Debulking Hepatectomy for Colorectal Liver Metastasis Conveys Survival Benefit.

(1) Background: Despite advances in surgical technique and systemic chemotherapy, some patients with multifocal, bilobar colorectal liver metastases (CRLM) remain unresectable. These patients may benefit from surgical debulking of liver tumors in combination with chemotherapy compared to chemotherapy alone. (2) Methods: A retrospective study including patients evaluated for curative intent resection of CRLM was performed. Patients were divided into three groups: those who underwent liver resection with recurrence within 6 months (subtotal debulked, SD), those who had the first stage only of a two-stage hepatectomy (partially debulked, PD), and those never debulked (ND). Kaplan-Meier survival curves and log-rank test were performed to assess the median survival of each group. (3) Results: 174 patients underwent liver resection, and 34 patients recurred within 6 months. Of the patients planned for two-stage hepatectomy, 35 underwent the first stage only. Thirty-two patients were never resected. Median survival of the SD, PD, and ND groups was 31 months, 31 months, and 19.5 months, respectively (p = 0.012); (4) Conclusions: Patients who underwent a debulking of CRLM demonstrated a survival benefit compared to patients who did not undergo any surgical resection. This study provides support for the evaluation of intentional debulking versus palliative chemotherapy alone in a randomized trial.

A Retrospective Study on the Role of Metformin in Colorectal Cancer Liver Metastases.

Colorectal cancer liver metastases (CRCLMs) have two main histopathological growth patterns (HPGs): desmoplastic (DHGP) and replacement (RHGP). The vascularization in DHGP tumours is angiogenic, while the RHGP tumours exert vessel co-option vasculature. The presence of vessel co-option tumours is associated with poor response to anti-angiogenic agents and chemotherapy, as well as a worse prognosis. Metformin has been shown to influence the progression and vasculature of tumours in different cancers. However, its role in CRCLM is poorly understood. Herein, we conducted a retrospective cohort study to examine the role of metformin in CRCLM. A dataset of 108 patients was screened, of which 20 patients used metformin. The metformin user patients did not use metformin as an anticancer agent. We noticed a significantly lower percentage of CRCLM patients with vessel co-opting RHGP tumours in the population that used metformin compared to CRCLM patients who did not use metformin. Similar results were obtained when we compared the ratio of recurrence and extrahepatic metastases incidence. Moreover, the metformin user patients had significantly higher survival outcome compared to nonusers. Collectively, our data suggest that metformin administration is likely associated with better prognosis of CRCLM.

Isolation of extracellular vesicles from human plasma samples: The importance of controls.

BACKGROUND: Extracellular vesicles (EV) are enriched with proteins and RNA cargo, promoting cell-to-cell communication. Biofluid derived EV cargo is used for discovering disease specific markers for diagnosis and disease monitoring. RATIONAL: Blood is a complex fluid with an abundance of protiens and thus isolation of EVs is challenging. Therefore, methods for EV isolation, including commercial kits use thromboplastin D (TP-D) for pretreatment of plasma to increase EV purity and yield. This pretreatment can introduce contaminants. METHOD AND RESULTS: We performed a comparative study to evaluate the effect of EV isolation methods focusing on (a) pretreatment of plasma with additives, which include: rabbit TP (rTP) versus human recombinant thromboplastin (huTP), to increase purity and yield (b) an additional centrifugation step prior to freezing plasma and (c) comparison of frozen versus fresh plasma EV isolations. Pretreatment with rTP generated a dynamic range of proteins, however, most of these proteins were contaminants, introduced from the rTP (99.1% purity). As an alternative, huTP was used, which did not introduce any significant contaminants, however, this did not increase yield or purity. Additionally, an extra 10,000 g centrifugation did not improve either EV yield or purity. Finally, comparison of fresh or frozen plasma showed no significant difference, an important factor when sourcing plasma from biobanks. CONCLUSION: Appropriate controlsare required when adding any additives during EV isolation as even a small percentage of contaminants can have a major effect on results. Furthermore, biobanked plasma can be used with no major changes to processing.

Histopathological growth patterns of liver metastasis: updated consensus guidelines for pattern scoring, perspectives and recent mechanistic insights.

