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Background: BRCA deficient Triple negative breast cancers (TNBC) are effectively treated with platinum agents but, upon relapse, resistance is common. A number of genes have been shown to mediate chemoresistance in vitro, but none have been clinically useful due to the heterogeneous mechanisms of in vivo tumor chemo-resilience. Methods: We attacked this problem by recapitulating the generation of in vivo resistance to cisplatin in 50 mice bearing a platinum sensitive TNBC patient derived xenograft (PDX) TM00099, a BRCA1 deficient type 1 tandem duplicator phenotype tumor. Untreated tumors were compared to residual tumors that were sampled after the first cycle of cisplatin, upon recovery, and after the second cycle of drug which generated platinum resistant and sensitive tumors. Our earlier work on TM00099 suggested the existence of two major subspecies, designated A and B, with shifts observed in their proportions post treatment (1). We deconvoluted the bulk tumors into their clonal components to assess the precise numerical fluxes after each treatment cycle to gain insight into the cellular basis of the emergence of in vivo resistance. Results: 55 single cell derived clones isolated from bulk TM00099 tumors were genomically characterized identifying five subclonal populations: B, CCR, and variations of the original A: A25, A33 and A50. SNP analyses indicated that these five subclones comprised the vast majority (~93%) of all TM00099 tumors. Lineage analysis revealed that all A clones were related but distinct from B. CCR however had both A and B SNP markers and relatively higher ploidy indicating that CCR is a fusion of ancestral A and B clones. We found that A50, A33, and CCR were ~1.9X more resistant to cisplatin (mean IC50=4.4 µM) compared to the sensitive clones A25 and B (mean IC50=2.3 µM; p=0.002) in vitro. Although B clones had similar IC50 to A25s, B had improved in vitro survival at higher concentrations of cisplatin suggesting a dormancy-like phenotype: the persistent B cells did not recover within 50 days after in vitro exposure to cisplatin. Using clonal markers in bulk tumors, we found excellent concordance with their in vitro phenotypic analysis: after the first cycle of cisplatin, there was a proportional decline in A25, an enrichment of B, and stable proportions of A50, A33, and CCR. After the second platinum cycle, the emerging resistant tumors were mostly devoid of A25, and had low proportions of B, but highly enriched for A50, A33, CCR and an uncharacterized resistant A clone. The sensitive tumor residuals after the second platinum dose were predominantly comprised of B. Genomic analysis of the clones did not reveal any genetic drivers of resistance. Transciptionally, the sensitive B clones were characterized as mesenchymal or basal-like 1 TNBC subtypes, while the resistant As were categorized as basal-like 2, which have enhanced growth factor signaling and is associated with poorer response to chemotherapy. Correspondingly, the MAPK and stress assosciated NF-κB signaling pathways were augmented in As which were indeed more sensitive to blockade of MEK, EGFR and NF-κB than the platinum sensitive B clone. A25s which are sensitive revertants of the otherwise resistant A group use a mechanism to bypass resistance likely driven by ZNF350 and ZNF93. Conclusions: Our clonal reconstruction of TM00099 showed that acquired resistance can emerge by enrichment of not one but a composite of multiple preexisting resistant clones. The origins and characteristics of these clones are complex and include epigenetically driven resistance (A50 and A33), cell fusion mediated resistance (CCR), reversion to sensitivity (A25), and dormancy despite initial sensitivity (B). These nuances would not be discerned using bulk tumor assessments pointing to single cell genomic analyses as the most precise way to deconvolute the capacity of TNBC tumors to develop resistance. References: 1. H. Kim et al., Sci Rep. 8, 17937 (2018). Citation Format: Pooja Kumar, Francesca Menghi, Robert Straub, Patience Mukashyaka, Michael W. Lloyd, Harshpreet Chandok, Joshy George, Jeffery Chuang, Edison Liu. Deconvoluting dynamics of acquired chemoresistance in Triple Negative Breast Cancer tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-10.

Three-dimensional (3D) culture models, such as organoids, are flexible systems to interrogate cellular growth and morphology, multicellular spatial architecture, and cell interactions in response to drug treatment. However, new computational methods to segment and analyze 3D models at cellular resolution with sufficiently high throughput are needed to realize these possibilities. Here we reportCellos(Cell and Organoid Segmentation), an accurate, high throughput image analysis pipeline for 3D organoid and nuclear segmentation analysis.Cellossegments organoids in 3D using classical algorithms and segments nuclei using a Stardist-3D convolutional neural network which we trained on a manually annotated dataset of 3,862 cells from 36 organoids confocally imaged at 5 μm z-resolution. To evaluate the capabilities ofCelloswe then analyzed 74,450 organoids with 1.65 million cells, from multiple experiments on triple negative breast cancer organoids containing clonal mixtures with complex cisplatin sensitivities.Celloswas able to accurately distinguish ratios of distinct fluorescently labelled cell populations in organoids, with Celloswas able to recapitulate traditional luminescence-based drug response quantifications by analyzing 3D images, including parallel analysis of multiple cancer clones in the same well. Moreover,Celloswas able to identify organoid and nuclear morphology feature changes associated with treatment. Finally,Cellosenables 3D analysis of cell spatial relationships, which we used to detect ecological affinity between cancer cells beyond what arises from local cell division or organoid composition.Cellosprovides powerful tools to perform high throughput analysis for pharmacological testing and biological investigation of organoids based on 3D imaging.

Abstract: The COVID-19 infection has already had a significant impact on the global health system. Nowadays, the second wave of the disease is surging in India. One significant difference between the two waves is that they are too virulent in terms of infection rate. During the surge of the second wave, the mortality rate is highest in the age group of 50-70 years old. In three age groups, below 10, 30 to 40 years, and above 80, the mortality rate is increased. Symptoms are also different in both waves. The second wave is becoming more dangerous due to the double mutant variant of the virus.

