Intent in Patent Infringement

In An Intentional Tort Theory of Patents , Professor Vishnubhakat makes two arguments. First, that liability for patent infringement should only be imposed upon defendants who intentionally make, use, or sell, patented inventions. And second, that if patent infringement includes such an intent requirement, it would no longer be a strict liability tort. This response agrees with the first thesis: patent infringement should require intentional conduct of a certain sort. However, the response disagrees with the second thesis: even if patent infringement requires such intent, liability would, in my view, still be “ strict.

Tetraploidy-associated genetic heterogeneity confers chemo-radiotherapy resistance to colorectal cancer cells

Altres ajuts: CG was supported by a contract from CIBEREHD and GEMCAD. Ö.G.G. was funded by Scientific and Technological Research Council of Turkey (TÜBİTAK) via 2214/A program. D.H. was supported by a Mildred Scheel postdoctoral scholarship of the German Cancer Aid (Deutsche Krebshilfe). This article is based on work from COST Action CA17118, supported by COST (European Cooperation in Science and Technology).Tetraploidy, or whole-genome duplication, is a common phenomenon in cancer and preludes chromosome instability, which strongly correlates with disease progression, metastasis, and treatment failure. Therefore, it is reasonable to hypothesize that tetraploidization confers multidrug resistance. Nevertheless, the contribution of whole-genome duplication to chemo-radiotherapy resistance remains unclear. Here, using isogenic diploid and near-tetraploid clones from three colorectal cancer cell lines and one non-transformed human epithelial cell line, we show a consistent growth impairment but a divergent tumorigenic potential of near-tetraploid cells. Next, we assessed the effects of first-line chemotherapeutic drugs, other commonly used agents and ionizing radiation, and found that whole-genome duplication promoted increased chemotherapy resistance and also conferred protection against irradiation. When testing the activation of apoptosis, we observed that tetraploid cells were less prone to caspase 3 activation after treatment with first-line chemotherapeutic agents. Furthermore, we found that pre-treatment with ataxia telangiectasia and Rad3 related (ATR) inhibitors, which targets response to replication stress, significantly enhanced the sensitivity of tetraploid cells to first-line chemotherapeutic agents as well as to ionizing radiation. Our findings provide further insight into how tetraploidy results in greater levels of tolerance to chemo-radiotherapeutic agents and, moreover, we show that ATR inhibitors can sensitize near-tetraploid cells to commonly used chemo-radiotherapy regimens

Intratumoral heterogeneity and clonal evolution induced by HPV integration

The human papillomavirus (HPV) genome is integrated into host DNA in most HPV-positive cancers, but the consequences for chromosomal integrity are unknown. Continuous long-read sequencing of oropharyngeal cancers and cancer cell lines identified a previously undescribed form of structural variation, "heterocateny," characterized by diverse, interrelated, and repetitive patterns of concatemerized virus and host DNA segments within a cancer. Unique breakpoints shared across structural variants facilitated stepwise reconstruction of their evolution from a common molecular ancestor. This analysis revealed that virus and virus-host concatemers are unstable and, upon insertion into and excision from chromosomes, facilitate capture, amplification, and recombination of host DNA and chromosomal rearrangements. Evidence of heterocateny was detected in extrachromosomal and intrachromosomal DNA. These findings indicate that heterocateny is driven by the dynamic, aberrant replication and recombination of an oncogenic DNA virus, thereby extending known consequences of HPV integration to include promotion of intratumoral heterogeneity and clonal evolution

The Evolution of Single Cell-derived Colorectal Cancer Cell Lines is Dominated by the Continued Selection of Tumor Specific Genomic Imbalances, Despite Random Chromosomal Instability

Intratumor heterogeneity is a major challenge in cancer treatment. To decipher patterns of chromosomal heterogeneity, we analyzed six colorectal cancer cell lines by multiplex interphase FISH (miFISH). The mismatch repair deficient cell lines DLD-1 and HCT116 had the most stable copy numbers, whereas aneuploid cell lines (HT-29, SW480, SW620 and H508) displayed a higher degree of instability. We subsequently assessed the clonal evolution of single cells in two CRC cell lines, SW480 and HT-29, which both have aneuploid karyotypes but different degrees of chromosomal instability. The clonal compositions of the single cell-derived daughter lines, as assessed by miFISH, differed for HT-29 and SW480. Daughters of HT-29 were stable, clonal, with little heterogeneity. Daughters of SW480 were more heterogeneous, with the single cell-derived daughter lines separating into two distinct populations with different ploidy (hyper-diploid and near-triploid), morphology, gene expression and tumorigenicity. To better understand the evolutionary trajectory for the two SW480 populations, we constructed phylogenetic trees which showed ongoing instability in the daughter lines. When analyzing the evolutionary development over time, most single cell-derived daughter lines maintained their major clonal pattern, with the exception of one daughter line that showed a switch involving a loss of APC. Our meticulous analysis of the clonal evolution and composition of these colorectal cancer models shows that all chromosomes are subject to segregation errors, however, specific net genomic imbalances are maintained. Karyotype evolution is driven by the necessity to arrive at and maintain a specific plateau of chromosomal copy numbers as the drivers of carcinogenesis

Molecular profiling for predicting tumor prognosis, treatment outcome and progression of squamous cell carcinoma

