Linda M. Murphy

Mutational spectrum of β-catenin, AXIN1, and AXIN2 in hepatocellular carcinomas and hepatoblastomas

Activation of Wnt signaling through β-catenin mutations contributes to the development of hepatocellular carcinoma (HCC) and hepatoblastoma (HB). To explore the contribution of additional Wnt pathway molecules to hepatocarcinogenesis, we examined β-catenin, AXIN1 and AXIN2 mutations in 73 HCCs and 27 HBs. β-catenin mutations were detected in 19.2% (14 out of 73) HCCs and 70.4% (19 out of 27) HBs. β-catenin mutations in HCCs were primarily point mutations, whereas more than half of the HBs had deletions. AXIN1 mutations occurred in seven (9.6%) HCCs and two (7.4%) HBs. The AXIN1 mutations included seven missense mutations, a 1 bp deletion, and a 12 bp insertion. The predominance of missense mutations found in the AXIN1 gene is different from the small deletions or nonsense mutations described previously. Loss of heterozygosity at the AXIN1 locus was present in four of five informative HCCs with AXIN1 mutations, suggesting a tumor suppressor function of this gene. AXIN2 mutations were found in two (2.7%) HCCs but not in HBs. Two HCCs had both AXIN1 and β-catenin mutations, and one HCC had both AXIN2 and β-catenin mutations. About half the HCCs with AXIN1 or AXIN2 mutations showed β-catenin accumulation in the nucleus, cytoplasm or membrane. Overall, these data indicate that besides the approximately 20% of HCCs and 80% of HBs with β-catenin mutations contributing to hepatocarcinogenesis, AXIN1 and AXIN2 mutations appear to be important in an additional 10% of HCCs and HBs.

Immunoreactivity of Hep Par 1 in Hepatic and Extrahepatic Tumors and Its Correlation With Albumin In Situ Hybridization in Hepatocellular Carcinoma

We evaluated the expression of Hep Par 1 (hepatocyte paraffin 1 monoclonal antibody) in 42 hepatocellular carcinomas (HCCs), 25 cholangiocarcinomas, 18 tumors metastatic to the liver, and 87 primary extrahepatic tumors. Albumin in situ hybridization (ISH) was performed in the HCC cases. Of 42 cases of HCC, 39 (93%) were positive for Hep Par 1. All cases of cholangiocarcinoma, renal cell carcinoma, adrenocortical carcinoma, and islet cell tumors were negative; 1 case each of primary urinary bladder (n = 10) and pancreatic (n = 10) adenocarcinoma and 3 of 11 cases of primary pulmonary adenocarcinoma showed focal positivity; 7 of 10 gastric and 6 of 8 esophageal adenocarcinomas were strongly positive. Albumin ISH was positive in 39 (93%) HCC cases. All cases of HCC were positive for Hep Par 1 or albumin ISH. Hep Par 1 immunoreactivity has high sensitivity in the diagnosis of HCC. Strong positive staining also occurs in gastroesophageal adenocarcinomas. Cholangiocarcinoma and carcinomas from most other sites are negative for Hep Par 1. Hep Par 1 immunoreactivity shows high correlation with albumin ISH; their combined use for diagnosis of HCC had a sensitivity of 100% in this population.

High expression levels of survivin protein independently predict a poor outcome for patients who undergo surgery for clear cell renal cell carcinoma.

In a previous study of gene array data, the authors identified survivin as a candidate marker of aggressiveness in clear cell renal cell carcinoma (ccRCC). What remained in question was whether survivin expression at the protein level is an independent predictor of disease progression and cancer‐specific survival.

Immunohistochemical expression of folate receptor α in colorectal carcinoma: patterns and biological significance

Folate receptor alpha (FRalpha) has emerged as a potential cancer therapy target with several folate-linked therapeutic agents currently undergoing clinical trials. In addition, FRalpha expression in tumors may offer prognostic significance. Most studies on FRalpha expression used reverse transcriptase polymerase chain reaction or cytofluorimetric assays. The applicability of such methods to paraffin-embedded tissues is limited. The aims of this study were to assess the feasibility of immunohistochemistry in detecting FRalpha expression and to assess the patterns and clinical significance of FRalpha expression in colorectal tissues. We used tissue microarrays containing 152 normal colorectal mucosa samples, 42 adenomas, 177 primary, and 52 metastatic colorectal carcinomas. Our results showed that staining for FRalpha on colorectal tissues was simple and easy to read. FRalpha positivity was more frequent in carcinomas (33% in primaries and 44% in metastases) than in normal mucosa or adenoma (7% in both) (P < .001). Positive staining in primary carcinomas correlated with younger age (n = 130) (P = .008), presence of distant metastasis (n = 130) (P = .043), and non-high-frequency microsatellite instability status (as detected by the standard polymerase chain reaction method using the 5 National Cancer Institute-recommended markers) (n = 77) (P = .006). Positive staining in primary carcinomas also correlated with a worse 5-year disease-specific survival (P = .04) on univariate but not multivariate analysis. Thus, our data show that there is selective expression of FRalpha in some colorectal cancers, providing a foundation for investigating the use of folate conjugates for imaging and therapy of colorectal tumors. Furthermore, our results suggest that a possible association exists between FRalpha expression and the microsatellite instability status in colorectal carcinoma. The significance of such an association as well as the prognostic value of FRalpha expression deserves further exploration.

