Fabiola Cecchi

Transdermal Delivery of Heparin Using Pulsed Current Iontophoresis

PurposeIn clinical practice heparin has to be administered by injection with obvious disadvantages; thus, transdermal delivery by electrically assisted methods have been studied. In this study we evaluated the efficacy of a Food and Drug Administration-approved pulsed current iontophoresis system in delivering heparin through living rat skin.MethodsFluorescent and radioactive heparin as well as a commercial heparin preparation were delivered through rat skin via a pulsed current iontophoresis system.ResultsPulsed current iontophoresis allowed fluorescent heparin to cross the stratum corneum localizing in epidermis and dermis. Unfractionated, high-, and low molecular weight fraction pools, obtained by fractionating [35S]-unfractionated heparin on a molecular weight sieve, were then separately tested. Pulsed current iontophoresis elicited the transdermal delivery of low molecular weight heparin, but not that of high molecular weight heparin. Finally, pulsed current iontophoresis of an unfractionated pharmaceutical heparin preparation significantly decreased plasmatic factor Xa activity.ConclusionsWe hypothesize that this technique could be used to administer low molecular weight heparin in a cost-efficient and safe manner without the need for syringes and needles.

Preclinical and correlative studies of cabozantinib (XL184) in urothelial cancer (UC).

314 Background: Mounting evidence supports Met as a target in urothelial cancer (UC). Activated Met can promote angiogenesis and tumor growth by upregulating VEGF and may play a role in UC pathogenesis. Cabozantinib inhibits both VEGFR2 and Met pathways. In this study, we assessed shed Met (sMet) levels in the urine and serum of UC patients (pts) and cabozantinibs effects on HGF-driven UC cell growth and invasion. METHODS sMet levels in serum and urine samples from 31 pts with UC (23 metastatic, 8 muscle-invasive) were correlated with stage, presence of visceral metastases and urinary source. The effects of cabozantinib on 4 human UC-derived cell lines were studied in vitro. Intact RT4, TCC-SUP, T24M2 and T24M3 cells at 80% confluence were serum deprived 16 h, then left untreated or treated with hepatocyte growth factor (HGF) and/or cabozantinib prior to analysis of Met, phospho- (p)Met, pAkt, Akt, pMAPK and MAPK by immunoassay or immunoblotting. Cabozantinib effects on basal and HGF-induced UC cell invasion, proliferation and soft agar growth were measured. RESULTS Median serum Met levels were modestly higher in pts with metastatic versus muscle-invasive disease. Urinary Met levels were clearly higher in pts with visceral metastasis (p=0.0111) and in urine from ileal conduits and neobladders compared to normally voided urine, regardless of stage (p=0.0489). Met content in UC cell lines was low in RT4 and higher in T24M2, T24M3 and TCC-SUP. Basal pMet content was universally low, increased significantly by HGF and this was reversed by cabozantinib. HGF-driven increases in pAkt/Akt and pMAPK/MAPK in all 4 cell lines were reversed by cabozantinib, as were HGF-enhanced UC cell invasion, proliferation and anchorage independent growth. CONCLUSIONS Median urinary sMet is significantly higher in pts with visceral metastasis and in specimens from ileal conduits and neobladders relative to normally voided urine. UC cell Met content in culture increased with disease grade; HGF stimulated activation of Met and known effectors, and enhanced invasion, growth rate and anchorage-independent growth; cabozantinib effectively reversed these HGF-driven effects. These data support evaluation of cabozantinib in pts with metastatic UC.

Targeted Therapies for Targeted Populations: Anti-EGFR Treatment for EGFR-Amplified Gastroesophageal Adenocarcinoma

