Lesley Lomo

Epidermal growth factor receptor as a biomarker for cervical cancer

This review focuses on the different modes of expression of the epidermal growth factor receptor (EGFR). All methods used to assess EGFR expression are critically analyzed and insights into the use of inhibitors of EGFR for treatment of cervical cancer are discussed. Currently, expression of EGFR as a biomarker for prognosis or for treatment of cervical cancer is not defined for clinical use.

Histologic and immunohistochemical decision-making in endometrial adenocarcinoma

Diffuse p53 immunostaining distinguishes 85% of serous (Type II) from endometrioid (Type I) carcinomas and is an independent marker for poor prognosis. Interobserver reproducibility for the diagnosis of these entities, as well as selection and prediction of p53 immunostaining results, is unknown. Reproducibility of three pathologists regarding: (1) a two (I and II) and (2) three part classification (I, II or indeterminate); (3) recommendation for p53 staining and (4) expectations of p53 staining results were computed with the kappa (k) statistic. All cases were immunostained for p53 and independently scored. A two and three tiered classification scheme achieved high (k=0.71) and moderate (k=0.49) reproducibility. Non-unanimous cases were more likely to be reclassified into an ‘indeterminate’ category (27 cases, 39% of passes) compared to those with unanimous (82 cases, 14% of passes) classification. Pathologists recommended p53 immunostaining with poor (k=0.28) reproducibility, but staining prediction was made with good concordance (69%, k=0.50). Moreover, p53 staining was more common in diagnostically discordant (46%) compared to concordant (16%) cases. A subset of endometrial cancers do not readily fit within a two-class system and can be culled from cases that (1) do not achieve interobserver concordance and (2) are more likely to be chosen for p53 immunostaining and (3) are more likely to stain positive for p53. Because p53 is an important marker for endometrial adenocarcinoma outcome, and cannot be predicted in advance in indeterminate cases, p53 immunostaining should be employed in cases with observer disagreement in a binary system.

High incidence of ErbB3, ErbB4, and MET expression in ovarian cancer.

Ovarian cancer is the leading cause of death from gynecologic cancers in the United States. Failure may be due to variable expression and/or complex interactions of growth factor receptors in individual tumors. As ErbB3-MET cooperativity is implicated in solid tumor resistance to EGFR/ErbB2 inhibitors, we evaluated expression of MET and all 4 ErbB family members in ovarian cancers. Tissue arrays were prepared from archival formalin-fixed paraffin-embedded tumor samples, including 202 ovarian carcinomas (Stage I–IV) and controls. Of 202 patient samples, only 25% were positive for EGFR and 35% for ErbB2 expression. ErbB3, ErbB4, and MET showed marked expression in 76%, 98%, and 96% of cases. Consistent with high incidence, there was no significant correlation for expression of ErbB3, ErbB4, or MET with outcome. On the basis of their high expression in the majority of cases, inhibitors targeting ErbB3, ErbB4, and/or MET may be broadly applicable as therapeutic agents in this disease.

Squamous morules are functionally inert elements of premalignant endometrial neoplasia

Squamous morules are a common component of premalignant glandular lesions that are followed by glandular, rather than squamous, carcinomas. We tested the hypothesis that the appearance of glands associated with morules predicts cancer risk, and undertook molecular testing to determine the clonal and hormonal response properties of admixed squamous and glandular elements. A total of 66 patients with squamous morules in an index endometrial biopsy had follow-up clinical data (average follow-up: interval 31 months, 2.5 biopsies) showing development of carcinoma in 11% (7/66) of cases. The histological appearance of morule-associated glands in the index biopsy was significantly associated with this clinical outcome, with the majority (71%, 5/7) of cancer occurrences following an overtly premalignant lesion (endometrial intraepithelial neoplasia) with squamous morules. Eight endometrial intraepithelial neoplasias with squamous morules were examined by immunohistochemistry for estrogen and progesterone receptors and mitotic activity (Ki-67 antigen percent stained). Glandular components had abundant estrogen and progesterone receptors, and high levels of mitotic activity in all cases. In sharp contrast, all squamous morules were devoid of sex hormone receptors and had undetectable or extremely low-proliferation rates. When mutated, the same specific PTEN mutation was detected in squamous and glandular elements, indicating that both are of common lineage. The clinical and laboratory data are consistent with a model of morule biology in which squamous morules are a hormonally incompetent subpopulation of endometrial glandular lesions. Isolated morules might result from artifactual displacement from their native glandular context, or selective hormonally induced regression of the glandular but not squamous components over time. Subsequent cancer risk, as promoted by estrogens, is greatest when the glandular component has the appearance of endometrial intraepithelial neoplasia. Even isolated morules should be carefully followed, however, to exclude a coexisting undersampled, or occult, glandular lesion.