Folate receptor in adenocarcinoma and squamous cell carcinoma of the lung: potential target for folate-linked therapeutic agents.

CONTEXT Lung cancer is the number one cause of cancer deaths in the United States and globally. The advent of targeted therapies has offered a new treatment paradigm for lung cancer, but currently validated and emerging drugs are effective in only a small minority of lung cancers, predominantly adenocarcinomas. Folate receptors can serve as targets for drugs attached to folate and are overexpressed in many cancers. OBJECTIVE To determine the frequency of folate receptor overexpression in lung cancers of different cell types as potential targets for folate-linked therapy. DESIGN High-density tissue microarrays were constructed from archival formalin-fixed, paraffin-embedded resection specimens from 188 primary stage I or stage II adenocarcinomas or squamous cell carcinomas of the lung with three 0.1-cm cores from each tumor. Tissue microarrays were immunostained for folate receptor α with mAb343 and the results scored (0 to 1+ = weak expression, 2+ to 3+ = strong expression). RESULTS Eighty-four of 117 (72%) of the adenocarcinomas were strongly positive for the folate receptor, and 36 of 71 (51%) of the squamous cell carcinomas were strongly positive for the folate receptor. CONCLUSIONS Our data indicate that a large percentage of lung cancers, including squamous cell carcinomas in addition to adenocarcinomas, strongly express folate receptor. This suggests that folate-linked targeted therapy can potentially be used to treat the majority of lung cancers, both adenocarcinomas and, particularly, squamous cell carcinomas, that do not respond to current targeted therapies.

Immune cell quantitation in normal breast tissue lobules with and without lobulitis

While the immune microenvironment has been investigated in breast cancers, little is known about its role in non-malignant breast tissues. Here we quantify and localize cellular immune components in normal breast tissue lobules, with and without visible immune infiltrates (lobulitis). Up to ten representative lobules each in eleven normal breast tissue samples were assessed for B cells (CD20), cytotoxic T cells (CD8), helper T cells (CD4), dendritic cells (CD11c), leukocytes (CD45), and monocytes/macrophages (CD68). Using digital image analysis, immune cell densities were measured and compared between lobules with/without lobulitis. 109 lobules in 11 normal breast tissue samples were evaluated; 31 with lobulitis and 78 without. Immune cells showed consistent patterns in all normal samples, predominantly localized to lobules rather than stroma. Regardless of lobulitis status, most lobules demonstrated CD8+, CD11c+, CD45+, and CD68+ cells, with lower densities of CD4+ and CD20+ cells. Both CD11c+ and CD8+ cells were consistently and intimately associated with the basal aspect of lobule epithelium. Significantly higher densities of CD4+, CD8+, CD20+, and CD45+ cells were observed in lobules with lobulitis. In contrast, densities of monocytes/macrophages and dendritic cells did not vary with lobulitis. In normal breast tissue, myeloid and lymphoid cells are present and localized to lobules, with cytotoxic T and dendritic cells directly integrated with epithelium. Lobules with lobulitis have significantly more adaptive immune (B and T) cells, but no increase in dendritic cells or monocytes/macrophages. These findings indicate an active and dynamic mucosal immune system in normal breast tissue.