The first consensus guidelines for scoring the histopathological growth patterns (HGPs) of liver metastases were established in 2017. Since then, numerous studies have applied these guidelines, have further substantiated the potential clinical value of the HGPs in patients with liver metastases from various tumour types and are starting to shed light on the biology of the distinct HGPs. In the present guidelines, we give an overview of these studies, discuss novel strategies for predicting the HGPs of liver metastases, such as deep-learning algorithms for whole-slide histopathology images and medical imaging, and highlight liver metastasis animal models that exhibit features of the different HGPs. Based on a pooled analysis of large cohorts of patients with liver-metastatic colorectal cancer, we propose a new cut-off to categorise patients according to the HGPs. An up-to-date standard method for HGP assessment within liver metastases is also presented with the aim of incorporating HGPs into the decision-making processes surrounding the treatment of patients with liver-metastatic cancer. Finally, we propose hypotheses on the cellular and molecular mechanisms that drive the biology of the different HGPs, opening some exciting preclinical and clinical research perspectives.

Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3.

Resistance to anti-angiogenic therapy is a major challenge in the treatment of colorectal cancer liver metastases (CRCLMs). Vessel co-option has been identified as a key contributor to anti-angiogenic therapy resistance in CRCLMs. Recently, we identified a positive correlation between the expression of Angiopoietin1 (Ang1) in the liver and the development of vessel co-opting CRCLM lesions in vivo. However, the mechanisms underlying its stimulation of vessel co-option are unclear. Herein, we demonstrated Ang1 as a positive regulator of actin-related protein 2/3 (ARP2/3) expression in cancer cells, in vitro and in vivo, which is known to be essential for the formation of vessel co-option in CRCLM. Significantly, Ang1-dependent ARP2/3 expression was impaired in the cancer cells upon Tie2 or PI3K/AKT inhibition in vitro. Taken together, our results suggest novel mechanisms by which Ang1 confers the development of vessel co-option in CRCLM, which, targeting this pathway, may serve as promising therapeutic targets to overcome the development of vessel co-option in CRCLM.

Loss of mitochondrial ATPase ATAD3A contributes to nonalcoholic fatty liver disease through accumulation of lipids and damaged mitochondria.

Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and nonalcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced nonalcoholic steatohepatitis and in human livers from patients diagnosed with NAFLD. We used CRISPR-Cas9 to delete ATAD3A in Huh7 human hepatocellular carcinoma cells and used RNAi to silence ATAD3A expression in human hepatocytes isolated from humanized liver-chimeric mice to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor of acetyl-CoA acetyltransferase. Our results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells and also that triglyceride levels were significantly increased in ATAD3A KO Huh7 cells following inhibition of lipolysis mediated by upregulation of lipid droplet-binding protein perilipin-2. Moreover, loss of ATAD3A upregulated autophagosome-associated light chain 3-II protein and p62 in Huh7 cells and fresh human hepatocytes through blockage of autophagosome degradation. Finally, we show the mitophagy mediator, PTEN-induced kinase 1, was downregulated in ATAD3A KO Huh7 cells, suggesting that ATAD3A KO inhibits mitophagy. These results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. Taken together, we conclude that loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, triglyceride, and damaged mitochondria in hepatocytes.

Cancer Cells Promote Phenotypic Alterations in Hepatocytes at the Edge of Cancer Cell Nests to Facilitate Vessel Co-Option Establishment in Colorectal Cancer Liver Metastases.

Vessel co-option is correlated with resistance against anti-angiogenic therapy in colorectal cancer liver metastases (CRCLM). Vessel co-opting lesions are characterized by highly motile cancer cells that move toward and along the pre-existing vessels in the surrounding nonmalignant tissue and co-opt them to gain access to nutrients. To access the sinusoidal vessels, the cancer cells in vessel co-opting lesions must displace the hepatocytes and occupy their space. However, the mechanisms underlying this displacement are unknown. Herein, we examined the involvement of apoptosis, autophagy, motility, and epithelial-mesenchymal transition (EMT) pathways in hepatocyte displacement by cancer cells. We demonstrate that cancer cells induce the expression of the proteins that are associated with the upregulation of apoptosis, motility, and EMT in adjacent hepatocytes in vitro and in vivo. Accordingly, we observe the upregulation of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and actin-related protein 2/3 (ARP2/3) in adjacent hepatocytes to cancer cell nests, while we notice a downregulation of E-cadherin. Importantly, the knockdown of runt-related transcription factor 1 (RUNX1) in cancer cells attenuates the function of cancer cells in hepatocytes alterations in vitro and in vivo. Altogether, our data suggest that cancer cells exploit various mechanisms to displace hepatocytes and access the sinusoidal vessels to establish vessel co-option.

