Faculty Bibliography

BACKGROUND:Patient derived xenografts (PDXs) represent an essential tool in oncologic research, and we sought to further expand our repertoire of head and neck squamous cell carcinoma (HNSCC) while determining potential boundaries for this system. METHODS:We consented new patients for PDX development and determined if a 24-h time delay from tumor excision to xenograft implantation affected PDX establishment. We developed a tissue microarray (TMA) from formalin fixed, paraffin embedded PDXs and their subsequent passages and carried out quantitative immunohistochemistry for EGFR, pEGFR, pAkt, pERK and ERCC1. First and last passaged PDXs were compared via a paired t-test to examine for the stability of protein expression across passages. We performed a similar comparison of the mutational profile of the patient tumor and resulting xenografts using a targeted sequencing approach. RESULTS:No patient/tumor characteristics influenced PDX take rate and the 24-h time delay from tumor excision to xenograft implantation did not affect PDX establishment, growth or histology. There was no significant difference in biomarker expression between the first and last passaged PDXs for EGFR, pEGFR, pAkt, and ERCC1. For pERK there was a significant difference (p=0.002), but further analysis demonstrated this only arose in three of 15 PDXs. Targeted sequencing revealed striking stability of passenger and likely driver mutations from patient to xenograft. CONCLUSIONS:The stability of protein expression across PDX passages will hopefully allow greater investigation of predictive biomarkers in order to identify ones for further pre-clinical and clinical investigation.

Head and neck squamous cell carcinoma (HNSCC) remains a challenging cancer to treat with overall 5-year survival on the order of 50-60%. Therefore, predictive biomarkers for this disease would be valuable to provide more effective and individualized therapeutic approaches for these patients. While prognostic biomarkers such as p16 expression correlate with outcome; to date, no predictive biomarkers have been clinically validated for HNSCC. We generated xenografts in immunocompromised mice from six established HNSCC cell lines and evaluated response to cisplatin, cetuximab, and radiation. Tissue microarrays were constructed from pre- and posttreatment tumor samples derived from each xenograft experiment. Quantitative immunohistochemistry was performed using a semiautomated imaging and analysis platform to determine the relative expression of five potential predictive biomarkers: epidermal growth factor receptor (EGFR), phospho-EGFR, phospho-Akt, phospho-ERK, and excision repair cross-complementation group 1 (ERCC1). Biomarker levels were compared between xenografts that were sensitive versus resistant to a specific therapy utilizing a two-sample t-test with equal standard deviations. Indeed the xenografts displayed heterogeneous responses to each treatment, and we linked a number of baseline biomarker levels to response. This included low ERCC1 being associated with cisplatin sensitivity, low phospho-Akt correlated with cetuximab sensitivity, and high total EGFR was related to radiation resistance. Overall, we developed a systematic approach to identifying predictive biomarkers and demonstrated several connections between biomarker levels and treatment response. Despite these promising initial results, this work requires additional preclinical validation, likely involving the use of patient-derived xenografts, prior to moving into the clinical realm for confirmation among patients with HNSCC.

Human melanoma contains multipotent stem cells that express the neural crest stem cell marker CD271. CD271-expressing melanoma cells in murine xenografts give rise to metastatic tumor. However, a comprehensive clinical investigation of its role in different stages of melanomagenesis has not been well studied. We studied CD271 expression with immunohistochemistry in 11 cases of banal melanocytic nevus, 9 cases of primary cutaneous melanoma, 10 cases of primary mucosal melanoma, 5 cases of metastatic melanoma in regional lymph nodes, and 11 cases of metastatic melanoma in the brain. In addition, 9 cases of metastatic, high-grade adenocarcinomas from breast and lung to the brain were studied as controls. The staining was scored based on the number of positive cells and analyzed by student t-test. All banal melanocytic nevi showed negative to equivocal staining. Primary cutaneous melanomas showed variable patterns, mucosal melanomas were mostly negative, and metastases to lymph nodes ranged from negative to moderate positivity. In contrast, all 11 cases of metastatic melanoma to the brain showed moderate (4 cases) to strong positivity (7 cases). Metastases from lung and breast origin were used as controls and showed negative to weakly positive staining in all but one case. Statistically, CD271 has significantly increased expression in metastatic melanoma to the brain when compared to the other groups studied (P < 0.05). The findings suggest that CD271 expression is specifically increased in metastatic melanoma to the brain. Further prospective study for the role of CD271 in prediction of melanoma brain metastasis as well as prognosis assessment will be of great clinical significance.

BACKGROUND: Patient derived xenografts (PDXs) for head and neck cancer (HNC) and other cancers represent powerful research platforms. Most groups implant patient tissue into immunodeficient mice immediately although the significance of this time interval is anecdotal. We tested the hypothesis that the time from tumor excision to implantation is crucial for PDX passaging and establishment. METHODS: We examined whether time or storage medium affected PDX viability for passaging two established HNC PDXs (UW-SCC34, UW-SCC52). Tumors were harvested, stored in ice-cold media or saline for 0-48 hours, and implanted into new mice. Tumor growth was compared by two-way ANOVA with respect to time and storage condition. Three new HNC PDXs (UW-SCC63-65) were generated by implanting patient tissue into mice immediately (Time 0) and 24 hours after receiving tissue from the operating room. RESULTS: Similar quantities of tumor were implanted into each mouse. At the end of the experiment, no significant difference was seen in mean tumor weight between the media and saline storage conditions for UW-SCC34 or UW-SCC52 (p = 0.650 and p = 0.177, respectively). No difference in tumor formation prevalence was seen on the basis of time from harvest to implantation (>/=13 of 16 tumors grew at every time point). Histological analysis showed strong similarity to the initial tumor across all groups. Tumors developed at both Time 0 and 24 hours for UW-SCC63 and UW-SCC64. CONCLUSIONS: We demonstrated that neither storage medium nor time from tumor excision to implantation (up to 48 hours) affected viability or histological differentiation in a subsequent passage for two HNC PDXs. Moreover, we revealed that fresh patient tissue is viable up to 24 hours post-resection. This information is important as it applies to the development and sharing of PDXs.

PURPOSE: To describe the presentation and fertility sparing treatment of a young woman found to have a steroid cell tumor not otherwise specified (NOS) and her spontaneous pregnancy and delivery shortly after surgery. METHODS: A 20-year-old Hispanic female presented with hirsuitism, virilization, and elevated androgen levels (testosterone 328 ng/dL) and was wrongly diagnosed with polycystic ovarian syndrome. Four months later she sought a second opinion. Her androgens were as follows: testosterone level 485 ng/dL, androstenedione 1,738 ng/dL and DHEA 1,459 ng/dL. She had normal levels of progesterone, estradiol, and DHEA-SO4. On transvaginal ultrasound she had a solid-appearing right ovarian mass. She underwent fertility sparing surgery with a laparoscopic right oophorectomy. RESULTS: Gross and histological pathology confirmed a benign steroid cell tumor NOS. She had rapid normalization of all androgens 13 days after surgery. She had spontaneous resumption of menses 4 months later. She conceived despite using emergency contraception approximately 9 months following surgery and delivered a healthy boy at term without complication. CONCLUSION: Prompt evaluation for an androgen producing tumor should be performed when testosterone levels are greater than 200 ng/dL. Pregnancy following removal of this rare tumor has not previously been reported.