Previous anti-EGFR trials in unselected patients with gastroesophageal adenocarcinoma (GEA) were resoundingly negative. We identified EGFR amplification in 5% (19/363) of patients at the University of Chicago, including 6% (8/140) who were prospectively screened with intention-to-treat using anti-EGFR therapy. Seven patients received ≥1 dose of treatment: three first-line FOLFOX plus ABT-806, one second-line FOLFIRI plus cetuximab, and three third/fourth-line cetuximab alone. Treatment achieved objective response in 58% (4/7) and disease control in 100% (7/7) with a median progression-free survival of 10 months. Pretreatment and posttreatment tumor next-generation sequencing (NGS), serial plasma circulating tumor DNA (ctDNA) NGS, and tumor IHC/FISH for EGFR revealed preexisting and/or acquired genomic events, including EGFR-negative clones, PTEN deletion, KRAS amplification/mutation, NRAS, MYC, and HER2 amplification, and GNAS mutations serving as mechanisms of resistance. Two evaluable patients demonstrated interval increase of CD3+ infiltrate, including one who demonstrated increased NKp46+, and PD-L1 IHC expression from baseline, suggesting an immune therapeutic mechanism of action. EGFR amplification predicted benefit from anti-EGFR therapy, albeit until various resistance mechanisms emerged.Significance: This paper highlights the role of EGFR inhibitors in EGFR-amplified GEA-despite negative results in prior unselected phase III trials. Using serial ctDNA and tissue NGS, we identified mechanisms of primary and acquired resistance in all patients, as well as potential contribution of antibody-dependent cell-mediated cytotoxicity to their clinical benefit. Cancer Discov; 8(6); 696-713. ©2018 AACR.See related commentary by Strickler, p. 679This article is highlighted in the In This Issue feature, p. 663.

Targeting the hepatocyte growth factor/Met pathway in cancer

Hepatocyte growth factor (HGF)-induced activation of its cell surface receptor, the Met tyrosine kinase, drives mitogenesis, motogenesis and morphogenesis in a wide spectrum of target cell types and embryologic, developmental and homeostatic contexts. Typical paracrine HGF/Met signaling is regulated by HGF activation at target cell surfaces, HGF binding-induced receptor activation, internalization and degradation. Despite these controls, HGF/Met signaling contributes to oncogenesis, tumor angiogenesis and invasiveness, and tumor metastasis in many types of cancer, leading to the rapid growth of pathway-targeted anticancer drug development programs. We review here HGF and Met structure and function, basic properties of HGF/Met pathway antagonists now in clinical development, and recent clinical trial results. Presently, the main challenges facing the effective use of HGF/Met-targeted antagonists for cancer treatment include optimal patient selection, diagnostic and pharmacodynamic biomarker development, and the identification and testing of effective therapy combinations. The wealth of basic information, analytical reagents and model systems available regarding normal and oncogenic HGF/Met signaling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective cancer treatment.

Heparin/heparan sulfate anticoagulant glycosaminoglycans in human plasma of healthy donors: Preliminary study on a small group of recruits

Glycosaminoglycans in normal human plasma, mainly represented by chondroitin sulfates and heparan sulfates/heparin (HSGAGs), show a specific distribution in the Cohn–Oncley fractions of human plasma. In the present study we investigated their effects on coagulation. Plasma was fractionated following the procedure of Cohn–Oncley, and each fraction was treated for extraction of glycosaminoglycans after extensive proteolysis; the anticoagulant activity in the extracted samples was measured by activated partial thromboplastin time (APTT). The effects of the samples containing HSGAGs on factor II and factor X activities, before and after treatment with heparinase I, were also measured. The molecular weight of HSGAGs was determined by polyacrylamide gel-electrophoresis. Cryoprecipitate and fraction I, fraction II+III, and fraction IV-1 (the fractions containing HSGAGs) prolonged the APTT, whereas fractions IV-4 and V had no effect on the APTT. Fractions containing HSGAGs showed effects on factor II and factor X activities that were sensitive to heparinase I treatment. The molecular weight of HSGAGs recovered in cryoprecipitate and fraction I was 15–18 kDa; that of HSGAGs recovered in fraction IV-1 was 12.0 kDa. In conclusion, these results demonstrate that HSGAGs of different molecular weight, endowed with anticoagulant activity, circulate in normal human plasma in association with specific proteins involved in the regulation of hemostasis; and that endogenous HSGAGs play a role in maintaining the antithrombotic/hemostatic balance in normal human plasma.

