Understanding how life-history strategies influence cancer susceptibility in dinosaurs requires a molecular-level analysis of preserved soft tissues. While previous research has largely focused on skeletal remains, the discovery of soft tissue structures in fossils, such as Telmatosaurus transsylvanicus, highlights the need for a new approach. Paleoproteomics offers a transformative opportunity to analyze ancient proteins, revealing the evolutionary trade-offs between growth, reproduction, and cancer suppression. This study argues that prioritizing fossil collection and soft tissue preservation is crucial, as future advances in molecular techniques will allow deeper insights into disease evolution. By integrating life-history theory with paleopathology, we can better understand the selective pressures that shaped cancer susceptibility in extinct species and identify potential mechanisms of tumor resistance. This commentary highlights the necessity of long-term fossil conservation efforts to support future breakthroughs in evolutionary biology and comparative oncology.

Cancer-promoting mutations are common in healthy tissue but rarely lead to tumour formation. A study of the mouse mammary gland reveals three protective mechanisms that limit the ability of cells to give rise to cancer. Three protective mechanisms limit the ability of cells to form a tumour.

The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland.

Background & Aims: While normal human liver is thought to be generally quiescent, clonal hepatocyte expansions have been observed, though neither their cellular source nor their expansion dynamics have been determined. Knowing the hepatocyte cell of origin, and their subsequent dynamics and trajectory within the human liver will provide an important basis to understand disease associated dysregulation.Methods: Herein, we use in vivo lineage tracing and methylation sequence analysis to demonstrate normal human hepatocyte ancestry. We exploit next-generation mitochondrial sequencing to determine hepatocyte clonal expansion dynamics across spatially distinct areas of laser-captured, microdissected, clones, in tandem with computational modelling in morphologically normal human liver.Results: Hepatocyte clones and rare SOX9+ hepatocyte progenitors commonly associate with portal tracts and we present evidence that clones can lineage-trace with cholangiocytes, indicating the presence of a bipotential common ancestor at this niche. Within clones, we demonstrate methylation CpG sequence diversity patterns indicative of periportal not pericentral ancestral origins, indicating a portal to central vein expansion trajectory. Using spatial analysis of mitochondrial DNA variants by next-generation sequencing coupled with mathematical modelling and Bayesian inference across the portal-central axis, we demonstrate that patterns of mitochondrial DNA variants reveal large numbers of spatially restricted mutations in conjunction with limited numbers of clonal mutations.Conclusions: These datasets support the existence of a periportal progenitor niche and indicate that clonal patches exhibit punctuated but slow growth, then quiesce, likely due to acute environmental stimuli. These findings crucially contribute to our understanding of hepatocyte dynamics in the normal human liver.& COPY; 2023 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Background: The potential for accumulation of somatic mutations in the healthy breast throughout life and pregnancy is poorly understood. In particular, the unique mutational landscape of both epithelial and stromal components of the mammary gland has not been investigated in depth. As cancer risk correlates with both age, age of first-time pregnancy and other factors including pregnancy itself, we wished to study mutational rate over time, using these landmarks. Methods: Here, using whole genome sequencing, we determined how the rate of mutations in both cancer drivers and passenger mutations are affected by both age and pregnancy. We aimed to describe for the first time how the mammary epithelium and stroma differ in their mutational burden. Results: Our analysis of epithelial and stromal laser-capture micro-dissected DNA from 25 normal breast samples of nulliparous and age-matched early- and late-parous women collected from Komen Tissue Bank, University of Indiana, shows that the mammary gland is characterised by known COSMIC signatures SBS1 and SBS5, both of which correlate with age (p Citation Format: Biancastella Cereser, Neha Tabassum, Lisa Del Bel Belluz, Sladjana Zagorac, Angela Yiu, Philip Carter, Cristian Miere, Alicia R Jeffries-Jones, Justin Stebbing. Mutational burden of the normal breast during age and pregnancy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD5-10.

