Farah Khalil

Annotation of human cancers with EGFR signaling-associated protein complexes using proximity ligation assays

A method for detecting receptor tyrosine kinase signaling in patient tumor samples may improve diagnosis and therapy. Visualizing Protein Complexes in Clinical Samples Aberrant activation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), plays a role in cancer initiation, progression, and acquired drug resistance. Genetic analysis may not always reveal aberrant activity of receptor tyrosine kinase signaling; thus, detecting active signaling through these kinases in clinical samples should improve prognostication and personalization of therapies. Diverse cancers, for example, those found in the lung, colon, or head and neck, can have aberrant activation of EGFR signaling, and EGFR-targeted therapies are used to treat these diseases. Smith et al. developed a proximity ligation assay (PLA) to detect the interaction between EGFR and the requisite signaling adaptor GRB2 (growth factor receptor–bound protein 2) in common clinical preparations. EGFR:GRB2 PLA recapitulated traditional readouts of active EGFR signaling in cultured cells, in a panel of tumor xenografts in mice derived from primary patient samples, and in samples from three large cohorts of human patients. Moreover, EGFR:GRB2 PLA detected increased EGFR signaling in tumors with wild-type EGFR, which would have been undetectable by genetic analysis, and was predictive of therapeutic response to EGFR inhibitors in both mice and humans. Thus, using PLA to detect signaling-associated protein complexes has the potential to improve current diagnostic measures. Strategies to measure functional signaling-associated protein complexes have the potential to augment current molecular biomarker assays, such as genotyping and expression profiling, used to annotate diseases. Aberrant activation of epidermal growth factor receptor (EGFR) signaling contributes to diverse cancers. We used a proximity ligation assay (PLA) to detect EGFR in a complex with growth factor receptor–bound protein 2 (GRB2), the major signaling adaptor for EGFR. We used multiple lung cancer cell lines to develop and characterize EGFR:GRB2 PLA and correlated this assay with established biochemical measures of EGFR signaling. In a panel of patient-derived xenografts in mice, the intensity of EGFR:GRB2 PLA correlated with the reduction in tumor size in response to the EGFR inhibitor cetuximab. In tumor biopsies from three cohorts of lung cancer patients, positive EGFR:GRB2 PLA was observed in patients with and without EGFR mutations, and the intensity of EGFR:GRB2 PLA was predictive of overall survival in an EGFR inhibitor–treated cohort. Thus, we established the feasibility of using PLA to measure EGFR signaling–associated protein complexes in patient-based materials, suggesting the potential for similar assays for a broader array of receptor tyrosine kinases and other key signaling molecules.

Immunohistochemical expression of survivin in cutaneous sebaceous lesions.

Background:Survivin is a member of the inhibitor of apoptosis family of proteins implicated in the inhibition of apoptosis and cell cycle control, both crucial in the progression to malignancy. Survivin overexpression has been demonstrated in numerous malignancies including cutaneous squamous cell carcinoma and melanoma. To date, there are no studies evaluating the expression of survivin in sebaceous neoplasms. Methods:Immunohistochemical expression of survivin was evaluated in a total of 20 extraocular sebaceous neoplasms: sebaceous hyperplasia (SH, 8), sebaceous adenoma (SA, 8), and sebaceous carcinoma (SC, 4). All the results were independently evaluated by a single dermatopathologist. Results:Nuclear expression of survivin was present in 1.4% of lesional SH cells, 8.2% of SA cells, and 12.5% of SC cells. A significant difference in survivin expression with the Student t test was noted between SH and SA (P = 0.01), SA and SC (P = 0.05), and SH and SC (P = 0.001). Conclusions:There is a statistically significant difference in survivin expression among SH, SA, and SC. These findings demonstrate the potential diagnostic utility of survivin, further assisting in the microscopic differentiation of benign and malignant sebaceous neoplasms. However, larger studies are needed to determine the significance of survivin expression as it relates to recurrence, metastatic potential, and outcome.