Triple-negative breast cancer (TNBC) and ovarian carcinomas (OvCas) with

Triple-negative breast cancer (TNBC) and ovarian carcinomas (OvCas) with BRCA1 promoter methylation ( BRCA1 meth) respond more poorly to alkylating agents compared to those bearing mutations in BRCA1 and BRCA2 ( BRCA mut). This is a conundrum given the biologically equivalent homologous recombination deficiency (HRD) induced by these genetic and epigenetic BRCA perturbations. We dissected this problem through detailed genomic analyses of TNBC and OvCa cohorts and experimentation with patient-derived xenografts and genetically engineered cell lines. We found that despite identical downstream genomic mutational signatures associated with BRCA1 meth and BRCA mut states, BRCA1 meth uniformly associates with poor outcomes. Exposure of BRCA1 meth TNBCs to platinum chemotherapy, either as clinical treatment of a patient or as experimental in vivo exposure of preclinical patient derived xenografts, resulted in allelic loss of BRCA1 methylation and increased BRCA1 expression and platinum resistance. These data suggest that, unlike BRCA mut cancers, where BRCA loss is a genetically “fixed” deficiency state, BRCA1 meth cancers are highly adaptive to genotoxin exposure and, through reversal of promoter methylation, recover BRCA1 expression and become resistant to therapy. We further found a specific augmented immune transcriptional signal associated with enhanced response to platinum chemotherapy but only in patients with BRCA-proficient cancers. We showed how integrating both this cancer immune signature and the presence of BRCA mutations results in more accurate predictions of patient response when compared to either HRD status or BRCA status alone. This underscores the importance of defining BRCA heterogeneity in optimizing the predictive precision of assigning response probabilities in TNBC and OvCa.

Background. Homologous recombination deficiency (HRD), induced by germline and somatic BRCA1 or BRCA2 gene mutations (BRCAmut) and by BRCA1 promoter methylation (BRCA1meth), has been associated with better response to platinum agents in both triple negative breast cancer (TNBC) and ovarian carcinoma (OvCa). A major conundrum arising from recent studies is why patients with BRCA1meth cancers do more poorly compared to those with BRCAmut cancers given the biologically equivalent HRD in both states. Here, we address this question by performing detailed genomic analyses of primary TNBC and OvCa cohorts and through patient-derived xenografts (PDXs) and their derivative cell lines. Methods. Both new and publicly available cohorts of primary TNBC and OvCa encompassing 499 individuals treated with a combination of platinum and taxane chemotherapy were analyzed by whole genome and transcriptome sequencing as well as limited epigenetic analyses. A cohort of 43 PDX models of TNBC was genomically characterized and responses to both single agent platinum and docetaxel were evaluated in vivo. PDX longitudinal studies were performed to assess the dynamics of BRCA1 methylation following treatment. Results. Genomic analyses revealed that BRCA1mut and BRCA1meth cancers share the same pattern of BRCA1-linked genomic rearrangements. Nonetheless, in all four primary cancer cohorts examined we found that patients with BRCAmut cancers, but not those with BRCA1meth cancers, had significantly better response outcomes compared to those with BRCA proficient cancers. A separate analysis of PDX TNBCs with BRCA1 promoter methylation showed that PDXs derived from treatment naïve cancers had complete methylation of the BRCA1 promoter, whereas those derived from post-treatment cancers invariably had only partial methylation. Compared to PDXs with complete methylation, those with partial methylation had a lower response rate to in vivo platinum-based therapy, but not to docetaxel. Using single cell clonal expansions from BRCA1meth PDX models, we confirmed that partial methylation was the result of demethylation of one of the BRCA1 promoter alleles and not of the outgrowth of a non-methylated clone. Exposure of TNBC PDXs with complete methylation to a single course of platinum therapy resulted in the emergence of an unmethylated BRCA1 promoter allele, which associated with an increase in BRCA1 expression. We confirmed that platinum treatment results in progressive loss of BRCA1 methylation and restoration of BRCA1 expression in the clinical setting, by studying the BRCA1 status of a longitudinal series of four TNBC PDX models established from the same patient at different stages of her clinical history. Differential gene expression analysis revealed an increased immune transcriptional signal, especially an elevated M1 macrophage signature, associated with enhanced response to platinum therapy only in patients with BRCA proficient cancers, in both TNBC and OvCa. Integrating both the strength of this cancer immune signature and the presence of BRCA mutations resulted in more accurate predictions of response when compared to either HRD or BRCA status alone. Conclusions. These results suggest that unlike BRCAmut cancers, where BRCA deficiency is more genetically stable, BRCA1meth cancers are highly adaptive to genotoxin exposure resulting in demethylation of one allele, recovery of BRCA1 expression and acquired insensitivity to platinum. On the other end, a high immune transcriptional signature identifies patients with BRCA proficient cancers that are more likely to benefit from platinum therapy. Taken together, our study underscores the importance of characterizing molecular heterogeneity to optimize predictive precision in assigning response probabilities in TNBC and OvCa. Citation Format: Francesca Menghi, Kalyan Banda, Pooja Kumar, Robert Straub, Lacey E. Dobrolecki, Isabel Rodriguez, Susan E. Yost, Harshpreet Chandok, Marc Radke, George Somlo, Yuan Yuan, Michael T. Lewis, Elizabeth Swisher, Edison Liu. PD5-01 Genomic and epigenomic BRCA alterations predict adaptive resistance and response to platinum-based therapy in triple negative breast cancer and ovarian carcinoma [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-01.