Runt related transcription factor-1 plays a central role in vessel co-option of colorectal cancer liver metastases.

Colorectal cancer liver metastasis (CRCLM) has two major histopathological growth patterns: angiogenic desmoplastic and non-angiogenic replacement. The replacement lesions obtain their blood supply through vessel co-option, wherein the cancer cells hijack pre-existing blood vessels of the surrounding liver tissue. Consequentially, anti-angiogenic therapies are less efficacious in CRCLM patients with replacement lesions. However, the mechanisms which drive vessel co-option in the replacement lesions are unknown. Here, we show that Runt Related Transcription Factor-1 (RUNX1) overexpression in the cancer cells of the replacement lesions drives cancer cell motility via ARP2/3 to achieve vessel co-option. Furthermore, overexpression of RUNX1 in the cancer cells is mediated by Transforming Growth Factor Beta-1 (TGFß1) and thrombospondin 1 (TSP1). Importantly, RUNX1 knockdown impaired the metastatic capability of colorectal cancer cells in vivo and induced the development of angiogenic lesions in liver. Our results confirm that RUNX1 may be a potential target to overcome vessel co-option in CRCLM.

Claudin-2 promotes colorectal cancer liver metastasis and is a biomarker of the replacement type growth pattern.

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that Claudin-2 is functionally required for colorectal cancer liver metastasis and that Claudin-2 expression in primary colorectal cancers is associated with poor overall and liver metastasis-free survival. We have examined the role of Claudin-2, and other claudin family members, as potential prognostic biomarkers of the desmoplastic and replacement histopathological growth pattern associated with colorectal cancer liver metastases. Immunohistochemical analysis revealed higher Claudin-2 levels in replacement type metastases when compared to those with desmoplastic features. In contrast, Claudin-8 was highly expressed in desmoplastic colorectal cancer liver metastases. Similar observations were made following immunohistochemical staining of patient-derived xenografts (PDXs) that we have established, which faithfully retain the histopathology of desmoplastic or replacement type colorectal cancer liver metastases. We provide evidence that Claudin-2 status in patient-derived extracellular vesicles may serve as a relevant prognostic biomarker to predict whether colorectal cancer patients have developed replacement type liver metastases. Such a biomarker will be a valuable tool in designing optimal treatment strategies to better manage patients with colorectal cancer liver metastases.

Cholesterol-binding translocator protein TSPO regulates steatosis and bile acid synthesis in nonalcoholic fatty liver disease.

Translocator protein (TSPO, 18 kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and protein kinase RNA-like ER kinase/ATF4/CCAAT-enhancer-binding protein homologous protein pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. A TSPO ligand 19-Atriol blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD.

Neutrophils expressing lysyl oxidase-like 4 protein are present in colorectal cancer liver metastases resistant to anti-angiogenic therapy.

Colorectal cancer liver metastases (CRCLM) that present with a replacement histopathological growth pattern (HGP) are resistant to neoadjuvant anti-angiogenic therapy. Surrogate biomarkers are not available to preoperatively identify patients with these tumors. Here we identify differentially expressed genes between CRCLM with a replacement HGP and those with a desmoplastic HGP using RNA sequencing. We demonstrate that LOXL4 is transcriptionally upregulated in replacement HGP CRCLM compared with desmoplastic HGP CRCLM and the adjacent normal liver. Interestingly, lysyl oxidase-like 4 (LOXL4) protein was expressed by neutrophils present in the tumor microenvironment in replacement HGP CRCLM. We further demonstrate that LOXL4 expression is higher in circulating neutrophils of cancer patients compared with healthy control patients and its expression can be induced by stimulation with lipopolysaccharide and TNF-α. Our study is the first to show the expression of LOXL4 in neutrophils and reveals the potential for LOXL4-expressing neutrophils to support the replacement HGP phenotype and to serve as a surrogate biomarker for this subtype of CRCLM. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Angiopoietin1 Deficiency in Hepatocytes Affects the Growth of Colorectal Cancer Liver Metastases (CRCLM).

