Cristiano Ferrario

Dasatinib sensitizes primary chronic lymphocytic leukaemia lymphocytes to chlorambucil and fludarabine in vitro

The dual c‐abl/Src kinase inhibitor, dasatinib, utilized to treat chronic myeloid leukaemia (CML) when used at clinically attainable sublethal concentrations, synergistically sensitized primary chronic lymphocytic leukaemia (CLL) lymphocytes to chlorambucil and fludarabine. In contrast, dasatinib alone demonstrated toxicity to CLL lymphocytes at concentrations that are generally not clinically attainable. Dasatinib resistance and poorer dasatinib‐mediated sensitization to chlorambucil and fludarabine was associated with higher expression of c‐abl protein levels. In contrast, chlorambucil and fludarabine resistance correlated with basal p53 protein levels. Moreover, Western blot analysis after in vitro treatment of primary CLL lymphocytes with dasatinib, chlorambucil and/or fludarabine, showed that dasatinib: (i) inhibited c‐abl function (e.g. downregulation of c‐abl protein levels and decreased the phosphorylation of a c‐abl downstream target, Dok2), (ii) decreased chlorambucil/fludarabine induced accumulation of p53 protein levels, (iii) altered the response to chlorambucil/fludarabine induced DNA‐damage as evidenced by an increase in chlorambucil/fludarabine‐induced H2AX phosphorylation, and (iv) accentuated the c‐abl downregulation induced by chlorambucil/fludarabine. Our results suggest that dasatinib in combination with chlorambucil or fludarabine may improve the therapy of CLL.

Intrathecal trastuzumab and thiotepa for leptomeningeal spread of breast cancer

A 31-year-old woman underwent a right segmental mastectomy in October 1999 for a pT2, pN2 invasive ductal carcinoma, negative for estrogen receptor/progesterone receptor, positive for amplification of human epidermal growth factor receptor-2 (HER-2). She received adjuvant chemotherapy (doxorubicin + cyclophosphamide, followed by paclitaxel) and local radiotherapy. Two and a half years after surgery, she developed multiple liver metastases. She received i.v. carboplatin, paclitaxel, and trastuzumab, followed by capecitabine and trastuzumab, with a radiological complete response. Approximately 14 months later (April 2004), a single brain lesion was detected on magnetic resonance imaging (MRI), in the right occipital lobe. The lesion was surgically resected, followed by external beam whole-brain radiation. In August 2006, the patient presented with visual impairment, ptosis of the right eye, right hemi-facial hypoesthesia, left foot drop, and paralysis of the left facial nerve. An MRI exam revealed a right occipital brain lesion (1.5 cm), diffuse leptomeningealmetastases (LM), a cervical extramedullary intradural enhancing lesion (C1– C3), an intramedullary lesion centered at T12, and a nodular area of enhancement on the roots of the cauda equina. Only a suspicious 6-mm lung nodule was found on systemic restaging. A diagnostic lumbar puncture was carried out, and cytology of the cerebro-spinal fluid (CSF) was positive for cancer cells (5%of malignant cells). CSF glucose, lactate dehydrogenase, and protein levels were always normal. After placement of an Ommaya reservoir, she was started on intrathecal (i.t.) trastuzumab, initially on a weekly schedule, with gradual dose increase from 20 to 30 mg (Table 1), later combined with i.t. methotrexate (10 mg) for two doses. To reduce the risk of anaphylaxis and neurotoxicity from the preservative agent [1], i.t. trastuzumab was administered without the diluent provided. Interestingly, after the first dose of i.t. trastuzumab, all subsequent CSF cytologies were negative for malignant cells. After the two first doses of i.t. trastuzumab, we also started i.v. trastuzumab, that was never interrupted afterwards, and i.v. pegylated liposomal doxorubicin (PLD; letters to the editor Annals of Oncology

Both t-Darpp and DARPP-32 can cause resistance to trastuzumab in breast cancer cells and are frequently expressed in primary breast cancers