A Correction to this paper has been published: https://doi.org/10.1038/s41388-021-01649-0

The majority of human liver research is disease-focused such that far less is known of cellular dynamics within normal human liver. We have leveraged cytochrome c oxidase deficiency as a marker of clonal hepatocyte populations in such tissues. We demonstrate these populations commonly associate with portal tracts and lineage-trace hepatocytes with cholangiocytes, indicating the presence of a bipotential common ancestor at this niche. We also observe rare periportal SOX9+ hepatocytes progenitor candidates in our human tissues. To understand clonal expansion dynamics, we measured methylation diversity and identified mtDNA variants by next-generation sequencing within spatially-defined clonal hepatocyte patches. We coupled our sequencing with mathematical modelling and Bayesian inference to compare spatial patterns of mtDNA variants under assumptions with or without faster expansion from a portal-associated niche. These datasets support the existence of a periportal progenitor niche and indicate that clonal patches slowly expand, perhaps due to acute environmental stimuli, then quiesce. These findings crucially contribute to our understanding of hepatocyte dynamics in normal human liver and provide a baseline for understanding how such dynamics may be modulated in diseased liver.

ABSTRACTThe accumulation of somatic mutations in the healthy breast throughout life and pregnancy is poorly understood1–10. Similarly, the mutational landscape of both epithelial and stromal components of the mammary gland has not been investigated. Both are relevant for breast cancer (BC), as the interplay between age, pregnancy, and cancer risk has not been fully characterized11. We describe whole genome sequencing analysis of epithelial and stromal compartments from the normal breast. We show that, in a similar way to other normal organs, the mutational burden of the mammary nulliparous epithelium significantly increases with age. In a nulliparous status, mutated clones are maintained at a consistently small size throughout the life of the individual; however, at parity, pre-existent clones significantly increase in size with age. Both epithelial and stromal compartments of the healthy breast contain pre-existing known cancer mutations, albeit at low rate, indicative of subsequent positive selection for mutations in tissue-specific driver genes. In line with this, both compartments also present gene enrichment in preferentially mutated cancer pathways. Our results show that mutational landscapes differ between the parous and nulliparous epithelium and suggest an explanation for both differential breast cancer risk and development of pregnancy-associated BC (PABC).

Ulcerative colitis (UC) is an idiopathic inflammation of the intestine with an increased risk of developing colitis-associated cancer (CAC). Currently, clinical trials are underway aiming to inhibit SMAD7 to ameliorate inflammation. While the direct effect of depleting SMAD7, an inhibitory molecule in the transforming growth factor-β1 (TGFβ1) pathway, may be therapeutic in UC, its indirect effect on CAC development is largely unknown. TGFβ1 is known to enhance late stages of sporadic colorectal cancers (CRC), where SMAD7 is also elevated. Therefore, we hypothesise that removing inhibition of this pathway by depleting SMAD7 may also be detrimental for CAC. We therefore evaluated the expression of SMAD7 in the colonic epithelium during the inflammation associated neoplastic process to determine a possible role of SMAD7 in CAC. The expression of SMAD7 protein and mRNA in colonic epithelia was assessed by immunohistochemistry (IHC) and in situ hybridisation (ISH),, respectively, in a cohort of 53 archival colon samples (17 CAC, 12 dysplastic, 12 inflammed, 12 non-neoplastic/non-inflammed) from patients who have undergone colectomies for UC and CAC. The expression within the epithelial cells was evaluated by both digital quantification and validated by blind scoring by a pathologist. Significant differences were tested with one-way ANOVA and Mann–Whitney U test. Cytoplasmic expression of SMAD7 protein is significantly higher in the inflammed epithelium compared with non-inflamed epithelium (p < 0.0001). Interestingly, a significant decrease of the same was detected in dysplasia (p = 0.01), although this group is characterised by a higher variability. SMAD7 levels are elevated in cancer compared with dysplasia, suggesting a biphasic expression (p = 0.009), which could be in part due to the different genetic composition. SMAD7 mRNA expression was not significantly different across different stages of CAC (p = 0.49). We hypothesise that the lack of correlation between mRNA and protein levels could be attributed to yet unknown post-transcriptional or post-translational regulations. In our cohort of UC affected colon tissues, SMAD7 demonstrated a biphasic expression pattern along the different stages of CAC with peaks during active inflammation and cancer. The increase in SMAD7 expression during neoplastic transformation, comparable to sporadic CRC, may be a protective response of the epithelium to inhibit the effect of TGFβ1. Although inhibiting SMAD7 as a therapy for UC may remit inflammation, we hypothesise it may exacerbate CAC due to further enhancement in TGFβ1 signalling. We envisage further mechanistic studies in vitro, in particular in organoids, could help in understanding the TGFβ superfamily pathway in CAC.