A Novel Pharmacologic Activity of Ketorolac for Therapeutic Benefit in Ovarian Cancer Patients

Purpose: We previously identified the R-enantiomer of ketorolac as an inhibitor of the Rho-family GTPases Rac1 and Cdc42. Rac1 and Cdc42 regulate cancer-relevant functions, including cytoskeleton remodeling necessary for tumor cell adhesion and migration. This study investigated whether administration of racemic (R,S) ketorolac after ovarian cancer surgery leads to peritoneal distribution of R-ketorolac, target GTPase inhibition in cells retrieved from the peritoneal cavity, and measureable impact on patient outcomes. Experimental Design: Eligible patients had suspected advanced-stage ovarian, fallopian tube or primary peritoneal cancer. Secondary eligibility was met when ovarian cancer was confirmed and optimally debulked, an intraperitoneal port was placed, and there were no contraindications for ketorolac administration. R- and S-ketorolac were measured in serum and peritoneal fluid, and GTPase activity was measured in peritoneal cells. A retrospective study correlated perioperative ketorolac and ovarian cancer–specific survival in ovarian cancer cases. Results: Elevated expression and activity of Rac1 and Cdc42 was detected in ovarian cancer patient tissues, confirming target relevance. Ketorolac in peritoneal fluids was enriched in the R-enantiomer and peritoneal cell GTPase activity was inhibited after ketorolac administration when R-ketorolac was at peak levels. After adjusting for age, AJCC stage, completion of chemotherapy, and neoadjuvant therapy, women given perioperative ketorolac had a lower hazard of death (HR, 0.30; 95% confidence interval, 0.11–0.88). Conclusions: Ketorolac has a novel pharmacologic activity conferred by the R-enantiomer and R-ketorolac achieves sufficient levels in the peritoneal cavity to inhibit Rac1 and Cdc42, potentially contributing to the observed survival benefit in women who received ketorolac. Clin Cancer Res; 21(22); 5064–72. ©2015 AACR.

Targeted molecular-genetic imaging and ligand-directed therapy in aggressive variant prostate cancer

Significance Aggressive variant prostate cancer (AVPC) is a clinically defined tumor with neuroendocrine or small-cell differentiation, visceral metastases, low prostate-specific antigen, androgen receptor insensitivity, and poor/brief responses to androgen-deprivation or platinum-based chemotherapy. AVPC incidence has markedly increased, yielding an unmet diagnostic/therapeutic need. Here we adapted a patient-derived xenograft model and tumor samples to demonstrate ligand-directed theranostics of AVPC in vivo. We engineered human Herpes simplex virus thymidine kinase type-1 as a noninvasive imaging reporter/suicide transgene into adeno-associated virus/phage (AAVP) particles displaying motif ligands to cell surface-associated glucose-regulated protein 78kD (GRP78), toward a clinic-ready system. Although individual components of the AAVP system have been extensively investigated, this study is evidence of successful application in relevant preclinical models of untreatable and hard to diagnose aggressive tumor variants. Aggressive variant prostate cancers (AVPC) are a clinically defined group of tumors of heterogeneous morphologies, characterized by poor patient survival and for which limited diagnostic and treatment options are currently available. We show that the cell surface 78-kDa glucose-regulated protein (GRP78), a receptor that binds to phage-display-selected ligands, such as the SNTRVAP motif, is a candidate target in AVPC. We report the presence and accessibility of this receptor in clinical specimens from index patients. We also demonstrate that human AVPC cells displaying GRP78 on their surface could be effectively targeted both in vitro and in vivo by SNTRVAP, which also enabled specific delivery of siRNA species to tumor xenografts in mice. Finally, we evaluated ligand-directed strategies based on SNTRVAP-displaying adeno-associated virus/phage (AAVP) particles in mice bearing MDA-PCa-118b, a patient-derived xenograft (PDX) of castration-resistant prostate cancer bone metastasis that we exploited as a model of AVPC. For theranostic (a merging of the terms therapeutic and diagnostic) studies, GRP78-targeting AAVP particles served to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which has a dual function as a molecular-genetic sensor/reporter and a cell suicide-inducing transgene. We observed specific and simultaneous PET imaging and treatment of tumors in this preclinical model of AVPC. Our findings demonstrate the feasibility of GPR78-targeting, ligand-directed theranostics for translational applications in AVPC.

Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes

Significance Inflammatory breast cancer (IBC) is defined clinically and pathologically. Dermal lymphatic invasion is typical but is neither necessary nor sufficient for diagnosis; sentinel lymph node biopsy is contraindicated, challenging multidisciplinary management with upfront chemotherapy, surgery, and postoperative radiotherapy. Here we applied a ligand-directed “theranostic” (a combination of therapeutic and diagnostic) enabling platform to target IBC based on adeno-associated virus/phage (AAVP)-Herpes simplex virus thymidine kinase type-1 (HSVtk) particles displaying ligands to cell surface-associated 78-kD glucose-regulated protein (GRP78). In a suite of preclinical models and human tumor samples, we show simultaneous noninvasive molecular serial PET/CT imaging and targeted suicide transgene therapy. This study shows that a tumor-specific promoter, human GRP78 (hGRP78), can drive the expression of an imaging/suicide transgene in IBC and aggressive breast cancer in vivo. Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.

Follicular lymphoma with bilateral testicular and epididymal involvement: Case report and review of the literature

Testicular involvement with indolent lymphoma is extremely rare, particularly in the absence of transformation to an aggressive histology. We report a case of a 64-year-old man who presented with cervical lymphadenopathy. Staging CT scans revealed extensive lymphadenopathy as well as bilateral testicular and epididymal masses. Histologic examination of lymph node, bone marrow, and testicular/epididymal biopsies revealed involvement with grade I follicular lymphoma. The patient was started on chemotherapy with cyclophosphamide, vincristine, prednisone, and rituximab in addition to intrathecal methotrexate and testicular radiation. He is now 6 months into therapy and responding well. A review of the literature demonstrated this to be the first confirmed case of testicular and epididymal involvement with grade I follicular lymphoma.