Targeting Folate Receptors to Treat Invasive Urinary Bladder Cancer

Folate receptors (FR) may be of use for targeted delivery of cytotoxic drugs in invasive urothelial carcinoma (iUC), for which improved therapy is needed. FR expression and function in iUC were explored and the antitumor activity and toxicity of a folate-targeted vinblastine conjugate were evaluated in dogs with naturally occurring iUC, an excellent model for human iUC. FR immunohistochemistry was carried out on iUC and normal human and dog bladder tissues together with nuclear scintigraphy in dogs to monitor iUC folate uptake. Dose escalation of a folate-targeted vinblastine compound, EC0905, was conducted in dogs with biopsy-confirmed, FR-positive iUC. FRs were detected by immunohistochemistry (PU17) in most primary iUC and many nodal and lung metastases from dogs, and scintigraphy confirmed folate uptake in both primary and metastatic lesions. The maximum tolerated dose of EC0905 in dogs was 0.25 mg/kg IV weekly, with neutropenia at higher doses. Tumor responses included partial remission (≥ 50% reduction in tumor volume) in five dogs and stable disease (<50% change in tumor volume) in four dogs. Immunoreactivity to PU17 was similar in humans (78% of primary iUC, 80% of nodal metastases). Less immunoreactivity to mab343 (22% of cases) occurred. FR-β was noted in 21% of human iUC cases. Our findings suggest folate-targeted therapy holds considerable promise for treating iUC, where FR-β may be important in addition to FR-α.

Folate receptor-α expression in resectable hepatic colorectal cancer metastases: patterns and significance.

Folate receptor alpha (FRα), encoded by folate receptor 1 (adult) gene, has emerged as a cancer biomarker and potential therapeutic target. In addition, its expression in tumors may offer prognostic information. The aim of this study was to assess the prognostic value of FRα expression and other common molecular markers in resected liver metastases from colorectal cancer. To maximize potential biological differences, we selected two groups of patients with markedly different outcomes as study subjects. Immunohistochemical analysis of FRα expression and other common markers (thymidylate synthase, p53, p27, BCL2, ki67, MLH1, MSH2 and MGMT) on tissue microarrays was carried out on samples from 160 patients; 56 patients survived at least 10 years following liver resection, and 104 died within 2 years of surgery. These markers were evaluated and compared with standard clinical predictors of outcome including a previously validated clinical risk score. Our results showed that in addition to known clinical risk factors, FRα positivity was significantly associated with the early death group (32% compared with 13%; P=0.03). None of the other common molecular markers were differentially expressed between the two groups. On multivariate analysis, clinical risk score, margin status and FRα expression were independently associated with outcome. Specific multivariate comparisons confirmed that FRα expression was associated with outcome independent of the clinical risk score and margin. These data demonstrate that FRα expression is present in a subset of resected hepatic colorectal cancer metastases, and this marker is independently associated with survival after hepatic resection. The prognostic value of FRα expression and the utility of FRα-targeted therapies in stage-IV colorectal cancer patients deserve further exploration.

Sulfatase 2 protects hepatocellular carcinoma cells against apoptosis induced by the PI3K inhibitor LY294002 and ERK and JNK kinase inhibitors.

Background: Sulfatase 2 (SULF2), an extracellular heparan sulphate 6‐O‐endosulphatase, has an oncogenic effect in hepatocellular carcinoma (HCC) that is partially mediated through glypican 3, which promotes heparin‐binding growth factor signalling and HCC cell growth. SULF2 also increases phosphorylation of the anti‐apoptotic Akt kinase substrate GSK3β and SULF2 expression is associated with a decreased apoptotic index in human HCCs.

Alterations in the immune cell composition in premalignant breast tissue that precede breast cancer development

Purpose: Little is known about the role of the immune system in the earliest stages of breast carcinogenesis. We studied quantitative differences in immune cell types between breast tissues from normal donors and those from women with benign breast disease (BBD). Experimental Design: A breast tissue matched case–control study was created from donors to the Susan G. Komen for the Cure Tissue Bank (KTB) and from women diagnosed with BBD at Mayo Clinic (Rochester, MN) who either subsequently developed cancer (BBD cases) or remained cancer-free (BBD controls). Serial tissue sections underwent immunostaining and digital quantification of cell number per mm2 for CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD68+ macrophages and quantification of positive pixel measure for CD11c (dendritic cells). Results: In 94 age-matched triplets, BBD lobules showed greater densities of CD8+ T cells, CD11c+ dendritic cells, CD20+ B cells, and CD68+ macrophages compared with KTB normals. Relative to BBD controls, BBD cases had lower CD20+ cell density (P = 0.04). Nearly 42% of BBD cases had no CD20+ B cells in evaluated lobules compared with 28% of BBD controls (P = 0.02). The absence of CD20+ cells versus the presence in all lobules showed an adjusted OR of 5.7 (95% confidence interval, 1.4–23.1) for subsequent breast cancer risk. Conclusions: Elevated infiltration of both innate and adaptive immune effectors in BBD tissues suggests an immunogenic microenvironment. The reduced B-cell infiltration in women with later breast cancer suggests a role for B cells in preventing disease progression and as a possible biomarker for breast cancer risk. Clin Cancer Res; 23(14); 3945–52. ©2017 AACR.