<p>Supplementary Figure S9: Human TNBC cell lines MDA-MB-231 and MDA-MB-468 were injected in the T4 mammary fat pad of 12 week old nude mice. When tumors reached size of 100mm3, mice were randomized in Vehicle and AZD9668 100mg/kg groups. Mice were then treated daily B.I.D until the endpoint of Vehicle tumors reaching 2000mm3. (A, B) Tumor growth curves of mice treated with either Vehicle or AZD9668 100mg/kg in the MDA-MB-231 and MDA- MB-468 models respectively. Endpoint tumors harvested were subject to BrdU staining. (C, D) Representative images BrdU staining and (E, F) their corresponding quantitation in the MDA- MB-231 and MDA-MB-468 models respectively. (G) Spontaneous tumors isolated from PyMT NE+/+ were implanted in FVB NE+/+ mice. When tumors reached a measurable size of 30- 50mm3, mice were randomized into Vehicle and AZD9668 100mg/kg group and treated B.I.D until 400-500mm3. Tumor growth curves, as a function of time are shown.</p>
<p>Supplementary Figure S7: Recurrence-free survival (A) and overall survival (C) for each individual gene used in the METABRIC patient cohort signature and progression-free survival (B) and overall survival (D) used in the and TCGA patient cohort signature.</p>
Salivary gland cancers (SGCs) are rare yet aggressive malignancies with significant histological heterogeneity, which has made prediction of prognosis and development of targeted therapies challenging. In majority of patients, local recurrence and/or distant metastasis are common and systemic treatments have minimal impact on survival. Therefore, identification of novel targets for treatment that can also be used as predictors of recurrence for multiple histological subtypes of SGCs is an area of unmet need. In this study, we developed a novel transgenic mouse model of SGC, efficiently recapitulating the major histological subtype (adenocarcinomas of the parotid gland) of human SGC. CDK2 knock out (KO) mice crossed with MMTV-low molecular weight forms of cyclin E (LMW-E) mice generated the transgenic mouse models of SGC, which arise in the parotid region of the salivary gland, similar to the common site of origin seen in human SGCs. To identify the CDK2 independent catalytic partner(s) of LMW-E, we used LMW-E expressing cell lines in mass spectrometric analysis and subsequent biochemical validation in pull down assays. These studies revealed that in the absence of CDK2, LMW-E preferentially binds to CDK5. Molecular targeting of CDK5, using siRNA, resulted in inhibition of cell proliferation of human SGCs overexpressing LMW-E. We also provide clinical evidence of significant association of LMW-E/CDK5 co-expression and decreased recurrence free survival in human SGC. Immunohistochemical analysis of LMW-E and CDK5 in 424 patients representing each of the four major histological subtypes of human salivary cancers (Aci, AdCC, MEC, and SDC) revealed that LMW-E and CDK5 are concordantly (positive/positive or negative/negative) expressed in 70% of these patients. The co-expression of LMW-E/CDK5 (both positive) robustly predicts the likelihood of recurrence, regardless of the histological classification of these tumors. Collectively, our results suggest that CDK5 is a novel and targetable biomarker for the treatment of patients with SGC presenting with LMW-E overexpressing tumors.
Abstract Objectives Actionable, solid tumor activating neurotrophic receptor tyrosine kinase ( NTRK ) fusions are best detected via nucleic acid-based assays, while Pan-TRK immunohistochemistry (IHC) serves as a reasonable screening modality. We describe a practical and cost-effective approach to validate pan-TRK and discuss challenges that may be encountered. Methods Pan-TRK Clone EPR17341 was validated in accordance with the 2014 consensus statements set forth by the College of American Pathologists. Confirmation of IHC results were guided by the European Society of Medical Oncology recommendations for standard methods to detect NTRK fusions. Results Within 36 samples, ETV6-NTRK3 (n = 8) and TPM4-NTRK3 (n = 1) fusions were confirmed. ETV6-NTRK3 fusion positive cases revealed cytoplasmic and nuclear staining. A TPM4-NTRK3 fusion positive high grade malignant peripheral nerve sheath tumor revealed diffuse cytoplasmic staining. A high grade ovarian serous carcinoma revealed focal punctate staining and revealed a non-actionable NTRK1 truncation at intron 2. Diffuse cytoplasmic staining was observed in a case of fusion-negative polymorphous adenocarcinoma. Wild-type expression of TRK in pulmonary meningothelial-like nodules was discovered following a false-positive IHC interpretation. Conclusion Pan-TRK IHC shows some utility as a diagnostic and surrogate marker for NTRK screening however, physiologic or non-specific expression may lead to false-positive results.
