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(A) Schematic of the gene expression assay protocol. (B) Representative five-point standard curves of FFPE tumor RNA (red and black lines) and universal RNA (blue line) run on the breast cancer gene expression assay (slope of line indicated). (C) Intra-chip reproducibility of FFPE tumor samples run on the same 96.96 Dynamic Array. (D) Inter-chip reproducibility of the breast cancer gene expression assay assessed by comparing Ct values of universal RNA across seven independent assay runs. R-squared values are indicated in boxes.
Source publication
Patients with newly diagnosed, early stage estrogen receptor positive (ER+) breast cancer often show disease free survival in excess of five years following surgery and systemic adjuvant therapy. As such, an important question is whether diagnostic tumor tissue from the primary lesion offers an accurate molecular portrait of the cancer post recurre...
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Citations
... PIK3CA mutations are renowned for instigating the PI3K-AKT-mTOR pathway, a pivotal regulator of cellular growth and survival frequently implicated in breast cancer [58]. The diminished occurrence of PIK3CA mutations in the high-risk demographic could imply that these mutations are primarily prevalent in the initial stages of breast carcinoma, such as ductal carcinoma in situ [59]. Alternatively, this observation might signify the pre-AGING existence of an activated PI3K-AKT-mTOR pathway in high-risk patients, thereby contributing to their heightened risk. ...
... The primers used for the qRT-PCR analysis were designed using the Primer-BLAST tool on the NCBI website (127) for the lpw30151 (JAC29 and JAC30), lpw06421 (JAC31 and JAC32), lpw03471 (JAC33 and JAC34), lpw27171 (JAC35 and JAC36), and lpw03481 (JAC37 and JAC38) genes and are listed in Table S6. The relative RNA abundance was determined using the threshold cycle (2DC T ) method (C T for reference -C T for gene of interest) (128), with gyrB and lpw16991 being used as reference genes (45,80,129,130). In follow-up experiments, bacteria were incubated in BYE broth at 30°C to mid-log phase (OD 660 of 1.8), and then the cultures were split into equal portions; cultures were incubated for an additional 30 min at either 30°C, 37°C, 45°C, or 50°C; and RNA was analyzed as above. ...
Previously, we demonstrated that Cas2 encoded within the CRISPR-Cas locus of Legionella pneumophila strain 130b promotes the ability of the Legionella pathogen to infect amoebal hosts. Given that L. pneumophila Cas2 has RNase activity, we posited that the cytoplasmic protein is regulating the expression of another Legionella gene(s) that fosters intracellular infection. Proteomics revealed 10 proteins at diminished levels in the cas2 mutant, and reverse transcription-quantitative (qRT-PCR) confirmed the reduced expression of a gene encoding putative small heat shock protein C2 (HspC2), among several others. As predicted, the gene was expressed more highly at 37°C to 50°C than that at 30°C, and an hspC2 mutant, but not its complemented derivative, displayed ~100-fold reduced CFU following heat shock at 55°C. Compatible with the effect of Cas2 on hspC2 expression, strains lacking Cas2 also had impaired thermal tolerance. The hspC2 mutant, like the cas2 mutant before it, was greatly impaired for infection of Acanthamoeba castellanii, a frequent host for legionellae in waters. HspC2 and Cas2 were not required for entry into these host cells but promoted the replicative phase of intracellular infection. Finally, the hspC2 mutant exhibited an additional defect during the infection of macrophages, which are the primary host for legionellae during lung infection. In summary, hspC2 is upregulated by the presence of Cas2, and HspC2 uniquely promotes both L. pneumophila extracellular survival at high temperatures and infection of amoebal and human host cells. To our knowledge, these findings also represent the first genetic proof linking Cas2 to thermotolerance, expanding the repertoire of noncanonical functions associated with CRISPR-Cas proteins.
... Focused on primary versus metastatic diversity, a study in NSCLC patients showed that EGFR expression was lower in metastatic tissues 14 . In contrast, primary and metastatic specimens from breast cancer patients had a high degree of concordance between the immunohistochemistry staining levels of estrogen receptor, progesterone receptor, HER-2, and Ki-67 15,16 . More recent work identified differences in TMB measurements between specimens from primary and metastatic biopsies 4 . ...
Background
Tumor mutational burden (TMB) has been proposed as a predictive biomarker of response to immunotherapy. Efforts to standardize TMB scores for use in the clinic and to identify the factors that could impact TMB scores are of high importance. However, the biopsy collection site has not been assessed as a factor that may influence TMB scores.
Methods
We examine a real-world cohort comprising 137,771 specimens across 47 tissues in 12 indications profiled by the FoundationOne assay (Foundation Medicine, Cambridge, MA) to assess the prevalence of biopsy sites for each indication and their TMB scores distribution.
Results
We observe a wide variety of biopsy sites from which specimens are sent for genomic testing and show that TMB scores differ in a cancer- and tissue-specific manner. For example, brain or adrenal gland specimens from NSCLC patients show higher TMB scores than local lung specimens (mean difference 3.31 mut/Mb; p < 0.01, 3.90 mut/Mb; p < 0.01, respectively), whereas bone specimens show no difference.
