Precision Medicine of Cancer

癌症的精准医学是多方面的，我们的研究，从实验室到床边的方法，已经导致了重大的发现，将推进生物标志物的诊断和预后利用，并阐明微生物组在肺癌发生中的作用和机制。此外，对健康差异的研究仍然是一个焦点(Graff， M. et al., Am J Hum Genet, 2021；肺癌152:58-65,2021)，建立在实验室的遗产上，该实验室是第一个在美国非洲裔人口中进行GWAS的实验室(Zanetti KA。等人，肺癌杂志98:33-42,2016)。癌症微生物组：最近对微生物群组成的研究表明，在几种曾经被认为是无菌的癌症类型中存在丰富的细菌（Greathouse KL. et al., Cancer Cell 38: 158- 160,2020）。我们已经在腺癌和鳞状细胞癌中发现了与TP53突变和吸烟相关的Acidovorax spp。通过小鼠肺癌模型，我们发现Acidovorax spp是肺癌发生的贡献者，而不是乘客，通过激活促炎肺中性粒细胞。我们的数据显示，与pbs灌注（假）对照小鼠相比，暴露于A. temperans （Acidovorax temperans）的小鼠具有更大的肿瘤负荷和更丰富的肺部病变。a . temperans对荷瘤小鼠的生存有害，反复暴露会加速肺癌的发展，因此，a . temperans在肺癌的发展中具有功能性作用。全组织RNA测序（RNAseq）比较了a . temperans灌注小鼠和假对照组的差异基因表达，结果显示，通过myd88依赖的信号通路，a . temperans产生了强烈的炎症反应，myd88依赖信号通路是启动先天和适应性免疫的必要途径，并有助于癌症进展。与Romina Goldszmid合作，我们研究了在滴注A. temperans后肿瘤免疫微环境的变化。我们的荧光活化细胞分选（FACS）分析结果显示，在嗜中性粒细胞、CD4+ve、CD8+ve、双阴性（DN） T细胞和γ - δ T细胞的驱动下，灌胃A. temperans小鼠肺部CD45+ve免疫群体增加。总的来说，我们的研究结果表明，A. temperan增加了促炎细胞的浸润，这种浸润持续一段时间，促进了肺腺癌的发展。嗜中性粒细胞的募集在KPC小鼠肺部受到嗜中性粒细胞的刺激而加剧。在输注A. temperans的小鼠中，浸润的中性粒细胞表型增加了CD44lowICAM1highSiglecF+PDL1+中性粒细胞的频率，这些中性粒细胞先前被描述为寿命长、成熟的促肿瘤性肿瘤相关中性粒细胞，对KPC小鼠的肺肿瘤进展很重要。在A. temperans挑战小鼠中，RNAseq对活化的和成熟的中性粒细胞表现出更高的特征得分。细菌暴露可能是中性粒细胞成熟的原因，因此我们比较了先前报道的致病性大肠杆菌（E. coli）感染的中性粒细胞标记分数，发现a . temperans灌注小鼠由于细菌感染而具有更高的中性粒细胞成熟标记分数。中性粒细胞是肺癌患者肿瘤微环境中的主要免疫细胞群，有证据表明，循环中性粒细胞与白细胞比例的增加与预后不良以及对包括免疫治疗在内的治疗反应较差有关。下一步是建立治疗性小鼠模型，并验证中性粒细胞在介导黄芪诱导的肺肿瘤发生中的作用。癌症代谢组：已鉴定的代谢物与特定癌症的相关性创建了生物标志物谱，可用于许多类型的人类癌症的非侵入性诊断和预后评估。尿液、血清和血浆液体活检用于质谱测定肺癌的四种生物标志物（肌酸核糖苷（CR）、n -乙酰氨基酸（NANA）、硫酸皮质醇（CS）和27 α -5 - β -胆甾醇-3 α、7 α、12 α、24 α、25 α戊糖醛酸（NCPG）） （Haznadar， M. et al., cancer epidemiology）。生物标志物，2016,25:978- 86,2016)。CR与其他已确定的尿液代谢物生物标志物（如NANA）配对可提高诊断能力和可靠性。这些基础研究验证了尿液代谢物筛选的使用，从而进一步研究生物标志物与人类癌症的关联。(帕特尔,DP。et al。尿代谢物生物标志物分析可用于肝内胆管癌（ICC）的诊断和预后评估。采用UPLC-MS/MS，用于定量代谢物CR、n -乙酰神经氨酸（NANA）、硫酸皮质醇和葡萄糖醛酸碎片离子561+的四种代谢物在HCC和ICC中显著增加，并且与临床使用的标记物CA19-9联合用于ICC分类。对NCI-MD队列进行研究，观察结果由TIGER-LC队列验证。我们确定了生物标志物在液体活检中基于CLIA的生物标志物测定中具有重要意义的特性。

Precision medicine in cancer is multifaceted and our research, following the bench to bedside approach, has led to significant discoveries that will advance the diagnostic and prognostic utilization of biomarkers and elucidate the role and mechanism the microbiome plays in lung carcinogenesis. In addition, researching health disparity remains a focus (Graff, M. et al., Am J Hum Genet. 108: 564-582, 2021; Jiang M. et al. Lung Cancer 152: 58-65, 2021) that build on the laboratory's legacy of having been the first to conduct GWAS in the US population of African heritage (Zanetti KA. et al., Lung Cancer 98: 33-42, 2016). Cancer Microbiome: A recent study of microbiota composition revealed an abundance of bacteria in several cancer types once thought to be sterile (Greathouse KL. et al., Cancer Cell 38: 158-160, 2020). We have discovered Acidovorax spp associated with TP53 mutations and smoking in both adenocarcinoma and squamous cell carcinoma. Using a mouse model of lung cancer, we show Acidovorax spp is a contributor, not a passenger, of lung carcinogenesis, via activation of proinflammatory lung neutrophils. Our data showed that mice exposed to A. temperans (Acidovorax temperans) had significantly larger tumor burden and more abundant lung lesions compared to PBS-instilled (sham) control mice. A. temperans instillation is deleterious to survival and repeated exposure accelerates lung tumor development in tumor-bearing mice, thus, A. temperans has a functional role in lung cancer development. Whole tissue RNA sequencing (RNAseq) comparing differential gene expression in A. temperans-instilled mice and sham controls showed a strong