SUMMARYLoss of homologous recombination repair (HRR) via germline and somaticBRCA1orBRCA2gene mutations and viaBRCA1promoter methylation has been associated with better response to platinum agents and PARP inhibitors, in both triple negative breast cancer (TNBC) and ovarian carcinoma (OvCa). A major conundrum arising from recent clinical studies is why cancers withBRCA1promoter methylation (BRCA1meth) respond more poorly as compared to those bearing mutations inBRCA1andBRCA2(BRCAmut), given the biologically equivalent HRR deficiency in both states. We dissected this problem through detailed genomic analyses of primary TNBC and OvCa cohorts, as well as experimentation with patient-derived xenograft (PDX) models and genetically engineered cell lines. Using the precise genomic scar of the tandem duplicator phenotype as a precise genomic indicator of BRCA1 deficiency, we found that, in all cohorts,BRCA1mut andBRCA1meth cancers share an equivalent degree of BRCA1-linked genomic rearrangements. Nonetheless, we consistently found that patients withBRCAmut cancers, but not those withBRCA1meth cancers, had significantly better response outcomes when compared to those withBRCAproficient cancers. When fully promoter methylatedBRCA1PDX TNBCs were exposed to a single short course of platinum chemotherapy an unmethylatedBRCA1promoter allele emerged in resultant tumors associated with an increase inBRCA1expression. A separate analysis of PDXs derived from treatment naïve TNBCs featured complete methylation of theBRCA1promoter, whereas those derived from post-chemotherapy TNBCs invariably had only partial methylation. PDXs with partial methylation were significantly associated with lower response rates toin vivoplatinum-based therapy compared to those with complete promoter methylation. Using single cell clonal expansions from a partiallyBRCA1meth PDX, we confirmed that the reduced level of methylation was due to the demethylation of one of theBRCA1promoter alleles and not to the outgrowth of a non-methylated clone. Clinically, analysis of primary OvCas confirmed that high levels ofBRCA1methylation were significantly associated with reducedBRCA1gene expression whereas cancers with lower levels ofBRCA1methylation had expression levels approaching those found inBRCA1proficient cancers. These data suggest that unlikeBRCAmut cancers, where HRR deficiency is achieved via mutations that are genetically ‘fixed’,BRCA1meth cancers are highly adaptive to genotoxin exposure and more likely to recoverBRCA1expression, which may explain their poorer therapeutic response. We further found that an increased immune transcriptional signal, especially an elevated M1 macrophage signature, is associated with enhanced response to platinum-based chemotherapy only in patients withBRCAproficient cancers, in both TNBC and OvCa cohorts underscoring the importance of characterizing molecular heterogeneity to enhance predictive precision in assigning response probabilities in TNBC and OvCa.

The use of big data analytics tools and Machine Learning techniques in identifying and predicting opioid use disorder is a relatively new and emerging area. Previous studies analyzing trends in the opioid crisis have identified an increasingly expensive and worsening epidemic. Many factors contribute to opioid use, abuse, and addiction. Opioid addiction is a complex disease involving various physiological pathways as well as environmental factors. There is some evidence to suggest that people with low education levels and high unemployment and poverty levels are at higher risk of opioid abuse. In this paper, we evaluated different conventional Machine Learning models including Support Vector Machines (SVM), Decision Tree, and Logistic Regression and advanced algorithms like Gradient Boosting. The models were developed to predict opioid abuse disorder using county-level education, poverty, and unemployment data. In contrast, the results suggest that populations with higher socioeconomic status are at greater risk of opioid abuse disorder compared to individuals with lowers. This can be attributed to underlying factors not previously captured increased availability of opioids and resources to acquire them. Identifying which areas and populations are at higher risk of opioid abuse disorder and underlying contributing factors will help inform judicious effective policies, programs, and solutions to tackle the worsening health crisis.

Context: Postoperative Nausea and Vomiting (PONV) is associated with many adverse events, which prolong and increase the healthcare cost. Aims: To compare the efficacy of Palanosetron with respect to Granisetron and Ondansetron in prevention of PONV. To study the need of rescue antiemetic and incidence of side effects. Settings and Design: This Prospective, Randomized study was carried out with 150 adult female patients of ASA Grade I & II posted for elective gynecological surgery under GA. Materials and Methods: Patients were divided into three groups of 50 patients each. Group O received injection ondansetron 8 mg IV, Group G received Granisetron 2.5 mg IV & Group P received Palanosetron 0.075 mg iv before GA induction. Episodes of nausea, vomiting, need for rescue antiemetic and side effects were observed for 72 hours in postoperative period. Statistical Analysis Used: Graphpad Software with consideration of significant P value Results: Incidence of nausea and vomiting was more with Ondansetron and minimal with Palanosetron with statistical significance of P Lowest incidence of nausea was found with Palanosetron during 3-24 hours postoperatively (P Complete Control of PONV (No PONV, No rescue drug required) was maximum in P group in all hours of study [P Conclusion: Palanosetron is safe, well tolerated and effective than Granisetron and Ondansetron in reducing the incidence of PONV with less need of rescue antiemetics. Key Messages: Palanosetron is proved better antiemetic than granisetron and ondansetron in prevention of PONV. Keywords: General Anaesthesia, Granisetron, Gynaecological surgery, Ondansetron, Palanosetron, Postoperative nausea and vomiting.