Colorectal cancer liver metastases (CRCLM) that receive their blood supply via vessel co-option are associated with a poor response to anti-angiogenic therapy. Angiopoietins (Ang1 and Ang2) with their Tyrosine-protein kinase receptor (Tie2) have been shown to support vessel co-option. We demonstrate significantly higher expression of Ang1 in hepatocytes adjacent to the tumor region of human chemonaïve and treated co-opting (replacement histopathological growth patterns: RHGP) tumors. To investigate the role of the host Ang1 expression, Ang1 knockout (KO) mice were injected intra-splenically with metastatic MC-38 colon cancer cells that develop co-opting liver metastases. We observed a reduction in the number of liver metastases and interestingly, for the first time, the development of angiogenic driven desmoplastic (DHGP) liver metastases. In addition, in-vitro, knockout of Ang1 in primary hepatocytes inhibited viability, migration and invasion ability of MC-38 cells. We also demonstrate that Ang 1 alone promotes the migration and growth of both human and mouse colon cancer cell lines These results provide evidence that high expression of Ang1 in the host liver is important to support vessel co-option (RHGP lesions) and when inhibited, favours the formation of angiogenic driven liver metastases (DHGP lesions).

Histologic features and genomic alterations of primary colorectal adenocarcinoma predict growth patterns of liver metastasis.

BACKGROUND: Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients' prognosis and response to antiangiogenic therapy. However, the relationship between HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer has not been well established. AIM: To assess whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis. METHODS: A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients' charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases were selected randomly from each group for whole exome sequencing (WES) analysis. RESULTS: In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn's disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5). CONCLUSION: The HGPs, TBS, and CDR of primary CRC as well as the presence of specific genetic mutations such as those in PIK3CA could be used to predict the HGPs of liver metastasis, response to therapy, and patients' prognosis.

Vascularization of colorectal carcinoma liver metastasis: insight into stratification of patients for anti-angiogenic therapies.

Current treatment for metastatic disease targets angiogenesis. With the increasing data demonstrating that cancer cells do not entirely rely on angiogenesis but hijack the existing vasculature through mechanisms such as co-option of existing blood vessels, identification of targets has become of utmost importance. Our study looks at the vasculature of chemonaïve and treated colorectal carcinoma liver metastases (CRCLMs) to obtain a basic understanding of the microvessel density, type of vasculature (mature versus immature), and correlation with histopathological growth patterns that demonstrate unique patterns of angiogenesis. We performed immunohistochemistry on chemonaïve sections of desmoplastic histopathological growth pattern (DHGP) and replacement histopathological growth patterns (RHGP) lesions with CD31 [endothelial cell (EC) marker] and CD34/Ki67 double staining, which denotes proliferating ECs. The CD31 stains demonstrated a lower microvascular CD31 +ve capillary density in the DHGP versus RHGP lesions; and integrating both immunostains with CD34/Ki67 staining on serial sections revealed proliferating vessels in DHGP lesions and co-option of mature existing blood vessels in RHGP lesions. Interestingly, upon treatment with chemotherapy and bevacizumab, the RHGP lesions showed no necrosis whereas the DHGP lesions had almost 100% necrosis of the cancer cells and in most cases there was a single layer of viable cancer cells, just under or within the desmoplastic ring. The survival of these cells may be directly related to spatial location and possibly a different microenvironment, which may involve adhesion to different extracellular matrix components and/or different oxygen/nutrient availability. This remains to be elucidated. We provide evidence that DHGP CRCLMs obtain their blood supply via sprouting angiogenesis whereas RHGP lesions obtain their blood supply via co-option of existing vasculature. Furthermore current treatment regimens do not affect RHGP lesions and although they kill the majority of the cancer cells in DHGP lesions, there are cells surviving within or adjacent to the desmoplastic ring which could potentially give rise to a growing lesion.

Next generation sequencing of progressive colorectal liver metastases after portal vein embolization.

