Clinical correlates of blood-derived circulating tumor DNA in pancreatic cancer.

BackgroundTreatment outcomes for patients with advanced pancreatic ductal adenocarcinoma (PDAC) remain dismal. There are unmet needs for understanding the biologic basis of this malignancy using novel next-generation sequencing technologies. Herein, we investigated the clinical utility of circulating tumor DNA (ctDNA) (the liquid biopsy) in this malignancy.MethodsctDNA was analyzed in 112 patients with PDAC (54-73 genes) and tissue DNA in 66 patients (315 genes) (both clinical-grade next-generation sequencing). Number of alterations, %ctDNA, concordance between ctDNA and tissue DNA, and correlation of ctDNA results with survival were assessed.ResultsThe most common genes altered in ctDNA were TP53 (46% of patients, N = 51) and KRAS (44%, N = 49). Median number of characterized ctDNA alterations per patient was 1 (range, 0-6), but patients with advanced PDAC had significantly higher numbers of ctDNA alterations than those with surgically resectable disease (median, 2 versus 0.5, P = 0.04). Overall, 75% (70/94) of advanced tumors had ≥ 1 ctDNA alteration. Concordance rate between ctDNA and tissue DNA alterations was 61% for TP53 and 52% for KRAS. Concordance for KRAS alterations between ctDNA and tissue DNA from metastatic sites was significantly higher than between ctDNA and primary tumor DNA (72% vs 39%, P = 0.01). Importantly, higher levels of total %ctDNA were an independent prognostic factor for worse survival (hazard ratio, 4.35; 95% confidence interval, 1.85-10.24 [multivariate, P = 0.001]). A patient with three ctDNA alterations affecting the MEK pathway (GNAS, KRAS, and NF1) attained a response to trametinib monotherapy ongoing at 6 months.ConclusionsOur findings showed that ctDNA often harbored unique alterations some of which may be targetable and that significantly greater numbers of ctDNA alterations occur in advanced versus resectable disease. Furthermore, higher ctDNA levels were a poor prognostic factor for survival

Detection rate of actionable mutations in diverse cancers using a biopsy-free (blood) circulating tumor cell DNA assay.

Analysis of cell-free DNA using next-generation sequencing (NGS) is a powerful tool for the detection/monitoring of alterations present in circulating tumor DNA (ctDNA). Plasma extracted from 171 patients with a variety of cancers was analyzed for ctDNA (54 genes and copy number variants (CNVs) in three genes (EGFR, ERBB2 and MET)). The most represented cancers were lung (23%), breast (23%), and glioblastoma (19%). Ninety-nine patients (58%) had at least one detectable alteration. The most frequent alterations were TP53 (29.8%), followed by EGFR (17.5%), MET (10.5%), PIK3CA (7%), and NOTCH1 (5.8%). In contrast, of 222 healthy volunteers, only one had an aberration (TP53). Ninety patients with non-brain tumors had a discernible aberration (65% of 138 patients; in 70% of non-brain tumor patients with an alteration, the anomaly was potentially actionable). Interestingly, nine of 33 patients (27%) with glioblastoma had an alteration (6/33 (18%) potentially actionable). Overall, sixty-nine patients had potentially actionable alterations (40% of total; 69.7% of patients (69/99) with alterations); 68 patients (40% of total; 69% of patients with alterations), by a Food and Drug Administration (FDA) approved drug. In summary, 65% of diverse cancers (as well as 27% of glioblastomas) had detectable ctDNA aberration(s), with the majority theoretically actionable by an approved agent

Revisiting Epidermal Growth Factor Receptor (EGFR) Amplification as a Target for Anti-EGFR Therapy: Analysis of Cell-Free Circulating Tumor DNA in Patients With Advanced Malignancies.