Triple negative breast cancer (TNBC) has long been characterized by having a high degree of intratumoral heterogeneity. While significant contributions have been made in understanding the components of TNBC tumors, examining the dynamics of interaction between subclones in TNBC is relatively unexplored. By using single-cell RNA sequencing (scRNA-seq) technology, this study aims to focus on understanding changes in gene expression of two TNBC subclones: the cisplatin-resistant subclone (A50) and cisplatin-sensitive subclone (B), when cocultured in 3D organoids in the presence and absence of cisplatin. We hypothesized that there will be significant differentially expressed genes (DEGs) due to various concentrations of cisplatin and mixing effect. We found DEGs (ACTB, FOS, EIF5, BRD2, MAFG, SF3B4, ID1, MGMT, DDIT4 and LPP) due to treatment with cisplatin in both subclones, with the largest clear effect present at a high concentration of cisplatin. Additionally, the network analysis showed that cisplatin was an upstream regulator for 11 DEGs. Even though, gene expression changes due to mixing are much more subtle than effects due to treatment, we saw some DEGs caused by the mixing effect like MT1X which is related to cisplatin resistance. Taken together, the findings of this project will help in future studies when deducing which genes to target in TNBC chemotherapy treatments based on predictive molecular biomarkers. Citation Format: Shadae Nicholas, Patience Mukashyaka, Pooja Kumar, Jeffrey Chuang, Edison Liu. Uncovering transcriptional changes related to cisplatin treatment and mixing of TNBC subclones using single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1157.

The rapid industrialisation and urbanisation in India over the past few decades has resulted in severe environmental destruction and loss of traditional livelihoods, as well as worsened economic and social inequalities. Ennore, a northern suburb of Chennai is a classic example of this phenomenon. This photo essay presents the deleterious impacts of urbanisation on the artisanal fisherfolk of Ennore. The inherently unsustainable nature of water extraction for industries in this region coupled with lax regulations first destroyed the Ennore creek and consequently fisherfolk livelihoods. Dwindling fish catches have created an artificial demand for jobs, thereby resulting in formerly independent communities being completely enslaved to the development paradigm. The state’s response has not been to fix environmental and community health problems in Ennore, but only to push for more growth and development. The essay argues that this toxic idea of growth needs to be dismantled first, if the degradation of livelihood commons is to be stopped.

Intratumoral heterogeneity is an essential aspect of cancer biology, as the diversity of cancer cells can increase challenges of designing effective treatment. Cancer cells within the same tumor evolve and compete for shared resources and may also respond differently to drug treatments. In previous work, by closely analyzing triple negative breast cancer xenografts, we have identified a system of closely-related subclonal populations within a tumor that respond differentially to chemotherapy. To explore the dynamics of competition and cooperation between these subclones, we derived cell lines from each and tagged them with fluorescent markers. We then co-cultured these cell lines in 3D organoids and tracked them using Opera Phenix high content screening system (3D confocal fluorescence microscopy). To determine the number of cells and the location of each subclones within organoids, we developed a 3D cell segmentation pipeline. Our pipeline relies on a two-step process where organoids are segmented first and then individual cells are detected within each organoid. Preliminary analysis of hundreds of organoids suggests that the two cell types preserve their relative ratio and sensitivity to treatment regardless of organoid size, suggesting that the system is in a state of ecological coexistence. We will present results on cellular mixing during growth and treatment and the implications for adaptive therapy ecologies in tumors. Citation Format: Patience Mukashyaka, Javad Noorbakhsh, Pooja Kumar, Elise Courtois, Olga Anczukow, Paul Robson, Edson Liu, Jeffrey Chuang. Intratumor heterogeneity quantification using 3D segmentation of organoids co-culture systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3144.

Kaufman oculocerebrofacial syndrome (KOS) is a recessive neurodevelopmental disorder characterized by intellectual disability and lack of speech. KOS is caused by inactivating mutations in UBE3B, but the underlying biological mechanisms are completely unknown. We found that loss of Ube3b in mice resulted in growth retardation, decreased grip strength, and loss of vocalization. The brains of Ube3b(−/−) mice had hypoplasia of the corpus callosum, enlarged ventricles, and decreased thickness of the somatosensory cortex. Ube3b(−/−) cortical neurons had abnormal dendritic morphology and synapses. We identified 22 UBE3B interactors and found that branched-chain α-ketoacid dehydrogenase kinase (BCKDK) is an in vivo UBE3B substrate. Since BCKDK targets several metabolic pathways, we profiled plasma and cortical metabolomes from Ube3b(−/−) mice. Nucleotide metabolism and the tricarboxylic acid cycle were among the pathways perturbed. Substrate-induced mitochondrial respiration was reduced in skeletal muscle but not in liver of Ube3b(−/−) mice. To assess the relevance of these findings to humans, we identified three KOS patients who had compound heterozygous UBE3B mutations. We discovered changes in metabolites from similar pathways in plasma from these patients. Collectively, our results implicate a disease mechanism in KOS, suggest that it is a metabolic encephalomyopathy, and provide an entry to targeted therapies.

The processes by which tumors evolve are essential to the efficacy of treatment, but quantitative understanding of intratumoral dynamics has been limited. Although intratumoral heterogeneity is common, quantification of evolution is difficult from clinical samples because treatment replicates cannot be performed and because matched serial samples are infrequently available. To circumvent these problems we derived and assayed large sets of human triple-negative breast cancer xenografts and cell cultures from two patients, including 86 xenografts from cyclophosphamide, doxorubicin, cisplatin, docetaxel, or vehicle treatment cohorts as well as 45 related cell cultures. We assayed these samples via exome-seq and/or high-resolution droplet digital PCR, allowing us to distinguish complex therapy-induced selection and drift processes among endogenous cancer subclones with cellularity uncertainty

The service line (SL) model has been proven to help shift health care toward value-based services, which is characterized by coordinated, multidisciplinary, high-quality, and cost-effective care. However, academic medical centers struggle with how to effectively set up SL structures that overcome the organizational and cultural challenges associated with simultaneously delivering the highest-value care for the patient and advancing the academic mission. In this article, the authors examine the evolution of UMass Memorial Health Care's heart and vascular service line (HVSL) from 2006 to 2011 and describe the impact on its success of multiple strategic decisions. These include key academic physician leadership recruitments and engagement via a matrixed governance and management model; development of multidisciplinary teams; empowerment of SL leadership through direct accountability and authority over programs and budgets; joint educational and training programs; incentives for academic achievement; and co-localization of faculty, personnel, and facilities. The authors also explore the barriers to success, including the need to overcome historical departmental-based silos, cultural and training differences among disciplines, confusion engendered by a matrixed reporting structure, and faculty's unfamiliarity with the financial and organizational skills required to operate a successful SL. Also described here is the impact that successful implementation of the SL has on creating high-quality services, increased profitability, and contribution to the financial stability and academic achievement of the academic medical center.