Abstract Background: Amongst the various risk factors for breast cancer (BC), the molecular basis which may explain the correlation between age at first full-term and breast cancer risks is still understudied. Epidemiology studies indicate that an early first full-term pregnancy (before 25 years of age) confers a significant level of protection towards the development of post-menopausal BC compared to the risk in nulliparous or late-parous women. On the other hand, any pregnancy relates to a higher risk in developing cancer during or within one year of pregnancy (pregnancy-associated breast cancer, PABC). Thus, the relation between age of pregnancy and breast cancer risk may be too difficult to explain using only epidemiology data. Aims of the study: With our research study, we aim to study a cohort of 60 normal breast samples of nulliparous and age-matched early- and late-parous women collected from Komen Tissue Bank, University of Indiana, and to create for the first time a mathematical model of cell clone expansion in the normal breast growth. This will allow us to determine how the rates of both cancer drivers, passenger mutations and genetic variations are affected by pregnancy. We then aim to translate this in cancer tissues, and to determine how the rate of the same mutations in both pregnancy and non pregnancy-associated cancers (post-menopausal). At the same time, we intend to create a mouse model which will be used to further validate our model, where driver mutations will be induced in the mammary epithelium of pregnant mice of different ages. This will allow us to test our model of growth of a mutated clone in a pregnancy environment, and to determine what are the molecular changes in the pregnant mammary gland which can trigger a different BC risk in the early-parous cohort. Results: To examine the mutational landscape in the normal parous and nulliparous women, we extracted DNA from laser-capture microdissected epithelium and the stroma, the latter of which will be used to eliminate germ line mutations. We are currently analysing the results from Whole Genome Sequencing at 30x 100pe on a MGISEQ2000 platform on a first set of samples (two nulliparous samples and two age-matched parous samples from both early and late pregnancy). Our procedure for processing and analysis of this data follows the Broad Institute's “GATK Best Practice Guidelines” for use of next generation sequencing (NGS) data. Based on the collected data, we plan to continue with targeted sequencing or whole genome sequencing on the remaining samples. Conclusions: Our study will provide novel information on which areas of the genome are mostly mutated or altered in the normal breast, and will indicate how mutated cells, including mutations in driver genes for breast cancer, and genetic alterations change in the contest of pregnancy. With the mathematical model of clone growth/extinction, we intend to explain how different ages of pregnancy can significantly alter the clone composition in the normal breast and result in a different probability of developing breast cancer. Citation Format: Cereser B, Tabassum N, Carter P, Del Bel Belluz L, Stebbing J. Study of the mutational landscape of normal and pregnant breast to predict pregnancy-associated breast cancer risk [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-04-06.

Objective The crypt population in the human intestine is dynamic: crypts can divide to produce two new daughter crypts through a process termed crypt fission, but whether this is balanced by a second process to remove crypts, as recently shown in mouse models, is uncertain. We examined whether crypt fusion (the process of two neighbouring crypts fusing into a single daughter crypt) occurs in the human colon. Design We used somatic alterations in the gene cytochrome c oxidase (CCO) as lineage tracing markers to assess the clonality of bifurcating colon crypts (n=309 bifurcating crypts from 13 patients). Mathematical modelling was used to determine whether the existence of crypt fusion can explain the experimental data, and how the process of fusion influences the rate of crypt fission. Results In 55% (21/38) of bifurcating crypts in which clonality could be assessed, we observed perfect segregation of clonal lineages to the respective crypt arms. Mathematical modelling showed that this frequency of perfect segregation could not be explained by fission alone (p<10(-20)). With the rates of fission and fusion taken to be approximately equal, we then used the distribution of CCO-deficient patch size to estimate the rate of crypt fission, finding a value of around 0.011 divisions/crypt/year. Conclusions We have provided the evidence that human colonic crypts undergo fusion, a potential homeostatic process to regulate total crypt number. The existence of crypt fusion in the human colon adds a new facet to our understanding of the highly dynamic and plastic phenotype of the colonic epithelium.

