift mutation of mTOR genes and amplification of MCL1 gene in CCTL suggest that these mutations might play a role in proliferation of CCTL.

Estimating copy number using next-generation sequencing to determine ERBB2 amplification status.

Assessing Erb-b2 receptor tyrosine kinase 2 (ERBB2) amplification status in breast and gastric cancer is necessary for deciding the best therapeutic strategy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently used for assessing protein levels and gene copy number (CN), respectively. The use of next-generation sequencing (NGS) to measure ERBB2 CN in breast cancer is approved by the United States Federal Drug Administration as a companion diagnostic. However, a CN of less than 8 is evaluated as "equivocal", which means that some ERBB2 amplification cases diagnosed as "HER2 negative" might be false-negative cases. We reviewed the results of gene profiling targeting 160 cancer-related genes in breast (N = 90) and non-breast (N = 19) cancer tissue, and compared the ERBB2 CN results with the IHC/FISH scores. We obtained an estimated CN from the measured CN by factoring in the histological proportion of tumor cells and found that an ERBB2-estimated CN of 3.2 or higher was concordant with the combined IHC/FISH outcome in 98.4% (88/90) of breast cancer cases, while this was not always evident among non-breast cancer cases. Therefore, NGS-estimated ERBB2 CN could be considered a diagnostic test for anti-HER2 therapy after the completion of adequate prospective clinical trials.

HER2 copy number as predictor of disease-free survival in HER2-positive resectable gastric adenocarcinoma.

PURPOSE: The identification of HER2 overexpression in a subset of gastric adenocarcinoma (GA) patients represents a significant step forward in unveiling the molecular complexity of this disease. The predictive and prognostic value of HER2 amplification in advanced HER2 inhibitor-treated GA patients has been investigated. However, its predictive value in resectable patients remains elusive. METHODS: We enrolled 98 treatment-naïve resectable Chinese GA patients with HER2 overexpression assessed using IHC. Capture-based targeted sequencing using a panel consisting of 41 gastrointestinal cancer-related genes was performed on tumor tissues. Furthermore, we also investigated the correlation between HER2 copy number (CN) and survival outcomes. RESULTS: Of the 98 HER2-overexpressed patients, 90 had HER2 CN amplification assessed using next-generation sequencing, achieving 92% concordance. The most commonly seen concurrent mutations were occurring in TP53, EGFR and PIK3CA. We found HER2 CN as a continuous variable was an independent predictor associated with DFS (p = 0.029). Our study revealed HER2 CN-high patients showed a trend of intestinal-type GA predominant (p = 0.075) and older age (p = 0.07). The median HER2 CN was 15.34, which was used to divide the cohort into CN-high and CN-low groups. Patients with high HER2 CN had a significantly shorter DFS than patients with low HER2 CN (p = 0.002). Furthermore, HER2 CN as a categorical variable was also an independent predictor associated with DFS in patients. CONCLUSION: We elucidated the mutation spectrum of HER2-positive resectable Chinese GA patients and the association between HER2 CN and DFS. Our work revealed HER2 CN as an independent risk factor predicted unfavorable prognosis in HER2-positive GA patients and allowed us to further stratify HER2-positive resectable GA patients for disease management.

Histopathological features of HER2 overexpression in uterine carcinosarcoma: proposal for requirements in HER2 testing for targeted therapy.

Uterine carcinosarcoma (UCS) is an uncommon and highly aggressive tumor. There is no HER2 testing protocol for UCS despite the development of HER2 antibody conjugates. We aimed to elucidate histopathological HER2 expression details in UCS, to compare HER2 scores between ASCO/CAP criteria for gastric and breast cancer, and to propose requirements for HER2 testing for UCS. Eighty-nine specimens from 84 patients with metastatic/recurrent UCS were prospectively collected from May 2018 to July 2020. We performed HER2 immunohistochemistry (IHC) for 89 specimens and FISH for 44 specimens. HER2 expression details and HER2 score were evaluated according to the latest ASCO/CAP criteria for gastric (2016) and breast cancer (2018). HER2 IHC scores according to the gastric cancer criteria were 0 in 31 cases (35%), 1+ in 26 (29%), 2+ in 22 (25%), and 3+ in 10 cases (11%) of the 89 specimens. A lateral/basolateral membranous staining pattern was observed in 28/32 (88%) specimens with HER2 scores of 2+/3+. HER2 intratumoral heterogeneity was identified in 28/32 (88%) of the specimens with HER2 scores of 2+/3+. The overall concordance rate of HER2 score was 70% between the gastric and breast criteria. FISH revealed HER2 gene amplification in 10/44 (23%) specimens containing only lateral/basolateral membranous staining pattern. Based on the histopathological features of HER2 expression in UCS, a scoring system that accepts lateral/basolateral staining patterns should be applied. Furthermore, we proposed specific requirements for UCS testing, including specimen selection, scoring system, and calculating the proportion of HER2-positive cells.

Diagnostic application of cyclin D1 fluorescent in situ hybridization for histologically undetermined early lesions of acral melanoma in situ: A case series.

Histologically undetermined early acral melanoma in situ (HUAMIS) is rare but a diagnostic challenge, being clinically and dermoscopically MIS (late onset, a large size (>7 mm), parallel ridges pattern) but microscopically without recognizable cytological atypia. Cyclin D1 (CCND1) gene amplification is a genetic aberration occurring in the early radial growth phase of AMs and could thus help determine malignancy for this disease. We determine the value of CCND1 amplification by FISH as a diagnostic marker for HUAMIS. CCND1 amplification was examined in paraffin-embedded skin biopsies and excisions using a dual-probes fluorescence in situ hybridization (FISH) (11q13 and CEP11). One FISH-negative case 6 was additionally examined by Mypath Melanoma (qRT-PCR). Seventeen cases (12 dysplastic nevi, 3 AMIS, and 2 invasive AM) were served as negative controls for FISH. All six patients (4 females and 2 males) were Hispanic. Pigment lesions were on the left plantar foot (4), right third finger palm (1), and right thumb subungual (1). All cases showed similar clinical and dermoscopical characteristics, including late onset (50 to 74 years old), long duration (from 2 to 15 years), large-sized pigments (from 16 to 40 mm), and a parallel ridge pattern. Junctional melanocytes with no or minimal atypia from five cases showed CCND1 amplifications. Four of 5 cases were received 1st or/and 2nd wide excisions, which demonstrated foci of histologically overt MIS. One FISH-negative case 6 demonstrated "likely malignancy" scores (>2) by Mypath Melanoma (qRT-PCR). None of negative controls showed the amplification. We propose here a simple CCND1 FISH is a practical diagnostic test to determine the malignancy of the very early progression phase of AM preceding histopathologically defined MIS. Cases presented here could be an indolent subtype of AMIS characterized by carrying a long latent radial growth phase without vertical growth, mimicking lentigo maligna.