The processes by which tumors evolve are essential to the efficacy of treatment, but quantitative understanding of intratumoral dynamics has been limited. Although intratumoral heterogeneity is common, quantification of evolution is difficult from clinical samples because treatment replicates cannot be performed and because matched serial samples are infrequently available. To circumvent these problems we derived and assayed large sets of human triple-negative breast cancer xenografts and cell cultures from two patients, including 86 xenografts from cyclophosphamide, doxorubicin, cisplatin, docetaxel, or vehicle treatment cohorts as well as 45 related cell cultures. We assayed these samples via exome-seq and/or high-resolution droplet digital PCR, allowing us to distinguish complex therapy-induced selection and drift processes among endogenous cancer subclones with cellularity uncertainty AUTHOR SUMMARYAn overarching challenge of cancer is that patients develop resistance to treatment -- an essentially evolutionary process. However, there is currently very little understanding of how tumor evolution can be exploited to improve treatment. One reason for this is that usually only 1-2 samples can be obtained per patient, so cancer evolutionary processes are still poorly understood. To solve this problem, we created many dozens of copies of the tumors from two breast cancer patients using xenografting and cell culture methods. We then compared the evolution in these tumor copies in response to different treatments, including four of the most common breast cancer chemotherapies. These studies present the most exhaustive comparisons of treatment-induced evolution that have yet been performed for individual cancer patients. Unexpectedly, high-resolution sequencing of these samples revealed a special dynamically treatable ecology in one tumor, in which tumor growth during platinum therapy was determined by the ecological balance of two tumor cell populations. Our work shows that ecologies that can be targeted by dynamic treatment strategies arise spontaneously in breast cancers. Population heterogeneity is common within cancers, and our work suggests how tracking of intratumoral evolution can be used to optimize treatment.

Background and objectives To prevent the spread of infections in all healthcare settings, hand hygiene must be routinely practiced. Appropriate hand hygiene techniques can go a long way in reducing nosocomial infections, cross-transmission of microorganisms and the risk of occupational exposure to infectious diseases. World Health Organisation (WHO) has taken an incredible approach called “My Five Moments for Hand Hygiene" which defines the key moments when health-care workers should perform hand hygiene. We thus carried out a survey to assess knowledge of hand hygiene practices among undergraduate medical students. Materials and methods A cross-sectional survey was conducted among 523 Indian medical undergraduates. The questionnaire used was adapted from the WHO hand hygiene knowledge questionnaire for health-care workers and was distributed both, in print and online formats. The response to each question was examined using percentages. Results Nearly 57% (n=298) of medical students who participated in this study did not receive any formal training in hand hygiene. Only 27% (n=141) students knew that the most frequent source of germs responsible for health-care associated infections were the germs already present on or within the patient. Nearly 68.6% (n= 359) students were unaware of the sequence of hand washing and hand rubbing. Although 71.9% (n=376 ) students claimed that they use an alcohol-based hand rub routinely, only 36.1% (n=189 ) students knew the time required for a hand rub to kill the germs on the hands. Overall hand hygiene knowledge was low in 6.9% (n=36), moderate in 80.9% (n=423) and good in 12.2% (n=23) of respondents. Conclusions The awareness about hand hygiene practices among medical students is low. Nearly 57% (n=298) of the respondents never received any formal training in hand hygiene throughout their course of medical undergraduate study. To prevent the spread of infections in healthcare settings, medical students should be given proper training in hand hygiene practices right from the first year of the medical curriculum. This should be done by running workshops and annual seminars on hand hygiene practices and making it a requisite for clinical skills assessment.

Objective: Utensils and tableware are food contact surfaces that have the potential to transmit disease-causing microorganisms if not washed, cleaned and sanitized correctly in a food service establishment. To prevent utensils and tableware from becoming vectors of disease, it is essential that operators and staff are able to adequately sanitize them and accurately test for it in a quick and convenient manner. It is also essential that Environmental Health officers are able to test whether adequate sanitization is occurring during their routine inspections. Currently there are no guidelines indicating the correct method of testing. Therefore, this study investigated two methods used to test residual sanitation concentration in a dishwasher. The purpose of this research was to determine if there is any difference in the two methods currently being used, and if so, which method is the more reliable one. Methods: LaMotte Chlorine Test Strips were used to detect the levels of chlorine in a commercial dishwasher. The chemically treated strips were dipped onto a freshly wet and washed utensil and directly in the rinse water of a dishwasher. Results: The difference in the mean of 60 samples from two independent groups was analyzed. Thirty samples were obtained from location one, the dishware l, and thirty samples were obtained from location two, the rinse water of the commercial dishwasher. The mean residual concentration was calculated and compared. The means demonstrated there is a significant difference (p = 0.035)between the two groups; the average residual concentration was lower for the dishware compared to the rinse water. Conclusion: Dishware is a vector capable of transmitting disease causing microorganisms if not sanitized adequately. Thus, it is important to ensure that dishes in a food service establishment have been thoroughly sanitized. The testing of that requires a consistent and reliable method. It is safe to assume that testing on the dishware is the best course of action to err on the side of caution.