Imaging the Met Receptor Tyrosine Kinase (Met) and Assessing Tumor Responses to a Met Tyrosine Kinase Inhibitor in Human Xenograft Mouse Models with a [99mTc] (AH-113018) or CY 5** (AH-112543) Labeled Peptide

Developing an imaging agent targeting the hepatocyte growth factor receptor protein (Met) status of cancerous lesions would aid in the diagnosis and monitoring of Met-targeted tyrosine kinase inhibitors (TKIS). A peptide targeting Met labeled with [99mTc] had high affinity in vitro (Kd = 3.3 nM) and detected relative changes in Met in human cancer cell lines. In vivo [99mTc]-Met peptide (AH-113018) was retained in Met-expressing tumors, and high-expressing Met tumors (MKN-45) were easily visualized and quantitated using singlephoton emission computed tomography or optical imaging. In further studies, MKN-45 mouse xenografts treated with PHA 665752 (Met TKI) or vehicle were monitored weekly for tumor responses by [99mTc]-Met peptide imaging and measurement of tumor volumes. Tumor uptake of [“mTc]-Met peptide was significantly decreased as early as 1 week after PHA 665752 treatment, corresponding to decreases in tumor volumes. These results were comparable to Cy5**-Met peptide (AH-112543) fluorescence imaging using the same treatment model. [99mTc] or Cy5**-Met peptide tumor uptake was further validated by histologic (necrosis, apoptosis) and immunoassay (total Met, p Met, and plasma shed Met) assessments in imaged and nonimaged cohorts. These data suggest that [99mTc] or Cy5**-Met peptide imaging may have clinical diagnostic, prognostic, and therapeutic monitoring applications.

Proteolysis of human plasma reveals the presence of complexes formed by endogenous heparin and peptides that stimulate angiogenesis

Human endogenous plasma heparin associates with proteins that mask its anticoagulant activity. This association persists after exhaustive proteolysis of plasma, and resulting peptide/heparin complexes have no anticoagulant activity. Looking for functions other than inhibition of coagulation, we considered that commercial preparations of heparin from bovine or porcine sources show alternative effects on angiogenesis, either stimulating or inhibiting the process. However, the effects of endogenous human heparin on angiogenesis are unknown. In this study, the fraction of plasma containing endogenous heparin was prepared by means of exhaustive proteolysis, either in the presence or in the absence of 35S-labeled heparin. Plasma from healthy donors was digested and the supernatant was precipitated with 66% ethanol, dialyzed, and submitted to basic and acidic ion-exchange chromatography. 35S-Labeled heparin as well as endogenous heparin bound plasmatic peptides, forming acidic, basic, and neutral complexes. Binding of peptides, eluting from both resins, impaired migration of heparin on cellulose acetate electrophoresis. Endogenous neutral complexes (i.e., those formed by human endogenous plasma heparin and peptides) were tested for angiogenic activity in chick embryo chorioallantoic membrane assay. Bovine heparin induced a moderate angiogenic response. Neutral complexes of human endogenous plasma heparin and basic plasma peptides induced a very strong angiogenic response. Treatment of neutral complexes with nitrous acid, which degrades heparin, abolished the angiogenic effect, thus demonstrating that it was due to the presence of heparin. These results demonstrate that proteolysis of human plasma generates angiogenic peptide/heparin complexes.

Personalized therapy based on sequential molecular analysis leads to 30 months of survival in a patient with diffuse unresectable gastric linitis plastica

Introduction: Diffuse gastric cancer is associated with poor prognosis. We report a patient with metastatic gastric linitis plastica harboring human epidermal growth factor receptor 2 (HER2) activating mutation and HER2 amplification. Case description: The patient received 5-fluorouracil/folinic acid and oxaliplatin combined with trastuzumab/pertuzumab, resulting in disease control for 8 months. Second-line therapy with nivolumab and trastuzumab/pertuzumab was well-tolerated, with macroscopic peritoneal response. Following ovarian progression and surgical resection of ovarian metastases, immunohistochemistry of PD-L1 was negative; proteomics demonstrated normal expression of HER2 and absence of PD-L1, while genomics showed HER2 amplification, suggesting mechanisms of escape to dual HER2 blockade by downregulation of HER2 and to nivolumab by the absence of PD-L1. Based upon this and nonexpression of biomarkers of taxane resistance, therapy was changed to paclitaxel. Two and a half years after diagnosis, the patient is undergoing treatment, with excellent performance status. Conclusions: Molecular analysis and personalized therapy can help optimize treatment in difficult-to-treat cancers.