PurposeTo date, evidence for tissue epidermal growth factor receptor (EGFR) overexpression as a biomarker for anti-EGFR therapies has been weak. We investigated the genomic landscape of EGFR amplification in blood-derived cell-free tumor DNA (cfDNA) across diverse cancers and the role of anti-EGFR therapies in achieving response.MethodsWe assessed EGFR amplification status among 28,584 patients with malignancies evaluated by clinical-grade next-generation sequencing (NGS) of blood-derived cfDNA (54- to 73-gene panel). Furthermore, we curated the clinical characteristics of 1,434 patients at the University of California San Diego who had cfDNA testing by this NGS test.ResultsOverall, EGFR amplification was detected in cfDNA from 8.5% of patients (2,423 of 28,584), most commonly in colorectal (16.3% [458 of 2,807]), non-small-cell lung (9.0% [1,096 of 12,197]), and genitourinary cancers (8.1% [170 of 2,104]). Most patients had genomic coalterations (96.9% [95 of 98]), frequently involving genes affecting other tyrosine kinases (72.4% [71 of 98]), mitogen-activated protein kinase cascades (56.1% [55 of 98]), cell-cycle-associated signals (52.0% [51 of 98]), and the phosphoinositide 3-kinase pathway (35.7% [35 of 98]). EGFR amplification emerged in serial cfDNA after various anticancer therapies (n = 6), including checkpoint inhibitors (n = 4), suggesting a possible role for these amplifications in acquired resistance. Nine evaluable patients with EGFR amplification were treated with anti-EGFR-based regimens; five (55.6%) achieved partial responses, including three patients whose tissue NGS lacked EGFR amplification.ConclusionEGFR amplification was detected in cfDNA among 8.5% of 28,584 diverse cancers. Most patients had coexisting alterations. Responses were observed in five of nine patients who received EGFR inhibitors. Incorporating EGFR inhibitors into the treatment regimens of patients harboring EGFR amplification in cfDNA merits additional study

Genomic Assessment of Blood-Derived Circulating Tumor DNA in Patients With Colorectal Cancers: Correlation With Tissue Sequencing, Therapeutic Response, and Survival.

PurposeGenomic alterations in blood-derived circulating tumor DNA (ctDNA) from patients with colorectal cancers were correlated with clinical outcomes.Patients and methodsNext-generation sequencing of ctDNA (54- to 73-gene panel) was performed in 94 patients with colorectal cancer.ResultsMost patients (96%) had metastatic or recurrent disease at the time of blood draw. The median number of nonsynonymous alterations per patient was three (range, zero to 30). The most frequently aberrant genes were TP53 (52.1% of patients), KRAS (34%), and APC (28.7%). Concordance between tissue and blood next-generation sequencing ranged from 63.2% (APC) to 85.5% (BRAF). Altogether, 74 patients (79%) had one or more nonsynonymous alterations, 69 (73%) had one or more potentially actionable alterations, and 61 (65%) had an alteration actionable by a drug approved by the US Food and Drug Administration (on or off label). Lung metastases correlated with improved survival from diagnosis in univariable analysis. ctDNA of 5% or more from blood tests as well as EGFR and ERBB2 (HER2) nonsynonymous alterations correlated with worse survival (but only ERBB2 remained significant in multivariable analysis). No two patients had identical molecular portfolios. Overall, 65% versus 31% of patients treated with matched (n = 17) versus unmatched therapy (n = 18) after ctDNA testing achieved stable disease for 6 months or more, partial response, or complete response (P = .045); progression-free survival, 6.1 versus 2.3 months (P = .08); and survival not reached versus 9.4 months (P = .146; all by multivariable analysis).ConclusionPatients with colorectal cancer have heterogeneous ctDNA profiles, and most harbor potentially actionable ctDNA alterations. Matched therapy yielded higher rates of stable disease for 6 months or more, partial response, or complete response. ctDNA assessment may have clinical utility and merits further investigation

BRAF mutations classes I, II, and III in NSCLC patients included in the SLLIP trial : The need for a new pre-clinical treatment rationale

BRAF V600 mutations have been found in 1-2% of non-small-cell lung cancer (NSCLC) patients, with Food and Drug Administration (FDA) approved treatment of dabrafenib plus trametinib and progression free survival (PFS) of 10.9 months. However, 50-80% of BRAF mutations in lung cancer are non-V600, and can be class II, with intermediate to high kinase activity and RAS independence, or class III, with impaired kinase activity, upstream signaling dependence, and consequently, sensitivity to receptor tyrosine kinase (RTK) inhibitors. Plasma cell-free DNA (cfDNA) of 185 newly diagnosed advanced lung adenocarcinoma patients (Spanish Lung Liquid versus Invasive Biopsy Program, SLLIP, NCT03248089) was examined for BRAF and other alterations with a targeted cfDNA next-generation sequencing (NGS) assay (Guardant360®, Guardant Health Inc., CA, USA), and results were correlated with patient outcome. Cell viability with single or combined RAF, MEK, and SHP2 inhibitors was assessed in cell lines with BRAF class I, II, and III mutations. Out of 185 patients, 22 had BRAF alterations (12%) of which seven patients harbored amplifications (32%) and 17 had BRAF mutations (77%). Of the BRAF mutations, four out of 22 (18%) were V600E and 18/22 (82%) were non-V600. In vitro results confirmed sensitivity of class III and resistance of class I and II BRAF mutations, and BRAF wild type cells to SHP2 inhibition. Concomitant MEK or RAF and SHP2 inhibition showed synergistic effects, especially in the class III BRAF-mutant cell line. Our study indicates that the class of the BRAF mutation may have clinical implications and therefore should be defined in the clinical practice and used to guide therapeutic decision