Next-generation sequencing studies have revealed genome-wide structural variation patterns in cancer, such as chromothripsis and chromoplexy, that do not engage a single discernable driver mutation, and whose clinical relevance is unclear. We devised a robust genomic metric able to identify cancers with a chromotype called tandem duplicator phenotype (TDP) characterized by frequent and distributed tandem duplications (TDs). Enriched only in triple-negative breast cancer (TNBC) and in ovarian, endometrial, and liver cancers, TDP tumors conjointly exhibit tumor protein p53 (TP53) mutations, disruption of breast cancer 1 (BRCA1), and increased expression of DNA replication genes pointing at rereplication in a defective checkpoint environment as a plausible causal mechanism. The resultant TDs in TDP augment global oncogene expression and disrupt tumor suppressor genes. Importantly, the TDP strongly correlates with cisplatin sensitivity in both TNBC cell lines and primary patient-derived xenografts. We conclude that the TDP is a common cancer chromotype that coordinately alters oncogene/tumor suppressor expression with potential as a marker for chemotherapeutic response.

The JAX® Onco-Hu® platform utilizes humanized mice engrafted with tumors to enable in vivo investigation of the interactions between the human immune system and human cancer. We have recently shown that humanized NOD-scid IL2Rγnull (NSG™) mice bearing patient-derived xenografts (PDX) allow efficacy studies of checkpoint inhibitors. A major avenue of our investigation is to generate murine humanized models containing a more complete human hematopoietic system and robust innate immune cell population. Next-generation NSG strains include triple transgenic NSG mice (NSG-SGM3) expressing myelosupportive human cytokines KITLG, CSF2, and IL-3. When engrafted with CD34+ human hematopoietic progenitor cells (HPCs) from CD3-depleted umbilical cord blood, NSG-SGM3 mice produce higher myeloid and Treg populations in the circulation as compared to NSG mice over 18 weeks post engraftment. We implanted an array of PDX tumors into humanized NSG-SGM3 mice at 2-3 months post engraftment. Tumors were dissociated and single-cell infiltrates were analyzed by multicolor flow cytometry with a focus on examining overall immune cell infiltration and the levels of hCD33+ myeloid cells. In the PS4050 melanoma PDX model, we found that hCD45+ cell infiltration was significantly increased in hu-NSG-SGM3 mice as compared to hu-NSG mice engrafted with the same HPC donor (3.7% vs. 1% of viable cells). The majority of tumor-infiltrating cells in hu-NSG-SGM3 mice expressed hCD33 (55% of hCD45+) and the percentage was significantly higher than that in hu-NSG mice (13%). hCD3+T cell infiltration level was similar between these two strains (~20% of hCD45+). PS4050-bearing hu-NSG-SGM3 mice treated with the anti-PD-1 antibody pembrolizumab (Keytruda) showed a significant reduction in tumor growth and the PD-1 levels in tumor-infiltrating T cells were greatly reduced by flow cytometry analysis. The overall hCD45+ cell infiltration and the frequencies of hCD4+T, hCD8+T, and hCD33+myeloid cells in tumors remained similar after treatment. Lastly, we observed that the effect of Keytruda on tumor growth reduction in hu-NSG-SGM3 mice is PD-L1-dependent using the human lung carcinoma cell line NCI-H460 depleted of PD-L1 expression by CRISPR. Keytruda treatment significantly reduced mock-transfected NCI-H460 cell growth. By comparison, PD-L1 KO NCI-H460 cells grew more slowly than the mock cells and lost the response to Keytruda. Together, these results indicate that PDX tumor-implanted hu-NSG-SGM3 mice serve as an important platform for understanding human immune system and tumor microenvironment interactions and for preclinical immuno-oncology efficacy studies. Citation Format: Li-Chin Yao, Mingshan Cheng, Ken-Edwin Aryee, Pooja Kumar, Nicole Walsh, Dale Greiner, Leonard Shultz, Edison T. Liu, Michael Brehm, James G. Keck. Patient-derived tumor xenografts in humanized NSG-SGM3 mice: An improved immuno-oncology platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5676.

Population heterogeneity within tumors is essential to the development of drug resistance. However, precise quantification of cellularity levels of subpopulations, and in particular how they evolve in response to treatment, has been challenging. Here we describe the high precision characterization of subclonal evolution within triple-negative breast cancer patient-derived xenografts (PDXs) generated from three patients. For each patient model, we established multiple PDXs and treated them in cohorts of 5-10 mice each for the therapies cyclophosphamide, doxorubicin, cisplatin, or docetaxel, with treatments lasting one month. In all three patient models, the average behavior across mice was a reduction in size in response to docetaxel, but growth under doxorubicin and cyclophosphamide. For cisplatin treatment, one of the three models showed tumor shrinkage while the other two models showed continued growth but at a rate lower than the doxorubicin or cyclophosphamide cohorts. To determine the evolutionary behaviors underlying these observations, we initially performed exome panel sequencing of 34 residual tumor samples from these and untreated xenografts. Computational mutation and copy number analysis indicated sample-specific differences in tumor populations both in response to treatment and due to genetic drift. However, they also revealed measurement uncertainties related to exome capture efficiency, locus-specific read counts, and computational copy number estimation that limited quantitative inference of evolutionary behaviors. To solve this problem we used droplet digital PCR (ddPCR) to measure variant allele frequencies and local copy number at selected loci from the prior residual tumors as well as additional samples from replicate cohort treatments and cultured conditionally reprogrammed progenitor cells. In total we performed 1665 ddPCR measurements across 150 cancer samples. These ddPCR measurements reduced sample-specific uncertainty in variant allele frequency to ~2% and copy number to ~0.2, allowing for precise identification of subclones and their cellularities in each sample. We observed several common modes of evolution within these tumors including selective sweeps, spatial diffusion, and stable coexistence between distinct subpopulations. In the samples from one patient model, we observed frequent symbiotic growth of two distinct subpopulations having differential cisplatin sensitivity, such that the degree of clonal selection during cisplatin treatment was proportional to the tumor volume change on a mouse-to-mouse basis. This study demonstrates how high precision genomic characterization across comparatively treated samples can reveal treatment-relevant subclonal ecology, as well as the mutations that distinguish populations with different behaviors. Citation Format: Hyunsoo Kim, Pooja Kumar, Francesca Menghi, Eliza Cerveira, Mallory Romanovitch, Guru Ananda, Joshy George, Henry Chen, Susan Mockus, Chengsheng Zhang, Yan Yang, James Keck, R. Krishna Murthy Karuturi, Carol Bult, Charles Lee, Edison Liu, Jeffrey H. Chuang. High-precision quantification of clonal evolution during comparative treatments of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2909. doi:10.1158/1538-7445.AM2017-2909