Conclusions
Our data shed light on the biopsied tissue as a driver of TMB measurement variability in clinical practice.
... Equally there are studies where inhibitors of these pathways have shown no significant improvement in TTP/PFS (71,72). The fact that the present study included hormone naïve patients and also used primary tumor tissue, where the PI3KCA/Akt/mTOR pathway may not be as disrupted as in metastases (73)(74)(75), may explain why no further markers reflective of the Akt pathway were intrinsically associated with TAM resistance. This therefore is one limitation of this study and it again highlights the importance of obtaining tumor tissue immediately prior to a new treatment especially within future studies. ...
... Tumor sections were macrodissected to maximize tumor content before DNA and RNA isolation. Genomic DNA and total RNA were purified using QIAamp DNA FFPE tissue kits (Qiagen) and High Pure FFPE RNA micro-kit (Roche Applied Sciences), respectively (15). ...
Purpose: Four consensus molecular subtypes (CMS1–4) of colorectal cancer were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for colorectal cancer–related mortality, we examined the molecular and clinical attributes of CMS in metastatic colorectal cancer (mCRC).
Experimental Design: We developed a colorectal cancer–focused NanoString-based CMS classifier that is ideally suited to interrogate archival tissues. We successfully used this panel in the CMS classification of formalin-fixed paraffin-embedded (FFPE) tissues from mCRC cohorts, one of which is composed of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modeling of colorectal cancer at relevant sites.
Results: Using our classifier, we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in nonmetastatic early-stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared with other CMS. Assigning CMS to primary tumors and their matched metastases reveals mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs reveals differences in stromal composition at each site. The development of two novel orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic colorectal cancer.
Conclusions: We describe the utility of a NanoString panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on colorectal cancer heterogeneity during disease progression.
... DNA yield was determined by droplet digital PCR targeting EFTUD2 and TRAK2 (ddPCR, Bio-Rad), with a range from 4-8000 ng and median yield of 59 ng. In addition, genomic DNA was extracted from available formalin-fixed paraffin-embedded (FFPE) tumor sections using the QIAamp DNA FFPE Tissue Kit (QIAGEN) as previously described (21). DNA from FFPE tissue was fragmented with Covaris S220, according to the manufacturer's instructions to around 150 bp with size confirmed by Bioanalyzer (Agilent). ...
Purpose:
We developed a method to monitor copy number variations (CNV) in plasma cell-free DNA (cfDNA) from patients with metastatic squamous non-small cell lung cancer (NSCLC). We aimed to explore the association between tumor-derived cfDNA and clinical outcomes, and sought CNVs that may suggest potential resistance mechanisms.
Experimental design:
Sensitivity and specificity of low-pass whole-genome sequencing (LP-WGS) were first determined using cell line DNA and cfDNA. LP-WGS was performed on baseline and longitudinal cfDNA of 152 patients with squamous NSCLC treated with chemotherapy, or in combination with pictilisib, a pan-PI3K inhibitor. cfDNA tumor fraction and detected CNVs were analyzed in association with clinical outcomes.
Results:
LP-WGS successfully detected CNVs in cfDNA with tumor fraction ≥10%, which represented approximately 30% of the first-line NSCLC patients in this study. The most frequent CNVs were gains in chromosome 3q, which harbors the PIK3CA and SOX2 oncogenes. The CNV landscape in cfDNA with a high tumor fraction generally matched that of corresponding tumor tissue. Tumor fraction in cfDNA was dynamic during treatment, and increases in tumor fraction and corresponding CNVs could be detected before radiographic progression in 7 of 12 patients. Recurrent CNVs, such as MYC amplification, were enriched in cfDNA from posttreatment samples compared with the baseline, suggesting a potential resistance mechanism to pictilisib.
Conclusions:
LP-WGS offers an unbiased and high-throughput way to investigate CNVs and tumor fraction in cfDNA of patients with cancer. It may also be valuable for monitoring treatment response, detecting disease progression early, and identifying emergent clones associated with therapeutic resistance.
... MammaTyper® may evolve as a valid alternative to IHC. This is supported by the substantial correlation that exists between protein and mRNA expression in general [15] and for breast cancer biomarkers in particular [16][17][18] and by the desire to increase the reproducibility of biomarker testing, in particular for the assessment of proliferative activity (i.e.Ki-67) [19]. Mam-maTyper® has shown excellent analytical performance, promising clinical validity both in the adjuvant and neoadjuvant setting, with concordance to IHC documented in more than 800 clinical samples [20][21][22]. ...
Background
Tissue heterogeneity in formalin-fixed paraffin-embedded (FFPE) breast cancer specimens may affect the accuracy of reverse transcription quantitative real-time PCR (RT-qPCR). Herein, we tested the impact of tissue heterogeneity of breast cancer specimen on the RT-qPCR-based gene expression assay MammaTyper®.