Portal vein embolization (PVE) can be required to stimulate liver regeneration before hepatectomy for colorectal liver metastasis (CRCLM), however PVE may also trigger CRCLM progression in patients initially exhibiting chemotherapy response. Using RNA-seq, we aimed to determine the molecular networks involved in metastatic progression in this context. A prospective study including all CRCLM patients undergoing PVE prior to hepatectomy was conducted. Paired biopsies of metastatic lesions were obtained prior to and after PVE and total RNA was isolated and used to prepare Illumina rRNA-depleted TruSeq stranded cDNA libraries for HiSeq 100 bp paired-end sequencing. Patients were classified with progression of disease (PDPVE) or stable disease (SDPVE) post-PVE using 3D-CT tumor volumetric analysis. RESULTS: Twenty patients were included, 13 (65.0%) in the PDPVE group (median 58.0% (18.6-234.3) increase in tumor volume) and 7 (35.0%) in the SDPVE group exhibiting continuous chemotherapy response (median -14.3% (-40.8 to -2.8) decrease in tumor volume) (p < 0.0001). Our results showed that progressive CRCLM after PVE undergo gene expression changes that indicate activation of core cancer pathways (IL-17 (p = 5.94 × 10-03), PI3K (p = 8.71 × 10-03), IL6 and IGF-1 signaling pathways), consistent with changes driven by cytokines and growth factors. Differential expression analysis in a paired model of progression (EdgeR, DeSeq) identified significantly dysregulated genes in the PDPVE group (FOS, FOSB, RAB20, IRS2). CONCLUSION: Differentially expressed genes and pathways with known links to cancer and metastasis were identified post-PVE in patients with disease progression. Highlighting these molecular changes is a crucial first step towards development of targeted therapeutic strategies that may mitigate the effects of PVE on tumor growth.

Loss of tumour-specific ATM protein expression is an independent prognostic factor in early resected NSCLC.

Ataxia-telangiectasia mutated (ATM) is critical in maintaining genomic integrity. In response to DNA double-strand breaks, ATM phosphorylates downstream proteins involved in cell-cycle checkpoint arrest, DNA repair, and apoptosis. Here we investigate the frequency, and influence of ATM deficiency on outcome, in early-resected non-small cell lung cancer (NSCLC). Tissue microarrays, containing 165 formalin-fixed, paraffin-embedded resected NSCLC tumours from patients diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, between 2003 and 2006, were analyzed for ATM expression using quantitative fluorescence immunohistochemistry. Both malignant cell-specific ATM expression and the ratio of ATM expression within malignant tumour cells compared to that in the surrounding tumour stroma, defined as the ATM expression index (ATM-EI), were measured and correlated with clinical outcome. ATM loss was identified in 21.8% of patients, and was unaffected by clinical pathological variables. Patients with low ATM-EI tumours had worse survival outcomes compared to those with high ATM-EI (p < 0.01). This effect was pronounced in stage II/III patients, even after adjusting for other clinical co-variates (p < 0.001). Additionally, we provide evidence that ATM-deficient patients may derive greater benefit from guideline-recommended adjuvant chemotherapy following surgical resection. Taken together, these results indicate that ATM loss seems to be an early event in NSCLC carcinogenesis and is an independent prognostic factor associated with worse survival in stage II/III patients.

Murine diet/tissue and human brain tumorigenesis alter Mthfr/MTHFR 5'-end methylation.

Polymorphisms and decreased activity of methylenetetrahydrofolate reductase (MTHFR) are linked to disease, including cancer. However, epigenetic regulation has not been thoroughly studied. Our goal was to generate DNA methylation profiles of murine/human MTHFR gene regions and examine methylation in brain and liver tumors. Pyrosequencing in four murine tissues revealed minimal DNA methylation in the CpG island. Higher methylation was seen in liver or intestine in the CpG island shore 5' to the upstream translational start site or in another region 3' to the downstream start site. In the latter region, there was negative correlation between expression and methylation. Three orthologous regions were investigated in human MTHFR, as well as a fourth region between the two translation start sites. We found significantly increased methylation in three regions (not the CpG island) in pediatric astrocytomas compared with control brain, with decreased expression in tumors. Methylation in hepatic carcinomas was also increased in the three regions compared with normal liver, but the difference was significant for only one CpG. This work, the first overview of the Mthfr/MTHFR epigenetic landscape, suggests regulation through methylation in some regions, demonstrates increased methylation/decreased expression in pediatric astrocytomas, and should serve as a resource for future epigenetic studies.

The Role of Prolactin in Bone Metastasis and Breast Cancer Cell-Mediated Osteoclast Differentiation.