The Jackson Laboratory PDX program engages 26 staff members, 5 PIs, and runs thousands of PDX models per year in pre-clinical experiments both for internal scientific experiments and external projects. Over the last 6 years of operation of the PDX program, we have gained a significant amount of experience in all aspects of the process. This experience includes the genomic analysis of the tumors and the establishment of an extensive database annotating many of the 446 PDX models in our US inventory. Herein, we describe some of the characteristics of the system that enhances successful experimentation in this platform. Several factors significantly improve the engraftment rate of tumors1: 1) the degree of immunodeficiency the host mouse - the NOD.Cg-Prkdcscid IL2rgtm1wjl/SzJ (aka, NSG), the NOD.CB17-Prkdcscid/J (aka,NOD-scid), or the NOD.Cg-Rag1tm1momIL2rgtm1wjl/SzJ (aka, NRG) engrafting better than the beige-scid and athymic nude mice; 2) the greater the amount of tissue engrafted, 3) the late or metastatic nature of the tumor, 4) the shorter time from surgery to implantation, 5) the absence of enzymatic dissociation, and 6) orthotopic implantation. Sequencing and expression analysis show the maintenance of the core genomic configuration between PDX and the primary tumors though some genetic differences are noted of indeterminate significance. Human stromal cells, however, tend to be replaced by murine stroma after the first passage. Tumor genetic heterogeneity is maintained through the fourth passage in NSG PDX models as confirmed by deep sequencing of bulk tumor and of individual progenitor clones. Most importantly, however, the tumor responses to systemic therapies appear to reflect the patient response2. Therefore, the use of PDX models to test novel agents may speed pre-clinical drug development. The most important use of PDX systems may be in immuno-oncology given that “humanized” PDX models where implantation of a primary human tumor is implanted in a mouse with a reconstituted human cellular immune system provides a powerful preclinical experimental platform to test new immune modulators in human cancers. We, and others, have shown that humanized NSG mice bearing human tumor xenografts exhibit dramatic responses to immune checkpoint inhibitors that are immune cell and drug dependent3. Immune reconstitution is more complex because of the requirement of human cytokines that are not substituted by their murine counterparts. Engineered mice expressing human cytokines, e.g., IL3, CSF2 (GM-CSF), and KITLG (stem cell factor) in the NSG background (aka, NSG-SGM3 mice), and those expressing CSF1 (M-CSF), IL3, CSF2, and THPO in the C;129S4-Rag2tm1.1Flv IL2rgtm1.1Flv/J background (aka, MITRG mice) after engraftment with human hematopoietic stem cells have been shown to support myeloid cells4 including macrophages absent in standard NSG5. It is anticipated that these “next generation” humanized mice will provide a more nuanced picture of the tumor-immune system interaction6. It is important to understand the challenges and limitations of the PDX platform that can be mitigated to a degree by quality control and study design. There are simple caveats. ~5 % of engrafted solid tumors give rise to EBV positive lymphomas and not the primary tumor. Moreover, ~5-10% of PDX tumors are overgrown by a transformed murine cell especially in late passaged. Thus, stringent histological quality control is necessary which includes the assessment of human cytokeratin, which provides an assessment of murine cancer incursion of solid human cancers. Another concern is that drug dosing for PDX experiments is often very different from that used in human studies. The NSG mice are more sensitive to certain DNA damaging agents and to radiation than NRG or Rag1null mice. Therefore, the structuring of combination studies using genotoxic agents is complicated and should be interpreted with appropriate care. In terms of study design, we have found that each PDX model from an individual patient will give rise to individual tumors in an NSG cohort with significant growth and response variations. Thus, for each treatment arm we have calculated that between 6-8 animals is the minimal number required to attain statistical power of 95-99% to identify efficacy between arms. Moreover, in any treatment arm, it is necessary to assess the response of each individual PDX bearing mouse since a few individual tumors in a cohort may be resistant to a drug whereas the average of the arm shows an overall response. Recently, Gao, et al7 presented an alternative way to conduct PDX studies for drug development where only one mouse per PDX model was used per drug. The overall data (not whether a drug specifically was efficacious in a specific disease type) provided important strategic information in development. They raised an important point and that is that PDX preclinical studies should be structured differently from classical clinical trials to make best use of the platform. Not only the trial design, but even how to call a response should be reexamined. The partial responses seen in PDX experiments that can be precisely quantified but that do not qualify using RECIST criteria provide potentially important information about drug efficacy. Taken together, the PDX platform using severely immunodeficient mice is a powerful tool that can significantly accelerate the development of new therapeutics by dramatically facilitating the advancement of innovative therapies with a high likelihood for success8. References: 1Shultz LD, Goodwin N, Ishikawa F, Hosur V, Lyons BL, Greiner DL. Human cancer growth and therapy in immunodeficient mouse models. Cold Spring Harbor Protoc. 2014 Jul 1;2014(7):694-708. 2Garralda E, Paz K, López-Casas PP, Jones S, Katz A, Kann LM, López- Rios F, Sarno F, Al-Shahrour F, Vasquez D, Bruckheimer E, Angiuoli SV, Calles A, Diaz LA, Velculescu VE, Valencia A, Sidransky D, Hidalgo M. Integrated next-generation sequencing and avatar mouse models for personalized cancer treatment. Clin Cancer Res. 2014 May 1;20(9):2476-84 Epub 2014 Mar 14. 3Wang M, Keck JG, Cheng M, Cai D, Shultz L, Palucka K, Banchereau J, Bult C, Huntress R. Patient-derived tumor xenografts in humanized NSG mice: a model to study immune responses in cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-050. 4Billerbeck E, Barry WT, Mu K, Dorner M, Rice CM, Ploss A. Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3- expressing NOD-SCID IL2Rγ(null) humanized mice. Blood. 2011 Mar 17;117(11):3076-86. 5Rongvaux A, Willinger T, Martinek J, Strowig T, Gearty SV, Teichmann LL, Saito Y, Marches F, Halene S, Palucka AK, Manz MG, Flavell RA. Development and function of human innate immune cells in a humanized mouse model. Nat Biotechnol. 2014 Apr;32(4):364-72. 6Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol. 2012 Nov;12(11):786-98. 7Gao H, Korn JM, Ferretti S, Monahan JE, Wang Y, Singh M, Zhang C, Schnell C, Yang G, Zhang Y, Balbin OA, Barbe S, Cai H, Casey F, Chatterjee S, Chiang DY, Chuai S, Cogan SM, Collins SD, Dammassa E, Ebel N, Embry M, Green J, Kauffmann A, Kowal C, Leary RJ, Lehar J, Liang Y, Loo A, Lorenzana E, Robert McDonald E 3rd, McLaughlin ME, Merkin J, Meyer R, Naylor TL, Patawaran M, Reddy A, Röelli C, Ruddy DA, Salangsang F, Santacroce F, Singh AP, Tang Y, Tinetto W, Tobler S, Velazquez R, Venkatesan K, Von Arx F, Wang HQ, Wang Z, Wiesmann M, Wyss D, Xu F, Bitter H, Atadja P, Lees E, Hofmann F, Li E, Keen N, Cozens R, Jensen MR, Pryer NK, Williams JA, Sellers WR. High- throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nat Med. 2015 Nov;21(11):1318-25. 8Gandara DR, Mack PC, Bult C, Li T, Lara PN Jr, Riess JW, Astrow SH, Gandour-Edwards R, Cooke DT, Yoneda KY, Moore EH, Pan CX, Burich RA, David EA, Keck JG, Airhart S, Goodwin N, de Vere White RW, Liu ET. Bridging tumor genomics to patient outcomes through an integrated patient-derived xenograft platform. Clin Lung Cancer. 2015 May;16(3):165- 72. Citation Format: Edison T. Liu, Carol Bult, Jeff Chuang, Pooja Kumar, Mingshan Cheng, R. Krishna Murthy Karuturi, Vivek Philip, James Keck, Karolina Palucka, Larry Shultz. Mice host selection for patient-derived xenograft (PDX) model development and other critical factors for success. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA29.