Detection of ERBB2 (HER2) Gene Amplification Events in Cell-Free DNA and Response to Anti-HER2 Agents in a Large Asian Cancer Patient Cohort

Background: HER2 antagonists have marked activity and are approved for the treatment of HER2 overexpressing breast and gastric cancers. Recent studies have shown that ERBB2 (HER2) gene amplification and overexpression may also be actionable in other tumor types. Inter- and intratumoral heterogeneity in HER2 status, however, poses a significant challenge in identifying patients that may benefit from HER2-targeted therapies. ERBB2 amplification as identified by circulating cell-free DNA (cfDNA), which circumvents tissue heterogeneity issues, is emerging as a robust biomarker predictive of response to anti-HER2 agents. Here, the prevalence and genomic landscape of ERBB2 alterations detectable by next-generation sequencing (NGS) of cfDNA was evaluated in a large cohort of Asian patients with advanced solid tumors.Methods: Results were queried for consecutive patients (n = 469) tested by a comprehensive 70/73-gene cfDNA NGS assay (Guardant360®) between November 2015 and June 2018. Patients with ERBB2 gene alterations including copy number amplifications (CNAs), single nucleotide variants (SNVs), and insertion-deletions (indels) were identified.Results:ERBB2 alterations were detected in 52 patients (11.1%); ERBB2 SNVs, CNAs, and indels were found in 27 (5.8%), 27 (5.8%), and 10 (2.1%) patients, respectively. ERBB2 amplification was most frequently identified in gastric (21.4%; 6/28), colorectal (11.1%; 5/45), lung (3.9%; 9/231), and breast (3.2%; 1/31) cancer patients. ERBB2 amplification was often mutually exclusive with other oncogenic alterations in gastric (83.3%; 5/6) and colorectal (60%; 3/5) cancer patients. ERBB2 copy number gains were also highest in gastric and colorectal cancers (median 4.8 and 6.6, respectively). We further report two cases of advanced gastric cancer patients, one treatment naïve, and the other having failed four lines of therapy, whose ERBB2 CNAs were identified by cfDNA and derived clinical benefit from HER2-based therapies.Conclusion: Our data indicate that ERBB2 amplification is a common event in solid tumors among Asian cancer patients. High ERBB2 incidence and copy number gains were observed in gastric and colorectal cancer patients, often in the absence of other oncogenic mutations, underscoring its likely role as the driver alteration in those settings. Finally, we show the potential of comprehensive cfDNA testing in identifying patients who are most likely to benefit from HER2-targeted therapies

Genomic profile of advanced breast cancer in circulating tumour DNA.

The genomics of advanced breast cancer (ABC) has been described through tumour tissue biopsy sequencing, although these approaches are limited by geographical and temporal heterogeneity. Here we use plasma circulating tumour DNA sequencing to interrogate the genomic profile of ABC in 800 patients in the plasmaMATCH trial. We demonstrate diverse subclonal resistance mutations, including enrichment of HER2 mutations in HER2 positive disease, co-occurring ESR1 and MAP kinase pathway mutations in HR + HER2- disease that associate with poor overall survival (p = 0.0092), and multiple PIK3CA mutations in HR + disease that associate with short progression free survival on fulvestrant (p = 0.0036). The fraction of cancer with a mutation, the clonal dominance of a mutation, varied between genes, and within hotspot mutations of ESR1 and PIK3CA. In ER-positive breast cancer subclonal mutations were enriched in an APOBEC mutational signature, with second hit PIK3CA mutations acquired subclonally and at sites characteristic of APOBEC mutagenesis. This study utilises circulating tumour DNA analysis in a large clinical trial to demonstrate the subclonal diversification of pre-treated advanced breast cancer, identifying distinct mutational processes in advanced ER-positive breast cancer, and novel therapeutic opportunities

Molecular Profiling of Hepatocellular Carcinoma Using Circulating Cell-Free DNA.