The clinical use of trastuzumab (Herceptin™), a humanized antibody against the HER2 growth factor receptor, has improved survival in patients with breast tumors with ERBB2 amplification and/or over-expression. However, most patients with advanced ERBB2 amplified breast cancers whose tumors initially respond to trastuzumab develop resistance to the drug, leading to tumor progression. To identify factors responsible for acquired resistance to trastuzumab, gene expression profiling was performed on subclones of an ERBB2 amplified breast cancer cell line, BT474, which had acquired resistance to trastuzumab. The most overexpressed gene in these subclones was PPP1R1B, encoding the DARPP-32 phosphatase inhibitor. Western analysis revealed that only the truncated isoform of the DARPP-32 protein, t-Darpp, was overexpressed in the trastuzumab resistant cells. Using gene silencing experiments, we confirmed that t-Darpp over-expression was required for trastuzumab resistance in these cells. Furthermore, transfecting t-Darpp in parental BT-474 cells conferred resistance to trastuzumab, suggesting that t-Darpp expression was sufficient for trastuzumab resistance. We also found that t-Darpp over-expression was associated with Akt activation and that the T75 residue in t-Darpp was required for both Akt activation and trastuzumab resistance. Finally, we found that full-length DARPP-32 and t-Darpp are expressed in a majority of primary breast tumors. Over-expression of full-length DARPP-32 can also confer resistance to trastuzumab and, moreover, is associated with a poor prognostic value in breast cancers. Thus, t-Darpp and DARPP-32 expression are novel prognostic and predictive biomarkers in breast cancer.

Endoplasmic reticulum stress induces PRNP prion protein gene expression in breast cancer

IntroductionHigh prion protein (PrP) levels are associated with breast, colon and gastric cancer resistance to treatment and with a poor prognosis for the patients. However, little is known about the underlying molecular mechanism(s) regulating human PrP gene (PRNP) expression in cancers. Because endoplasmic reticulum (ER) stress is associated with solid tumors, we investigated a possible regulation of PRNP gene expression by ER stress.MethodsPublished microarray databases of breast cancer tissues and breast carcinoma cell lines were analyzed for PrP mRNA and ER stress marker immunoglobulin heavy chain binding protein (BiP) levels. Breast cancer tissue microarrays (TMA) were immunostained for BiP and PrP. Breast carcinoma MCF-7, MDA-MB-231, HS578T and HCC1500 cells were treated with three different ER stressors - Brefeldin A, Tunicamycin, Thapsigargin - and levels of PrP mRNA or protein assessed by RT-PCR and Western blot analyses. A human PRNP promoter-luciferase reporter was used to assess transcriptional activation by ER stressors. Site-directed mutagenesis identified the ER stress response elements (ERSE). Chromatin immunoprecipitation (ChIP) analyses were done to identify the ER stress-mediated transcriptional regulators. The role of cleaved activating transcription factor 6α (ΔATF6α) and spliced X-box protein-1 (sXBP1) in PRNP gene expression was assessed with over-expression or silencing techniques. The role of PrP protection against ER stress was assessed with PrP siRNA and by using Prnp null cell lines.ResultsWe find that mRNA levels of BiP correlated with PrP transcript levels in breast cancer tissues and breast carcinoma cell lines. PrP mRNA levels were enriched in the basal subtype and were associated with poor prognosis in breast cancer patients. Higher PrP and BiP levels correlated with increasing tumor grade in TMA. ER stress was a positive regulator of PRNP gene transcription in MCF-7 cells and luciferase reporter assays identified one ER stress response element (ERSE) conserved among primates and rodents and three primate-specific ERSEs that regulated PRNP gene expression. Among the various transactivators of the ER stress-regulated unfolded protein response (UPR), ATF6α and XBP1 transactivated PRNP gene expression, but the ability of these varied in different cell types. Functionally, PrP delayed ER stress-induced cell death.ConclusionsThese results establish PRNP as a novel ER stress-regulated gene that could increase survival in breast cancers.

Advances in the approach to novel drug clinical development for breast cancer.

Introduction: In the post-genomic era clinical development of new agents to treat breast cancer (BC) can be a real challenge. Different from chemotherapy agents, with a broad but not specific spectrum of activity, novel drugs are being developed as ‘targeted’ agents, potentially benefiting a subgroup of patients. In BC, different clinically identifiable subtypes are now separately addressed in specific clinical trials. Areas covered: In this review, the authors discuss the clinical development of targeted drugs that have become part of the current treatment of BC. They also highlight the challenges that in other cases determined the failure of promising compounds. Furthermore, the article reports on how combinations of targeted agents have emerged as valid strategies to overcome acquired resistance. It also provides discussion of how ‘old’ therapies can be retargeted to certain patient populations or ‘reinvented’ as safer and more effective with the creation of drug conjugates. They also discuss how novel clinical trial designs are emerging to accelerate the successful matching of targeted drugs to the right patient population. Expert opinion: It is important not to forget that the development of BC therapeutics is a ‘moving target’, as its biology evolves in time under the pressure of ongoing treatments. There are currently a finite number of resources available for the development of new therapeutics, which means that resources need to be carefully allocated. There is also a need to prioritize clinical trials that can reduce the number of patients who are candidates for expensive treatments.