Individual tumors, including the aggressive and difficult to treat triple-negative (ER-/PR-/HER2-) breast cancers (TNBCs) are heterogeneous collections of cells with multiple subclonal populations each contributing to the tumor. While subclonal heterogeneity is likely responsible for the development of drug resistance, identification of how tumor cell populations change over time has been difficult, largely because of the challenges in resampling tumor tissue at close time points. Here we quantify tumor evolution in human patient-derived xenografts implanted into NSG mice, which we use to test subclonal heterogeneity as a function of location within a tumor, propagation time, and drug treatment. We used high-depth (∼400x) sequencing of a targeted panel of 358 genes to quantify somatic mutation allele frequencies from 6 spatially-separated and 8 temporally-propagated xenograft samples derived from the same TNBC patient tumors. Samples ranged in age from 2-4 months post-engraftment. Although we observed a few low frequency mutations distinguishing samples, overall we found that allele frequencies of somatic mutations were well-preserved on this time scale. We then generated replicate xenografts from the same patient tumor and treated them respectively with cisplatin, doxorubicin, cyclophosphamide, docetaxel, or vehicle control for 25 days. Although again somatic mutations showed few differences in allele frequency across samples, substantial variations were seen when data were analyzed for copy number alterations. To confirm these effects we repeated the treatments for xenografts derived from two additional TNBC patients. Again we observed strong changes in tumor heterogeneity at the copy number level. This effect was particularly, but not exclusively, apparent in tumors with the greatest response to therapy. We further verified these measurements through Sanger and digital PCR sequencing on the treated mice and other mice in the same cohorts. Using a multi-sample xenograft propagation, dissection, sequencing, and computational analysis protocol, we have shown that tumor subpopulation changes in response to treatment can be quantified and distinguished from spatial or temporal effects, even for treatment time courses as short as 1 month. In triple negative breast cancer these variations are most apparent at the level of copy number variation. Our study demonstrates how patient-derived xenografts can provide detailed resolution of tumor population evolution during the manifestation of resistance. Citation Format: Hyunsoo Kim, Pooja Kumar, Francesca Menghi, Joshy George, Guru Ananda, Susan Mockus, Chengsheng Zhang, Nicholas Larson, Henry C. Chen, Yan Yang, James Keck, R. Krishnamurthy Karuturi, Charles Lee, Carol Bult, Edison Liu, Jeffrey H. Chuang. Evolution during propagation and treatment of patient-derived triple negative breast cancer xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2411.