Methods
MammaTyper® quantifies the mRNA expression of the four biomarkers ERBB2, ESR1, PGR, and MKI67. Based on pre-defined cut-off values, this molecular in vitro diagnostic assay permits binary marker classification and determination of breast cancer subtypes as defined by St Gallen 2013. In this study, we compared data from whole FFPE sections with data obtained in paired RNA samples after enrichment for invasive carcinoma via macro- or laser-capture micro-dissection.
Results
Compared to whole sections, removal of surrounding adipose tissue by macrodissection generated mean absolute 40-ddCq differences of 0.28–0.32 cycles for all four markers, with ≥90% concordant binary classifications. The mean raw marker Cq values in the adipose tissue were delayed by 6 to 7 cycles compared with the tumor-enriched sections, adding a trivial linear fold change of 1.0078 to 1.0156. Comparison of specimens enriched for invasive tumor with whole sections with as few as 20% tumor cell content resulted in mean absolute differences that remained on average below 0.59 Cq. The mean absolute difference between whole sections containing up to 60% ductal carcinoma in situ (DCIS) and specimens after dissection of DCIS was only 0.16–0.25 cycles, although there was a tendency for higher gene expression in DCIS. Observed variations were related to small size of samples and proximity of values to the limit of detection.
Conclusion
Expression of ESR1, PGR, ERBB2 and MKI67 by MammaTyper® is robust in clinical FFPE samples. Assay performance was unaffected by adipose tissue and was stable in samples with as few as 20% tumor cell content and up to 60% DCIS.
... DNA and RNA Isolation from FFPE Tumor Tissue. DNA and RNA isolation was performed as described previously (41). Briefly, tumor tissue from FFPE sec- tions was lysed using tumor lysis buffer and proteinase K to allow for complete digestion and release of nucleic acids. ...
Significance
Programmed death-ligand 1 (PD-L1) expression on tumor cells and tumor-infiltrating immune cells is regulated by distinct mechanisms and has nonredundant roles in regulating anticancer immunity, and PD-L1 on both cell types is important for predicting best response to atezolizumab in non-small cell lung cancer.
... Pretreatment tumor samples were indeed analyzed with the Fluidigm-based gene-expression platform. 48 CD8A, GZMA, GZMB, IFN-γ, EOMES, CXCL9, CXCL10, and TBX21, defining the Teff and IFN-γ signatures as associated with activated T-cells, cytolytic activity and IFN-γ expression, were shown to have high co-expression in tumor specimens of POPLAR patients. 19 ...
Francesco Facchinetti, Paola Bordi, Alessandro Leonetti, Sebastiano Buti, Marcello Tiseo Medical Oncology Unit, University Hospital of Parma, Parma, Italy Abstract: Programed cell death-1/programed death ligand-1 (PD-1/PD-L1) blockade represents an affirmed reality in the treatment of advanced non-small-cell lung cancer (NSCLC) patients. Atezolizumab, an anti-PD-L1 agent, figures among the drugs that provide previously unenvisaged outcomes in the pretreated setting of metastatic NSCLC. Increasing evidence vouches for the early administration of PD-1/PD-L1 blockers in untreated patients, encompassing atezolizumab combinations with chemotherapy and the anti-angiogenic agent bevacizumab. Moreover, the development of atezolizumab allowed to derive several hints regarding clinical and immunological factors predictive of its activity and efficacy, some of them exclusive among this class of drugs. This review provides an overview of atezolizumab development throughout clinical trials toward its applicability in the routine practice, with a particular focus on patient selection based on clinical and immune-related factors. Keywords: non-small cell lung cancer, NSCLC, immune checkpoint blockers, ICB, PD-1, PD-L1, atezolizumab development, biomarkers
... Locally activated T cells have several potential targets in the metastatic setting (assuming overlap in antigenicity between a primary tumor and the metastases it seeds [21][22][23] , and can influence growth dynamics systemically. For a locally activated effector cell to extravasate at a particular anatomical target site it must first traverse the circulatory system to reach the tumor, influenced by both the blood flow to that region and activation site-specific homing cues 24 . ...
Complex interactions occur between tumor and host immune system at each site in the metastatic setting, the outcome of which can determine behavior ranging from dormancy to rapid growth. An additional layer of complexity arises from the understanding that cytotoxic T cells can traffic through the host circulatory system. Coupling mathematical models of local tumor-immune dynamics and systemic T cell trafficking allows us to simulate the evolution of tumor and immune cell populations in anatomically distant sites following local therapy and thus computationally evaluate immune interconnectivity. Results suggest that the presence of a secondary site may either inhibit or promote growth of the primary, depending on the capacity for immune recruitment of each tumor and the resulting systemic redistribution of T cells. Treatment such as surgical resection and radiotherapy can be simulated to estimate both the decrease in tumor volume at the local treatment-targeted site, and the change in overall tumor burden and tumor growth trajectories across all sites. Qualitatively similar responses of distant tumors to local therapy (positive and negative abscopal effects) to those reported in the clinical setting were observed. Such findings may facilitate an improved understanding of general disease kinetics in the metastatic setting: if metastatic sites are interconnected through the immune system, truly local therapy does not exist.