The trastuzumab and vinorelbine combination: an alternative to taxane-based chemotherapy for early-stage and locally advanced her2-positive breast cancer.

BACKGROUNDnAnthracyclines and taxanes have historically constituted the backbone of chemotherapy regimens for patients with breast cancer positive for the human epidermal growth factor receptor 2 (her2). For a subset of patients who categorically refuse alopecia, or for those with a contraindication to those drugs, there is an urgent need to define alternative regimens. Here, we report our institutional experience with trastuzumab and vinorelbine (tv), a combination with good clinical activity and a good side effect profile for patients with her2-positive breast cancer.nnnMETHODSnIn a retrospective analysis, outcomes data were extracted for patients receiving tv as their only chemotherapy in the non-metastatic setting at the Jewish General Hospital. For the most part, tv was administered weekly for 6 months, followed by trastuzumab for 6 months.nnnRESULTSnThe analysis identified 46 patients (mean age: 64 years) who received tv between 2003 and 2012 (n = 36 adjuvant, n = 10 neoadjuvant). Of the patients in the adjuvant group, 81% had stage i disease. In the neoadjuvant group, 3 patients experienced a complete pathologic response. Only 1 patient experienced local recurrence after a short course (3 months) of adjuvant tv. Overall survival and breast cancer-specific survival were 94% and 98% respectively at a median 5 years of follow-up. Febrile neutropenia-induced sepsis resulted in the death of 1 patient with significant medical comorbidities; 2 other patients died of comorbidities unrelated to their cancer or treatment. Grades 3 or 4 adverse events included neutropenia (23%), febrile neutropenia (10%), fatigue (2%), and anemia (2%).nnnCONCLUSIONSnFor patients with non-metastatic breast cancer refusing alopecia, or for patients who are not candidates for standard chemotherapy, tv is a reasonable alternative to standard adjuvant chemotherapy.

Aromatase inhibition in relapsing low malignant potential serous tumours of the ovary

Low malignant potential serous tumours (LMPSTs) of the ovary represent an indolent disease, with an excellent prognosis in a majority of patients. Patients with recurrent LMPSTs tend to develop widespread disease with a mortality rate as high as 70%. These tumours tend to have a very poor response to standard chemotherapy, and the management of primary and recurrent disease beyond surgical resection is not well defined. The majority of LMPST have been reported to express oestrogen and progesterone hormone receptors. However, only three reported cases of antihormonal treatment in this setting, and only one using aromatase inhibitors (AI), have been previously reported. We herein report long-term complete remission of two patients with relapsed, chemotherapy-resistant LMPSTs, treated with long-term AI (anastrozole 1u2005mg daily) as per negative MRI and positron emission tomography scans. Our results warrant further investigation for the use of AIs for metastatic recurrent LMPSTs.

Abstract P4-14-19: ONT-380 in the treatment of HER2+ breast cancer central nervous system (CNS) metastases (mets)