Squamous cell carcinoma is the most common histological tumor type in the cervix uteri and oral tongue. Although both cancers are diagnosed at an early stage in the majority of cases, cervical cancer has a better prognosis despite similarities in treatment. The aim of this thesis is to increase our knowledge of tumor progression in squamous cell carcinoma at the molecular level, and to use this knowledge to explore the clinical implications of this knowledge in the development of therapeutic regimens. We collected archived tissues from squamous cell carcinoma of the cervix uteri and oral tongue (OTSCC) and applied immunohistochemistry (IHC), DNA cytometry and fluorescence in situ hybridization (FISH) to paraffin-embedded tissues. Proliferative activity and genomic instability are two important factors in tumor progression. To identify patients with a high risk for locoregional recurrences we investigated Ki-67 expression (by means of IHC) and DNA ploidy (using DNA image cytometry) in 76 pretreatment OTSCC biopsy specimens. We found Ki-67 expression to be associated with an increased risk for locoregional recurrence in surgically-treated Stage I cancer patients (P=0.028). Ninety-seven percent of OTSCC specimens were aneuploid. Overexpression of epidermal growth factor receptor (EGFR) is associated with poor prognosis in head and neck cancer, but information on EGFR status in OTSCC is limited. We analyzed EGFR protein expression (IHC) and gene copy number (FISH) in 78 pretreatment OTSCC samples. We found EGFR gene copy numbers to be significantly associated with EGFR protein expression (P=0.002). EGFR was overexpressed in all OTSCC, suggesting that patients with this cancer type may benefit from EGFR targeting treatment. Non-smokers showed higher EGFR gene copy numbers and protein overexpression than did smokers. The presence of lymph node metastases is a strong prognostic factor in early stage cervical cancer and OTSCC. LAMP3, PROX1, PRKAA1 and CCND1 are genes associated with carcinogenesis. We analyzed these gene copy numbers using FISH probes in pretreatment cervical biopsies from LN positive and LN negative Stage IB-IIA cervical cancer patients (N=31) to explore their role in predicting LN metastasis. A combined marker panel consisting of amplified probes for LAMP3, PROX1 and PRKAA1 provided a significant (P=0.001) predictor for LN metastasis and needs to be evaluated in larger studies. To further explore genetic alterations in OTSCC, and inspired by the association between smoking habits and EGFR gene copy numbers, we applied five FISH probe markers (TERC, CCND1, EGFR, P53, CEP®4) to 65 pretreatment OTSCC specimens. CCND1 displayed the highest copy number of all markers and highest levels of this gene correlated significantly with better prognosis in Stage II OTSCC (P=0.03). Non-smoking habits were significantly related to higher copy numbers in all five markers (P=0.002)

An Improved Breast Epithelial Sampling Method for Molecular Profiling and Biomarker Analysis in Women at Risk for Breast Cancer

Background There is a strong need to define the molecular changes in normal at-risk breast epithelium to identify biomarkers and new targets for breast cancer prevention and to develop a molecular signature for risk assessment. Improved methods of breast epithelial sampling are needed to promote whole-genome molecular profiling, increase ductal epithelial cell yield, and reduce sample cell heterogeneity. Methods We developed an improved method of breast ductal sampling with ductal lavage through a 22-gauge catheter and collection of ductal samples with a microaspirator. Women at normal risk or increased risk for breast cancer were studied. Ductal epithelial samples were analyzed for cytopathologic changes, cellular yield, epithelial cell purity, quality and quantity of DNA and RNA, and use in multiple downstream molecular applications. Results We studied 50 subjects, including 40 subjects at normal risk for breast cancer and 37 subjects with non-nipple aspirate fluid-yielding ducts. This method provided multiple 1.0 mL samples of high ductal epithelial cell content (median ≥8 samples per subject of ≥5,000 cells per sample) with 80%–100% epithelial cell purity. Extraction of a single intact ductal sample (fluid and cells) or the separate frozen cellular component provided DNA and RNA for multiple downstream studies, including quantitative reverse transcription-polymerase chain reaction (PCR) for microRNA, quantitative PCR for the human telomerase reverse transcriptase gene, whole-genome DNA amplification, and array comparative genomic hybridization analysis. Conclusion An improved breast epithelial sampling method has been developed, which should significantly expand the acquisition and biomarker analysis of breast ductal epithelium in women at risk for breast cancer

Single Chromosome Aneuploidy Induces Genome-Wide Perturbation of Nuclear Organization and Gene Expression

Chromosomal aneuploidy is a defining feature of carcinomas and results in tumor-entity specific genomic imbalances. For instance, most sporadic colorectal carcinomas carry extra copies of chromosome 7, an aneuploidy that emerges already in premalignant adenomas, and is maintained throughout tumor progression and in derived cell lines. A comprehensive understanding on how chromosomal aneuploidy affects nuclear organization and gene expression, i.e., the nucleome, remains elusive. We now analyzed a cell line established from healthy colon mucosa with a normal karyotype (46,XY) and its isogenic derived cell line that acquired an extra copy of chromosome 7 as its sole anomaly (47,XY,+7). We studied structure/function relationships consequent to aneuploidization using genome-wide chromosome conformation capture (Hi-C), RNA sequencing and protein profiling. The gain of chromosome 7 resulted in an increase of transcript levels of resident genes as well as genome-wide gene and protein expression changes. The Hi-C analysis showed that the extra copy of chromosome 7 is reflected in more interchromosomal contacts between the triploid chromosomes. Chromatin organization changes are observed genome-wide, as determined by changes in A/B compartmentalization and topologically associating domain (TAD) boundaries. Most notably, chromosome 4 shows a profound loss of chromatin organization, and chromosome 14 contains a large A/B compartment switch region, concurrent with resident gene expression changes. No changes to the nuclear position of the additional chromosome 7 territory were observed when measuring distances of chromosome painting probes by interphase FISH. Genome and protein data showed enrichment in signaling pathways crucial for malignant transformation, such as the HGF/MET-axis. We conclude that a specific chromosomal aneuploidy has profound impact on nuclear structure and function, both locally and genome-wide. Our study provides a benchmark for the analysis of cancer nucleomes with complex karyotypes