Verrucous psoriasis : A distinctive clinicopathologic variant of psoriasis

Psoriasis is capable of presenting in a variety of clinical and pathologic guises including a rarely described variant variably termed hypertrophic or verrucous psoriasis. Herein, we describe the clinical and pathologic attributes of a large series of patients with this unusual variant of psoriasis and distinguish it from other entities in the differential diagnosis. The histopathologic features and clinical and demographic attributes of a series of 12 cases from 12 patients were reviewed by a single dermatopathologist (MM). The 12 patients consisted of 7 males and 5 females with an average age of 61.8 years (males 38-93 years, females 41-71 years). Eight of the patients were Caucasian, 3 Hispanic and 1 African-American. Six of the lesions were located on the knees, 4 involved the elbows, and 2 were seen on the dorsum of the hands (metacarpal-phalangeal joint). The clinical appearance of the lesions consisted of flesh-toned to white mammillated plaques (8 cases) and coalesced papules (4 cases). Each of the biopsies showed regular psoriasiform epidermal hyperplasia with acanthosis, hyperkeratosis, and either spongiform neutrophilic or Munro micro-abscesses. In addition, each showed papillomatosis with bowing of the peripheral rete ridges toward the center of the lesion (buttressing). At high power, epidermal neutrophils were seen in particular surmounting the tips of the suprapapillary plates with accompanying serum. Hypergranulosis and koilocytic change were not observed in any of the lesions. Human papilloma virus (HPV) immunostaining and periodic acid Schiff (PAS) special staining for fungi were negative. Verrucous psoriasis is a distinctive variant of psoriasis with overlapping clinical and pathologic features that might prompt consideration of verruca vulgaris. The presence of epidermal papillomatosis and epidermal buttressing seen in these lesions is reminiscent of the histologic features of verruca vulgaris. Similarly, the presence of coalesced papules might prompt clinical consideration of verruca vulgaris as well. It is likely that this under recognized clinicopathologic entity represents a patterned response of the epithelium to repeated trauma/irritation typical of the anatomic locations that were encountered in this series. Recognition of this entity should preempt confusion with verruca vulgaris or other entities capable of producing wart-like epidermal changes.

Evaluation of invasive breast cancer samples using a 12-chemokine gene expression score: correlation with clinical outcomes

BackgroundA unique 12-chemokine gene expression score (CS) accurately predicted the presence of tumor-localized, ectopic lymph node-like structures (TL-ELNs) and improved overall survival (OS) in primary colorectal cancer and metastatic melanoma. We analyzed the correlation between CS, clinicopathological variables, molecular data, and 366 survival in Moffitt Cancer Center’s Total Cancer Care (TCC) patients with non-metastatic breast cancer.MethodsAffymetrix gene expression profiles were used to interrogate the CS by the principal component method. Breast tumors were classified as high or low score based on median split, and correlations between clinicopathologic variables, PAM50 molecular subtype, and ELN formation were analyzed using the TCC dataset. Differences in overall survival (OS) and recurrence-free survival (RFS) in the larger KM Plot breast cancer public datasets were compared using Kaplan-Meier curves.ResultsWe divided the Total Cancer Care (TCC) breast cancer patients into two groups of high or low CS. Mean CS was 0.24 (range, 2.2–2.1). Patients with higher CS were more likely to be white (172 vs. 159; p = 0.03), had poorly differentiated tumors (112 vs. 59; p <0.0001), ER/PR negative (41 vs. 26) and HER2 positive (36 vs. 19; p = 0.001), and contain TL-ELNs. Higher CS scores were also seen in the basal and HER2+ molecular subtypes. In the KM Plot breast cancer datasets higher CS patients demonstrated superior OS (HR = 0.73, p = 0.008) and RFS (HR 0.76, p = <0.0001), especially in basal and HER2+ patients.ConclusionsHigh CS breast tumors tend to be higher grade, basal or HER2+, and present more frequently in Caucasians. However, this group of patients also shows the presence of TL-ELNs within the tumor microenvironment and has better survival outcomes. The CS is a novel tool that can identify breast cancer patients with tumors of a unique intratumoral immune composition and better prognosis. Whether or not the CS is a predictive response marker in breast cancer patients undergoing immunotherapy remains to be determined.

Expression of insulin-like growth factor-I receptor in primary cutaneous carcinomas

Background:  Insulin‐like growth factor‐I (IGF‐I) is the principal mediator of growth hormone, exerting its effects through binding of the insulin‐like growth factor‐I receptor (IGF‐IR). Post‐receptor activation leads to the production of transcription factors involved in cell proliferation, differentiation, transformation, and survival. Data indicate that IGF‐IR is involved in tumorigenesis. To our knowledge, this receptor has not been previously studied in primary cutaneous carcinomas.