Abstract 4044: R-ketorolac targets Cdc42 and Rac1 GTPases and alters ovarian tumor cell behaviors critical for invasion and metastasis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Ovarian cancer (OvCa) is the 5th leading cause of cancer death in women in the US with a 5-year survival rate of 44.6%. About 70% patients are diagnosed at advanced stages with intraperitoneal dissemination, therefore identifying intracellular targets and developing effective molecules to reduce tumor metastasis have great significance for ovarian cancer therapy. Cdc42 and Rac1 are small Rho GTPase and function as molecular switches of actin reorganization which are crucial to tumor cell adhesion, migration and invasion. In previous work, our group found Cdc42 and the constitutively active Rac1b are overexpressed in primary ovarian tumor tissues and ovarian cancer cell lines. Lead identification through a high-throughput screen combined with a computational shape homology approach, identified R-ketorolac as a Cdc42 and Rac1 regulator, an activity that is distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. RhoA was unaffected by R-ketorolac. A bead-based flow cytometry assay identified R-ketorolac reduced Cdc42 and Rac1 GTPase nucleotide binding in vitro and inhibition was an allosteric mechanism of action. In cell-based assays, using Skov3ip cells, and in ascites-derived ovarian tumor cells, R-ketorolac was found to inhibit the activities of Cdc42 and Rac1 and their direct downstream effectors, the phosphorylation of p21-activated kinases (PAKs). R-ketorolac, but not S-ketorolac impeded Cdc42 mediated filopodia formation, measured based on both the length and numbers of filopodia in Skov3ip and primary OvCa cells. Cell behavior assays showed that R-ketorolac but not S-ketorolac strikingly inhibited cell adhesion, migration and invasion. In a xenograft mouse model a 50% reduction in tumor cell number and decreased total tumor burden was observed with R-ketorolac as compared to S-ketorolac treatment. Finally, in a ‘phase 0′ clinical study we found Cdc42 and Rac1 activities were reduced in a time-dependent manner after a single IV dose of racemic ketorolac treatment. In sum, we established R-ketorolac inhibition on Cdc42 and Rac1 activities and subsequent physiological consequences which are critical to tumor metastasis. Our findings provide the first demonstration of selective inhibition of Cdc42 and Rac1 GTPases by an FDA approved drug in humans. Citation Format: Yuna Guo, S. Ray Kenney, Larry A. Sklar, Tione Buranda, Tudor I. Oprea, Oleg Ursu, Sarah F. Adams, Teresa Rutledge, Carolyn Muller, Lesley Lomo, Laurie G. Hudson, Angela Wandinger-Ness. R-ketorolac targets Cdc42 and Rac1 GTPases and alters ovarian tumor cell behaviors critical for invasion and metastasis. [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 4044. doi:10.1158/1538-7445.AM2015-4044

Abstract P5-10-08: Estrogen receptor quantitative measures and differences in breast cancer survival