[This corrects the article DOI: 10.18632/oncotarget.24257.].

In this study we utilized data on patient responses to guided treatments, and we evaluated their benefit for a non-small cell lung cancer cohort. The recommended therapies used were predicted using tumor molecular profiles that involved a range of biomarkers but primarily used immunohistochemistry markers. A dataset describing 91 lung non-small cell lung cancer patients was retrospectively split into two. The first group's drugs were consistent with a treatment plan whereby all drugs received agreed with their tumor's molecular profile. The second group each received one or more drug that was expected to lack benefit. We found that there was no significant difference in overall survival or mortality between the two groups. Patients whose treatments were predicted to be of benefit survived for an average of 402 days, compared to 382 days for those that did not (P = 0.7934). In the matched treatment group, 48% of patients were deceased by the time monitoring had finished compared to 53% in the unmatched group (P = 0.6094). The immunohistochemistry biomarker for the ERCC1 receptor was found to be a marker that could be used to predict future survival; ERCC1 loss was found to be predictive of poor survival.

We used data obtained by Caris Life Sciences, to evaluate the benefits of tailoring treatments for a breast carcinoma cohort by using tumor molecular profiles to inform decisions. Data for 92 breast cancer patients from the commercial Caris Molecular Intelligence database was retrospectively divided into two groups, so that the first always followed treatment recommendations, whereas in the second group all patients received at least one drug after profiling that was predicted to lack benefit. The biomarker and drug associations were based on tests including fluorescent in situ hybridization and DNA sequencing, although immunohistochemistry was the main test used. Patients whose drugs matched those recommended according to their tumor profile had an average overall survival of 667 days, compared to 510 days for patients that did not (P=0.0316). In the matched treatment group, 26% of patients were deceased by the last time of monitoring, whereas this was 41% in the unmatched group (P=0.1257). We therefore confirm the ability of tumor molecular profiling to improve survival of breast cancer patients. Immunohistochemistry biomarkers for the androgen, estrogen and progesterone receptors were found to be prognostic for survival.

[This corrects the article DOI: 10.18632/oncotarget.23675.].

We evaluated the effect of tailoring treatments based on predictions informed by tumor molecular profiles across a range of cancers, using data from Caris Life Sciences. These included breast carcinoma, colorectal adenocarcinoma, female genital tract malignancy, lung non-small cell lung cancer, neuroendocrine tumors, ovarian surface epithelial carcinomas, and urinary tract cancers. Molecular profiles using mostly immunohistochemistry (IHC) and DNA sequencing for tumors from 841 patients had been previously used to recommend treatments; some physicians followed the suggestions completely while some did not. This information was assessed to find out if the outcome was better for the patients where their received drugs matched recommendations. The IHC biomarker for the progesterone receptor and for the androgen receptor were found to be most prognostic for survival overall. The IHC biomarkers for P-glycoprotein (PGP), tyrosine-protein kinase Met (cMET) and the DNA excision repair protein ERCC1 were also shown to be significant predictors of outcome. Patients whose treatments matched those predicted to be of benefit survived for an average of 512 days, compared to 468 days for those that did not (P = 0.0684). In the matched treatment group, 34% of patients were deceased at the completion of monitoring, whereas this was 47% in the unmatched group (P = 0.0001).

It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser‐capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi‐lineage differentiation within CCO‐deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO‐deficient areas appeared large within areas of ductal carcinoma in situ , suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Small mothers against decapentaplegic (SMAD) proteins are a family of signal transduction molecules in transforming growth factor β (TGFβ) ligand pathways that have been found to have a key role in the pathogenesis of inflammatory bowel disease (IBD). Long standing IBD predisposes individuals to colitis-associated colorectal cancer (CAC), an entity that possess unique characteristics compared to hereditary and sporadic cancer. The ligands of the TGFβ super family along with SMADs have also been implicated in several aspects of colorectal cancer formation. SMAD proteins are shown to be involved in a number of potentially carcinogenic mechanisms such as altering gene transcription, controlling stem cell differentiation to causing epigenetic changes. Modulation of these proteins has emerged as a novel therapeutic intervention for IBD although its effect on carcinogenesis remains elusive. This account reviews available evidence linking SMAD proteins to CAC and explores the potential areas for future research in this area.