Targeted data-independent acquisition for mass spectrometric detection of RAS mutations in formalin-fixed, paraffin-embedded tumor biopsies

Genomic testing for KRAS and NRAS mutations in clinical biopsies of various cancers is routinely performed to predict futility of anti-epidermal growth factor receptor (anti-EGFR) therapies. We hypothesized that RAS mutations could be detected and quantified at the protein level for diagnostic purposes using data-independent acquisition (DIA)-based mass spectrometry in formalin-fixed, paraffin-embedded (FFPE) tumor samples. We developed a targeted DIA assay that surveys the specific mass range of all possible peptides harboring activating mutations in KRAS exon 2. When the assay was applied to tumor samples with known KRAS or NRAS mutations (G12A, G12D, G12V, and G13D), RAS-mutant and wild-type peptides were successfully detected in 11 of 13 biopsy samples. Mutation statuses obtained by DIA were concordant with those obtained by DNA sequencing, and yields of mutant peptide (mutant peptide/[mutant + wild-type peptides]) exhibited linear correlation with yields of RAS-mutant mRNA. When applied to biopsy samples with failed DNA testing results, the DIA assay identified an additional RAS-mutated sample. SIGNIFICANCE: Proteomic detection of RAS mutations by DIA in tumor biopsies can provide solid evidence of mutant RAS protein regardless of the mutation types and sites in exon 2. This robust method could rescue samples that fail genomic testing due to insufficient tumor tissue or lack of sequenceable DNA. It can be used to explore the relationship between protein expression level of mutant RAS and therapeutic outcome.

Abstract 735: HER2 protein quantification in multiple cancer indications identifies candidates for HER2 targeted therapies

Background: Anti-HER2 therapy in cancer indications other than breast and gastric cancers is the subject of ongoing clinical trials. We previously used mass spectrometry to identify HER2 protein expression level cutoffs that correlate with standard measures of HER2 positivity (740 and 750 amol/µg in breast and gastric cancers, respectively). These studies also demonstrated that trastuzumab-treated patients whose tumors express high levels of HER2 (≥ 2200 and ≥ 1825 amol/µg in breast and gastric cancers, respectively) survived much longer than patients with lower HER2 levels. We hypothesized that targeted proteomics would reveal high HER2 levels in indications other than breast and gastric cancers, thus identifying patients likely to benefit from anti-HER2 therapy. Methods: We summarized results from samples processed in our CAP/CLIA-certified laboratory. Tumor areas from FFPE tissue blocks (N=3828) representing multiple cancer indications were marked by a pathologist, microdissected, and solubilized to tryptic peptides. In each liquefied tumor sample, HER2 and other protein targets were quantified with mass spectrometry-based proteomic analysis. Results: HER2 superexpression (> 2200 amol/µg) was found in 0.64% (12/1891) of patients with non-breast, non-gastroesophageal cancers. Among indications with > 50 patients tested, the highest rates of HER2 superexpression were in gynecological cancers (3/124; 2.4%) and bladder cancer (1/51; 2.0%). Treatment and outcome data are largely unavailable, but we are aware of 3 anecdotes: In a 74-year-old male with invasive adenocarcinoma of the gallbladder who had disease progression on gemcitabine + cisplatin, proteomic testing found high HER2 protein expression (3105 amol/µg). The patient responded to trastuzumab + FOLFIRI for 5 months. In a uterine cancer patient whose HER2 status was equivocal by genomic analysis, proteomics found high HER2 expression (4995 amol/µg). Proteomics also revealed the absence of a resistance marker for taxane (TUBB3) and high levels of the response marker for antifolate agents (FRalpha = 10500 amol/µg). The patient responded to trastuzumab + taxol for 9 months before developing resistance and responding to trastuzumab + antifolate. Lastly, a cervical cancer patient whose disease had progressed on chemotherapy showed HER2 superexpression (11322 amol/µg). She was treated with anti-HER2 combinations for > 12 months. Conclusions: Patients with high HER2 protein expression as measured by targeted mass spectrometry in multiple cancer types have benefitted from anti-HER2 therapy. Only small numbers of patients with non-breast, non-gastric tumors have HER2 protein levels indicative of survival benefit from anti-HER2 therapy. However, multiplexed targeted proteomics offers simultaneous, precise quantification of other biomarkers (eg, ERCC1, TUBB3, FRalpha) to guide therapy selection for multiple cancer types. Citation Format: Shankar Sellappan, Sarit Schwartz, Ellen Wertheimer, Fabiola Cecchi, Steven W. Mamus, Daniel VT Catenacci, Todd Hembrough. HER2 protein quantification in multiple cancer indications identifies candidates for HER2 targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 735. doi:10.1158/1538-7445.AM2017-735