Introduction: In New Mexico, Hispanic women have a 1.7-fold increased risk of breast cancer-specific death compared to non-Hispanic white women. In previous studies, race/ethnic minority women have had larger survival disparities in estrogen receptor positive (ER+) than ER- disease, suggesting some aspect of ER may mediate survival outcomes. We thus conducted an extensive assessment of ER quantitative measures. Objective: To determine whether ER percent positive and intensity differs by ethnicity, and to evaluate whether that potential difference might account for a proportion of survival disparities. Methods: We conducted a population-based case-cohort study of first invasive breast cancer diagnosed in white females from 1997-2009 in six NM counties, identified through Surveillance Epidemiology End Results (SEER). We selected 15% of breast cancer cases and all breast cancer deaths through 2012. After IRB approval, pathology reports and tissue microarrays served as sources of ER, PR, and Her2 information. Tumors were classified according to modified intrinsic subtypes based on immunohistochemistry. Data were analyzed using Cox proportional hazards models adapted for case-cohort with weighted estimates (cohort weighted by 6.67x), and estimated hazard ratios (HR) and 95% confidence intervals (CI) using the robust variance and alpha=.05. The proportional hazards assumption was verified by Schoenfeld residuals. All analyses were adjusted for age. Interaction was assessed by inclusion of main effects and a product term (subtype* exposure). Results: ER and intrinsic subtype information was available for 76% of the cohort (867/1143) and 70% of breast cancer deaths (689/991). Median follow up was 7.8 years. In analyses stratified by intrinsic subtype, Hispanic women experienced elevated mortality relative to non-Hispanic whites for luminal A (HR 1.9;95% CI 1.4-2.6), Luminal B (HR 2.9;95% CI 1.5-5.7), and TN tumors (HR 1.9;95% CI 1.0-3.6) but not Her2+ER- disease (HR 1.1;95% 0.3-3.4). Overall ER Quantitative measures: Among ER+ women, breast cancer mortality decreased with increasing ER+ staining, measured by percent (p-trend=.004) or quartile (p-trend=.002). After adjustment for ER percent(ER%+), women with increased ER intensity (score>2) had reduced mortality, relative to score=1 (HR 0.6;95% CI 0.4-.1.0). Results did not differ by Luminal A or B subtype (p interaction> .05). Ethnicity-specific ER quantitative measures: ER%+ distribution did not differ by Hispanic ethnicity. However, among Hispanic women, interaction terms for ER%+ (p=.04) or quartile (p=.08) by subtype in relation to breast cancer survival suggest that Hispanic women with increasing ER staining have a reduced risk of mortality in Luminal A but not Luminal B tumors. Such differences were not evident among non-Hispanic white women. In multivariate models, inclusion of ER%+ and staining intensity did not alter Hispanic survival disparity overall, but mediated 8.6% in Luminal B. Conclusion: After inclusion of ER%+, ER staining intensity is an independent risk factor for breast cancer survival. Differences in ER quantitative measures appear to account for only a small proportion of survival disparities. Survival gaps in ER+ breast cancer may be attributable to host or other tumor factors. Introduction: In New Mexico, Hispanic women have a 1.7-fold increased risk of breast cancer-specific death compared to non-Hispanic white women. In previous studies, race/ethnic minority women have had larger survival disparities in estrogen receptor positive (ER+) than ER- disease, suggesting some aspect of ER may mediate survival outcomes. We thus conducted an extensive assessment of ER quantitative measures. Objective: To determine whether ER percent positive and intensity differs by ethnicity, and to evaluate whether that potential difference might account for a proportion of survival disparities. Methods: We conducted a population-based case-cohort study of first invasive breast cancer diagnosed in white females from 1997-2009 in six NM counties, identified through Surveillance Epidemiology End Results (SEER). We selected 15% of breast cancer cases and all breast cancer deaths through 2012. After IRB approval, pathology reports and tissue microarrays served as sources of ER, PR, and Her2 information. Tumors were classified according to modified intrinsic subtypes based on immunohistochemistry. Data were analyzed using Cox proportional hazards models adapted for case-cohort with weighted estimates (cohort weighted by 6.67x), and estimated hazard ratios (HR) and 95% confidence intervals (CI) using the robust variance and alpha=.05. The proportional hazards assumption was verified by Schoenfeld residuals. All analyses were adjusted for age. Interaction was assessed by inclusion of main effects and a product term (subtype* exposure). Results: ER and intrinsic subtype information was available for 76% of the cohort (867/1143) and 70% of breast cancer deaths (689/991). Median follow up was 7.8 years. In analyses stratified by intrinsic subtype, Hispanic women experienced elevated mortality relative to non-Hispanic whites for luminal A (HR 1.9;95% CI 1.4-2.6), Luminal B (HR 2.9;95% CI 1.5-5.7), and TN tumors (HR 1.9;95% CI 1.0-3.6) but not Her2+ER- disease (HR 1.1;95% 0.3-3.4). Overall ER Quantitative measures: Among ER+ women, breast cancer mortality decreased with increasing ER+ staining, measured by percent (p-trend=.004) or quartile (p-trend=.002). After adjustment for ER percent(ER%+), women with increased ER intensity (score>2) had reduced mortality, relative to score=1 (HR 0.6;95% CI 0.4-.1.0). Results did not differ by Luminal A or B subtype (p interaction> .05). Ethnicity-specific ER quantitative measures: ER%+ distribution did not differ by Hispanic ethnicity. However, among Hispanic women, interaction terms for ER%+ (p=.04) or quartile (p=.08) by subtype in relation to breast cancer survival suggest that Hispanic women with increasing ER staining have a reduced risk of mortality in Luminal A but not Luminal B tumors. Such differences were not evident among non-Hispanic white women. In multivariate models, inclusion of ER%+ and staining intensity did not alter Hispanic survival disparity overall, but mediated 8.6% in Luminal B. Conclusion: After inclusion of ER%+, ER staining intensity is an independent risk factor for breast cancer survival. Differences in ER quantitative measures appear to account for only a small proportion of survival disparities. Survival gaps in ER+ breast cancer may be attributable to host or other tumor factors. Citation Format: Hill D, Royce M, Lomo L, Barry M, Kang H, Wiggins C, Prossnitz E. Estrogen receptor quantitative measures and differences in breast cancer survival [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-10-08.