Hamartin and tuberin immunohistochemical expression in cutaneous fibroepithelial polyps

Background:  Hamartin and tuberin are inactivating tumor suppressor proteins implicated in the development of gastrointestinal polyps and sporadic and tuberous sclerosis‐associated cutaneous angiofibromas. The pattern of expression of these peptides has not been studied in fibroepithelial polyps (FEPs).

Cell-surface marker discovery for lung cancer

Lung cancer is the leading cause of cancer deaths in the United States. Novel lung cancer targeted therapeutic and molecular imaging agents are needed to improve outcomes and enable personalized care. Since these agents typically cannot cross the plasma membrane while carrying cytotoxic payload or imaging contrast, discovery of cell-surface targets is a necessary initial step. Herein, we report the discovery and characterization of lung cancer cell-surface markers for use in development of targeted agents. To identify putative cell-surface markers, existing microarray gene expression data from patient specimens were analyzed to select markers with differential expression in lung cancer compared to normal lung. Greater than 200 putative cell-surface markers were identified as being overexpressed in lung cancers. Ten cell-surface markers (CA9, CA12, CXorf61, DSG3, FAT2, GPR87, KISS1R, LYPD3, SLC7A11 and TMPRSS4) were selected based on differential mRNA expression in lung tumors vs. non-neoplastic lung samples and other normal tissues, and other considerations involving known biology and targeting moieties. Protein expression was confirmed by immunohistochemistry (IHC) staining and scoring of patient tumor and normal tissue samples. As further validation, marker expression was determined in lung cancer cell lines using microarray data and Kaplan–Meier survival analyses were performed for each of the markers using patient clinical data. High expression for six of the markers (CA9, CA12, CXorf61, GPR87, LYPD3, and SLC7A11) was significantly associated with worse survival. These markers should be useful for the development of novel targeted imaging probes or therapeutics for use in personalized care of lung cancer patients.

Bif-1 and Bax expression in cutaneous Merkel cell carcinoma

Background:  Bax‐interacting factor‐1 (Bif‐1) binds to Bax, which in turn activates this proapoptotic protein. In the absence of Bif‐1, the ability to induce apoptosis through the intrinsic pathway is greatly reduced. Merkel cell carcinoma (MCC) classically shows an aggressive behavior and lack of response to chemotherapy, which remains unexplained. Previous studies have documented the presence of Bax in MCC, but Bif‐1 expression has not been evaluated. Herein, the expression of Bif‐1 and Bax in cutaneous MCC is examined.

Advances in EGFR as a Predictive Marker in Lung Adenocarcinoma.

BACKGROUND Worldwide, lung cancer is the most common cause of mortality. Toxins from tobacco smoke are known to increase the risk of lung cancer; however, up to 15% of lung cancer-related deaths in men and up to 50% of lung cancer-related deaths in women occur in people who do not smoke. Despite the fact that chemotherapy generally provides a survival benefit for non-small-cell lung cancer, not every patient will respond to therapy and many experience therapy-related adverse events. Thus, predictive markers are used to determine which patients are more likely to respond to a given regimen. METHODS We reviewed the current medical literature in English relating to predictive markers that may be positive, such as the presence of an activating EGFR mutation. RESULTS The advances in using EGFR as a molecular predictive marker were summarized. This biomarker influences therapeutic response in patients with lung adenocarcinoma. Clinical evidence supporting its value is also reviewed. CONCLUSIONS The use of EGFR as a predictive factor in lung adenocarcinoma may help target therapy to individual tumors to achieve the best likelihood for long-term survival and to avoid adverse events from medications unlikely to be effective.

NUT Midline Carcinoma: A Rare Malignancy.

Nuclear protein of the testis (NUT) midline carcinoma can present in the head, neck, and mediastinum. In general, it presents in young adult men and has a poor prognosis. We report on a case of NUT midline carcinoma of the mediastinum in a man 27 years of age without any prior malignancy. Due to the location of the tumor, mediastinal lymphoma and germ cell tumor were initially considered; however, immunohistochemistry was performed using NUT antibody that revealed it to be NUT midline carcinoma. Although guidelines exist for squamous cell carcinoma of the head, neck, and mediastinum, no such specific guidelines are available for NUT midline carcinoma, which looks morphologically similar to squamous cell carcinoma but behaves more aggressively and carries a poor prognosis.