BACKGROUND: Metastasis to the bone is a deleterious aspect of breast cancer and is a preferred site that results in bone loss. Hormones such as prolactin (PRL) have not yet been studied for their role in modulating the secondary tumor bone microenvironment. METHODS: We used quantitative immunohistochemistry with 134 samples of human primary breast cancer and 17 matched primary breast cancers and bone metastases. A Cox proportional hazards regression model was fitted to evaluate the associations between high prolactin receptor (PRLR) expression and time to bone metastasis, adjusting for estrogen receptor status, lymph node status, and chemotherapy status. We assessed osteoclast differentiation, osteoclast size, and measured pit formation in dentine slices. Statistical tests were two-sided. RESULTS: High PRLR expression in the primary breast tumor was associated with a shorter time to metastasis that includes bone (PRLRAQUA Max-per 100 unit hazard ratio = 1.04, 95% confidence interval = 1.00 to 1.07, P = .03). We observed the PRLR in rare samples of bone metastases and matched primary breast cancer. PRL treatment of breast cancer cells induced osteoclast differentiation and bone lysis via secreted factors and was abrogated by a PRLR antagonist (delta1-9-G129R-hPRL). We demonstrated that sonic hedgehog is a PRL-regulated cytokine in breast cancer cells and part of the mechanism that induces osteoclast differentiation. CONCLUSIONS: Our evidence indicates that PRL-PRLR can escalate the impact of breast cancer on bone metastasis and that the presence of the PRLR in the tumor microenvironment of breast cancer bone metastasis has the potential to modulate the microenvironment to induce lytic osteoclast formation.

ATM, THMS, and RRM1 protein expression in nasopharyngeal carcinomas treated with curative intent.

BACKGROUND: In advanced nasopharyngeal carcinoma (NPC), biomarkers may help predict survival. METHODS: Tumoral expression of ataxia-telangiectasia mutated (ATM), thymidylate synthetase (THMS), and ribonucleotide reductase subunit M1 (RRM1), was correlated with survival in patients with nonmetastatic NPC using quantitative fluorescence immunohistochemistry with automated quantitative digital image analysis. RESULTS: Of the 146 patients included, 58 patients (40%) received concurrent chemoradiation therapy; the remainder was treated with radiation. Overall survival (OS) at 5 years was 71% (95% confidence interval [CI], 62% to 78%); disease-free survival (DFS) was 48% (95% CI, 39% to 57%). OS worsened for increasing values of ATM (hazard ratio [HR], 2.83; 95% CI, 1.01-7.94; p = .049) for values greater than the 75th percentile compared to less than the 25th percentile, but improved for tumors with higher THMS levels (HR, 0.44; 95% CI, 0.20-0.94; p = .033) for values greater than the 25th percentile compared to less than or equal to the 25th percentile. RRM1 was not associated with OS (p = .748). No biomarkers were associated with DFS. CONCLUSION: In our cohort, relative overexpression of ATM and low THMS levels were associated with worse OS. © 2015 Wiley Periodicals, Inc. Head Neck 38: E384-E391, 2016.

The prognostic value of HPV status and p16 expression in patients with carcinoma of the anal canal.

BACKGROUND: In anal cancer studies, the detection frequency of high-risk HPV (human papillomavirus) is variable, depending on the method used. There are limited data reporting results of different HPV detection techniques in the same clinical series, and very few correlating results with clinical outcome. OBJECTIVES: To evaluate tumor expression of p16/HPV16 using three different methods, and to determine their association with clinical outcome in patients with anal canal squamous cell carcinomas (SCC). DESIGN: This retrospective study included patients with anal canal SCC treated with definitive radiotherapy or chemoradiotherapy at a single institution between 1992 and 2005. Formalin-fixed paraffin-embedded tumor samples from 53 of the 89 (60%) patient pre-treatment biopsies were adequate for tissue microarray construction. HPV status was determined using: p16 expression by conventional immunohistochemistry (IHC) and quantitative IHC (AQUA), HPV genotype analysis by chromogenic in situ hybridization (CISH) and HPV linear array sub-typing. Expression status was correlated with clinical outcome. RESULTS: 80% (28/35) of patient tumors had high p16 expression using conventional IHC. HPV16 CISH was positive in 81% (34/42) of tumors, and 78% (28/36) of tumors were HPV subtype 16. HPV16 CISH correlated with p16 evaluated by conventional IHC (correlation coefficient 0.46; p = 0.01) and by p16 AQUA score (correlation coefficient 0.49; p = 0.001). A subset of cases (15%) had very high p16 quantitative IHC scores (>244) and were associated with a higher incidence of local or distant recurrence (p = 0.04). CONCLUSIONS: The vast majority (80%) of anal canal SCC in our series were positive for HPV16/p16, regardless of the testing method used. The exploratory analysis of automated quantitative IHC scoring was the only technique to define a subset of patients with a worse prognosis by p16 expression status on univariate analysis. Further exploration of the molecular mechanisms of treatment resistance in association with very high p16 expression is warranted.