Background: The risk of CNS involvement in patients with HER2+ metastatic breast cancer (MBC) is high. The natural history of HER2+ CNS metastases (mets) is different from other breast cancer subtypes; there is a longer time from metastatic diagnosis to CNS relapse, greater control of extracranial disease at the time of CNS relapse, and longer median overall survival from the time of CNS relapse. Local treatments, i.e., surgery and/or radiation, remain the mainstay of treatment for HER2+ CNS disease as standard systemic therapy options have limited efficacy. ONT-380, a potent HER2 selective tyrosine kinase inhibitor with minimal EGFR-like side effects, has been associated with increased survival compared to lapatinib or neratinib in an animal model of HER2+ CNS disease. Here, we describe 24 pts from two studies with response-evaluable CNS mets treated with ONT-380 in combination with other systemic therapies. Methods: Pts with asymptomatic untreated or post-treatment progressive CNS mets were enrolled in ongoing phase 1b studies of ONT-380 + ado-trastuzumab emtansine (T-DM1) or ONT-380 ± trastuzumab (T) ±capecitabine (C). All pts received treatment in 21 day cycles including ONT-380 300 mg PO BID and approved doses of either T-DM1, T or C alone, or T+C. Eligibility criteria for all pts included prior treatment with T and a taxane, and for pts receiving T and/or C, prior T-DM1. Prior lapatinib was allowed. Assessments included safety, systemic tumor response per RECIST 1.1, and CNS tumor response by MRI every 2 cycles per modified RECIST 1.1. Results: 24 pts (10 with untreated CNS mets and 14 with progressive CNS mets after local therapy) received ONT-380 plus T-DM1 (n =14), C (n=1), T (n = 5) or T+C (n = 4) for 1-8 cycles. Of these 24 pts, 14 are evaluable for response in the CNS (at least one follow-up MRI); 7 pts have not yet been rescanned, and 3 are non-evaluable. In the 14 response-evaluable pts, best CNS response has been: 1 CNS CR (T-DM1), 4 CNS PR (T-DM1 n = 2; T+C n = 1; C n=1) and 9 CNS SD (T-DM1 n = 5; T n = 3; T+C n=1). Pts with CR or PR (prior lapatinib n=2; prior pertuzumab n=2; prior T-DM1 n=2) all had > 50% decrease in CNS target lesions. One CNS non-evaluable pt (T) had a 15% increase in their target lesion and underwent surgical resection; pathology, however, revealed no viable tumor with only necrotic tissue present. Two additional CNS non-evaluable pts (T-DM1 n=1; C+T n=1) were taken off study due to systemic PD after treatment was held for AEs (Gr 3 AST/ALT elevation [n=1]; Gr 4 edema in thalamic lesion [n=1]). No other ≥ Gr 3 ONT-380 related events were reported. Systemic disease control was also seen, with a best response in 17 evaluable pts of 6 PR, 9 SD, and 2 PD. Conclusions: This case series demonstrates early clinical signs of activity of ONT-380 against HER2+ CNS mets in combination with other systemic agents. Further study of the CNS activity of ONT-380 is ongoing and further studies are being planned. Updated results will be reported. Clinical trial information: NCT01983501, NCT02025192. Citation Format: Murthy RK, Hamilton E, Borges VF, Moulder S, Aucoin N, Welch S, Chaves J, Falkson CI, Walker L, Ferrario C. ONT-380 in the treatment of HER2+ breast cancer central nervous system (CNS) metastases (mets). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-19.

Tucatinib Combined With Ado-Trastuzumab Emtansine in Advanced ERBB2/HER2-Positive Metastatic Breast Cancer: A Phase 1b Clinical Trial

Importance Treatment options for patients with disease progression after treatment with trastuzumab, pertuzumab, and ado-trastuzumab emtansine (T-DM1) are limited. Tucatinib is an oral, potent, human epidermal growth factor receptor 2 (HER2)-specific tyrosine kinase inhibitor (TKI) being developed as a novel treatment for ERBB2/HER2-positive breast cancer. Objective To determine the maximum tolerated dosage of tucatinib in combination with T-DM1 in the treatment of patients with ERBB2/HER2-positive metastatic breast cancer with and without brain metastases. Design, Setting, and Participants In this phase 1b open-label, multicenter, clinical trial, 57 participants enrolled between January 22, 2014, and June 22, 2015, were 18 years of age or older with ERBB2/HER2-positive metastatic breast cancer previously treated with trastuzumab and a taxane. Data were analyzed between January and March 2018. Interventions Tucatinib 300 mg or 350 mg administered orally twice per day for 21 days and T-DM1 3.6 mg/kg administered intravenously once every 21 days. Main Outcomes and Measures Safety assessments, pharmacokinetics, and response were assessed using RECIST 1.1 every 2 cycles for 6 cycles, followed by every 3 cycles. Results Fifty-seven T-DM1-naive patients (median [IQR] 51 [44.0-63.0] years of age) who had undergone a median of 2 earlier HER2 therapies (range, 1-3) were treated. The tucatinib maximum tolerated dosage was determined to be 300 mg administered twice per day with dose-limiting toxic reactions seen at 350 mg twice per day. Pharmacokinetic analysis showed that there was no drug-drug interaction with T-DM1. Adverse events seen among the 50 patients treated at the maximum tolerated dosage regardless of causality included nausea (36 patients; 72%), diarrhea (30 patients; 60%), fatigue (28 patients; 56%), epistaxis (22 patients; 44%), headache (22 patients; 44%), vomiting (21 patients; 42%), constipation (21 patients; 42%), and decreased appetite (20 patients; 40%); the majority of adverse events were grade 1 or 2. Tucatinib-related toxic reactions that were grade 3 and above included thrombocytopenia (7 patients; 14%) and hepatic transaminitis (6 patients; 12%). Conclusions and Relevance In this study, tucatinib in combination with T-DM1 appeared to have acceptable toxicity and to show preliminary antitumor activity among heavily pretreated patients with ERBB2/HER2-positive metastatic breast cancer with and without brain metastases. Trial Registration ClinicalTrials.gov Identifier: NCT01983501