PurposeMolecular profiling has been used to select patients for targeted therapy and determine prognosis. Noninvasive strategies are critical to hepatocellular carcinoma (HCC) given the challenge of obtaining liver tissue biopsies.Experimental designWe analyzed blood samples from 206 patients with HCC using comprehensive genomic testing (Guardant Health) of circulating tumor DNA (ctDNA).ResultsA total of 153/206 (74.3%) were men; median age, 62 years (range, 18-91 years). A total of 181/206 patients had ≥1 alteration. The total number of alterations was 680 (nonunique); median number of alterations/patient was three (range, 1-13); median mutant allele frequency (% cfDNA), 0.49% (range, 0.06%-55.03%). TP53 was the common altered gene [>120 alterations (non-unique)] followed by EGFR, MET, ARID1A, MYC, NF1, BRAF, and ERBB2 [20-38 alterations (nonunique)/gene]. Of the patients with alterations, 56.9% (103/181) had ≥1 actionable alterations, most commonly in MYC, EGFR, ERBB2, BRAF, CCNE1, MET, PIK3CA, ARID1A, CDK6, and KRAS. In these genes, amplifications occurred more frequently than mutations. Hepatitis B (HBV)-positive patients were more likely to have ERBB2 alterations, 35.7% (5/14) versus 8.8% HBV-negative (P = 0.04).ConclusionsThis study represents the first large-scale analysis of blood-derived ctDNA in HCC in United States. The genomic distinction based on HCC risk factors and the high percentage of potentially actionable genomic alterations suggests potential clinical utility for this technology

STUDI ALTERNATIF PERENCANAAAN SISTEM RANGKA PEMIKUL MOMEN KHUSUS (SRPMK) DENGAN KOLOM BULAT PADA KANTOR PUSAT ESTIKES KEPANJEN MALANG

Kolom merupakam elemen vertikal suatu struktur yang berfungsi menahan beban aksial dan momen sebagai akibat beban gravitasi dan beban lateral yang bekerja pada struktur. Oleh karena itu, kolom memegan penampang penting pada keutuhan struktur, apabila kolom mengalami kegagalan akan berakibat pada keruntuhan struktur bangunan atas gedung. perbedaan kolom bulat dan kolom persegi sangkat mendasar. Jika ditinjau dari tulangan sengkang, kolom bulat perpenampang spiral memiliki jarak sengkang yang berdekatan diabnding dengan kolom dengan kolom persegi yang mempunya bentuk sengkang tunggal dan jarak antara yang relatif besaar. Kolom bulat yang menghasilkan kapsitas penampang, gaya – gaya dalam seperti gaya aksial; gaya geser; gaya momen, dan simpangan (maximum displacement) sehingga dalam skripsi ini untuk mengetahui desain kolom bulat yang efisien dan efikas didalam perencanaan. Hasil yang diperoleh dari perenanaan struktur gedung dengan kolom bulat pada gedung kantor pusat stikes kepanjen malang dengan program bantu STAAD PRO V 8, yang ditinjau dari kapasitas penampang dengan luas mutu beton kolom bulat menghasilkan ØPn (aksial nominal) = 3988,690 kN, ØMn ( Momen nominal ) = 478,812 kNm, Vn (Geser nominal) = 681629,848 N. Sehingga kolom bulat memiliki kapasitas penampang yang lebih besar dan efektif. Ditinjau dari rasio dan gaya-gaya dalam struktur, kolom bulat memiliki gaya aksial = 0,021, kekakuan struktur pada kolom bulat memiliki simpangan (maximum displacements) yang lebih besar dari kolom persegi. Sehingga kekakuan pada kolom bulat lebih tinggi dengan kolom persegi

HIV Capsid is a Tractable Target for Small Molecule Therapeutic Intervention

Despite a high current standard of care in antiretroviral therapy for HIV, multidrug-resistant strains continue to emerge, underscoring the need for additional novel mechanism inhibitors that will offer expanded therapeutic options in the clinic. We report a new class of small molecule antiretroviral compounds that directly target HIV-1 capsid (CA) via a novel mechanism of action. The compounds exhibit potent antiviral activity against HIV-1 laboratory strains, clinical isolates, and HIV-2, and inhibit both early and late events in the viral replication cycle. We present mechanistic studies indicating that these early and late activities result from the compound affecting viral uncoating and assembly, respectively. We show that amino acid substitutions in the N-terminal domain of HIV-1 CA are sufficient to confer resistance to this class of compounds, identifying CA as the target in infected cells. A high-resolution co-crystal structure of the compound bound to HIV-1 CA reveals a novel binding pocket in the N-terminal domain of the protein. Our data demonstrate that broad-spectrum antiviral activity can be achieved by targeting this new binding site and reveal HIV CA as a tractable drug target for HIV therapy