inflammatory response to A. temperans through MyD88-dependent signaling, a pathway essential to launch innate and adaptive immunity, and contributes to cancer progression. In collaboration with Romina Goldszmid, we investigated the changes in the tumor immune microenvironment upon instillations with A. temperans. Our fluorescence activated cell sorting (FACS) analysis results showed an increase of CD45+ve immune populations in the lungs of A. temperans-instilled mice driven by neutrophils, CD4+ve, CD8+ve, double negative (DN) T-cells as well as an increase in gamma-delta T cells. Overall, our results indicate that A. temperans increases infiltration of proinflammatory cells, which sustained over time, promote lung adenocarcinoma development. Neutrophil recruitment is exacerbated in the lungs of KPC mice upon challenge with A. temperans. The phenotype of the infiltrated neutrophils in A. temperans -instilled mice have increased frequency of CD44lowICAM1highSiglecF+PDL1+ neutrophils, previously characterized as long-lived, mature pro-tumorigenic tumor-associated neutrophils, important for lung tumor progression in KPC mice. RNAseq showed higher signature scores for activated and mature neutrophils in A. temperans-challenged mice. Bacterial exposure is likely the reason for neutrophil maturation, so we compared scores for a previously reported neutrophil signature of pathogenic Escherichia coli (E. coli) infection and found that A. temperans-instilled mice had higher signature scores for neutrophil maturation due to bacterial infection. Neutrophils are the dominant immune cell population in the tumor microenvironment of lung cancer patients and evidence shows that an increased ratio of circulating neutrophils to leukocytes is associated with poor prognosis, and worse response to therapy, including immunotherapy. Next is to develop a therapeutic mouse model and to validate the role of neutrophils in mediating A. temperans-induced lung tumorigenesis. Cancer Metabolome: Correlation of identified metabolites with specific cancers created biomarker profiles that can be utilized for non-invasion diagnostic and prognostic evaluation of many types of human cancer. Liquid biopsy of urine, serum and plasma are used to measure four biomarkers (creatine riboside (CR), N-acetylneuminic acid (NANA), cortisol sulfate (CS), and 27alpha-nor-5beta-cholestane-3aplpha, 7alpha, 12alpha 24alpha, 25alpha Pentol glucuronide (NCPG) of lung cancer by mass spectrometry (Haznadar, M. et al., Cancer Epidemiol. Biomarker Prev. 25:978-86, 2016). CR paired with other identified urinary metabolite biomarkers such as (NANA) improve diagnostic capability and reliability. These foundational studies validated the use of urinary metabolite screening leading to further investigation into biomarker association with human cancer. (Patel, DP. et al. J Pharm Biomed Anal. 191: 113596, 2020) And as mentioned in the 2020 annual report, urinary metabolite biomarker profiling could offer diagnostic and prognostic evaluation of intrahepatic cholangiocarcinoma (ICC). Employing UPLC-MS/MS, four metabolites, for the quantitation of metabolites CR, N-acetylneuraminic acid (NANA), cortisol sulfate, and a glucuronide fragmented ion designated as 561+, are significantly increased in HCC and ICC and are robust at classifying ICC in combination with a clinically utilized marker CA19-9. NCI-MD cohort were studied and observations verified by the TIGER-LC cohort. We determined properties that are significant for a biomarker to its use in CLIA based assays of biomarkers in liquid biopsy.