Abstract CT098: Phase 1 first-in-human study of anti-clusterin antibody AB-16B5 in patients with advanced solid malignancies

Background. Secreted clusterin (sCLU) promotes survival and stimulates epithelial to mesenchymal transition (EMT) in cancer cells, leading to tumor invasion, metastasis and chemoresistance. AB-16B5 is a humanized IgG2 mAb specific for the EMT-inducing region in some isoforms of sCLU produced by tumor cells, with much less affinity for sCLU found in healthy individuals. Pre-clinical models showed that AB-16B5 inhibits EMT and displays synergy with chemotherapeutic agents. We performed a first-in-human phase I trial to assess safety and tolerability of AB-16B5 and to determine a recommended dose for phase II studies. Methods. Patients (pts) with evaluable and refractory advanced solid malignancies were eligible with adequate organ and bone marrow functions and an ECOG of 0-2. AB-16B5 was administered as a 60-min IV weekly infusion (21-day cycles). Dose escalation followed an accelerated scheme for the 1.5 and 3 mg/kg dose levels and a standard 3 + 3 design for subsequent levels (6, 9 and 12 mg/kg). All pts underwent collection of blood samples for PK analysis. Pre-treatment and post-treatment (end of cycle 2) tumor biopsies were collected if feasible in pts dosed at 9 and 12 mg/kg. Biopsies were used to evaluate EMT status and to investigate the presence of AB-16B5. Radiological imaging was performed every 6 weeks for the first 8 cycles, then every 9 weeks thereafter. Results. 15 pts (13 carcinomas of various origins, 1 melanoma and 1 sarcoma) were enrolled from May 2015 to October 2016. Pts received between 1 and 53 weekly doses (median: 9 doses). The most frequently reported adverse events (AEs, all causalities) were nausea, abdominal pain, constipation, vomiting, back pain, pruritus and dyspnea. Most of the AEs were of Grade 1 or 2. Among the AEs ≥ Grade 3, only 2 (Grade 3 infusion related reaction and rash) were judged related to AB-16B5. No dose-limiting toxicities were identified during the first cycle of treatment for any pt. 5 serious AEs were reported (sepsis, pyrexia, dyspnea, intra-abdominal hemorrhage and main stem bronchus obstruction), none of which were judged related to the study treatment. PK analysis across all dose levels confirmed that systemic exposure to AB-16B5 increased in a dose proportional manner. The presence of AB-16B5 at the tumor site was confirmed in all 5 pts where a post-treatment tumor lysate could be generated. Biomarker analysis in paired tumor biopsies provided some evidence for EMT inhibition as seen by increased E-cadherin expression after treatment with AB-16B5 in 2 pts. In one of these 2 pts with advanced gastric cancer, this was also accompanied by a loss of vimentin expression. This pt had stable disease (SD) with clinical benefit and remained on treatment for 24 weeks. Another pt with follicular thyroid cancer had SD for almost 1 year. Conclusion. Weekly infusions of AB-16B5 are well tolerated up to 12 mg/kg. This dose level is the recommended phase II dose for future trials. Early correlative studies on tumor tissue provide evidence of molecular modulation of the tumor environment in humans. Clinical trial registry number: NCT02412462 Citation Format: Cristiano Ferrario, Julie Laurin, Leon Van Kempen, Caroline Lambert, Alan Spatz, Oksana Markova, Gerald Batist, Adrian Langleben, Mario Filion, Jacques Jolivet. Phase 1 first-in-human study of anti-clusterin antibody AB-16B5 in patients with advanced solid malignancies [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 CT098. doi:10.1158/1538-7445.AM2017-CT098