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The aim of this article is to describe and analyze the principal aspects of a phenomenon that periodically affects medicines and medical devices all around the world in all contexts of the hospitals and the community, i.e. their shortage. Recently in Italy there has been a prolonged shortage of some medicines such as antibiotics, symptomatic remedies for fever and pain, pediatric medicines and inhalers but the phenomenon is not rare. We want to analyze some causal aspects, the principal practical consequences and also some possible strategies to remedy the problem. Technical pharmacological aspects of the individual classes of drugs mentioned will not be taken into consideration.

Supplementary Table 3: EPHA2 expression in tumor samples with HSCORE

Supplementary Figure 1. A: Phosphoarray analysis of SW48 and SW48-CR cell lines. B: Silencing of EPHA2 decreases the sensitivity to ALW-II-41-27 in HCT15 and SW48-CR cell lines.

The EPHA2 tyrosine kinase receptor is implicated in tumor progression and targeted therapies resistance. We evaluated EPHA2 as a potential resistance marker to the antiepidermal growth factor receptor (EGFR) monoclonal antibody cetuximab in colorectal cancer. We studied activation of EPHA2 in a panel of human colorectal cancer cell lines sensitive or resistant to anti-EGFR drugs. The in vitro and in vivo effects of ALW-II-41-27 (an EPHA2 inhibitor) and/or cetuximab treatment were tested. Formalin-fixed paraffin-embedded tumor specimens from 82 RAS wild-type (WT) metastatic colorectal cancer patients treated with FOLFIRI + cetuximab as first-line therapy in the CAPRI-GOIM trial were assessed for EPHA2 expression by immunohistochemistry and correlated with treatment efficacy. EPHA2 was differentially activated in colorectal cancer cell lines. Combined treatment with ALW-II-41-27 plus cetuximab reverted primary and acquired resistance to cetuximab, causing cell growth inhibition, inducing apoptosis and cell-cycle G1–G2 arrest. In tumor xenograft models, upon progression to cetuximab, ALW-II-41-27 addition significantly inhibited tumor growth. EPHA2 protein expression was detected in 55 of 82 tumor samples, frequently expressed in less-differentiated and left-sided tumors. High levels of EPHA2 significantly correlated with worse progression-free survival [8.6 months; confidence interval (CI) 95%, 6.4–10.8; vs. 12.3 months; CI 95%, 10.4–14.2; P = 0.03] and with increased progression rate (29% vs. 9%, P = 0.02). A specific EPHA2 inhibitor reverts in vitro and in vivo primary and acquired resistance to anti-EGFR therapy. EPHA2 levels are significantly associated with worse outcome in patients treated with FOLFIRI + cetuximab. These results highlight EPHA2 as a potential therapeutic target in metastatic colorectal cancer.

Supplementary Figure 2. A-B: Synergistic anti-proliferative effects of the combination of ALW-II-41-27 and cetuximab.C: Effect of Epha2 gene knockdown on cetuximab sensitivity in HCT116 and LOVO cell lines. D: Effects of combination treatment with ALW-II-41-27 and cetuximab on induction of apoptosis. E: Cell cycle distribution for HCT15 and SW48-CR following treatment with ALW-II-41-27 and/or cetuximab for 24, 48 and 72 hrs at indicated doses. F: Effects of EPHA2 blockade alone and in combination with cetuximab on intracellular signalling pathways of cell proliferation and survival.

Supplementary materials and methods

Supplementary Table 2: EPHA2 expression in tumor samples of 82 RAS WT colorectal cancer patients from the GOIM-CAPRI trial

Supplementary Table 1: Treatment with ALW-II-41-27 or cetuximab as single agents

Supplementary Figure 2 - PDF file 194K, Supplementary Figure 2. Effects on the downstream pathways by metformin treatment in A549 and H460

Purpose: Resistance to tyrosine kinase inhibitors (TKI) of EGF receptor (EGFR) is often related to activation of other signaling pathways and evolution through a mesenchymal phenotype.Experimental Design: Because the Hedgehog (Hh) pathway has emerged as an important mediator of epithelial-to-mesenchymal transition (EMT), we studied the activation of Hh signaling in models of EGFR-TKIs intrinsic or acquired resistance from both EGFR-mutated and wild-type (WT) non–small cell lung cancer (NSCLC) cell lines.Results: Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-mutated HCC827-GR cells, we found SMO (the Hh receptor) gene amplification, MET activation, and the functional interaction of these two signaling pathways. In HCC827-GR cells, inhibition of SMO or downregulation of GLI1 (the most important Hh-induced transcription factor) expression in combination with MET inhibition exerted significant antitumor activity.In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation. In addition, the inhibition of SMO by the use of LDE225 sensitizes EGFR-WT NSCLC cells to standard chemotherapy.Conclusions:This result supports the role of the Hh pathway in mediating resistance to anti-EGFR-TKIs through the induction of EMT and suggests new opportunities to design new treatment strategies in lung cancer. Clin Cancer Res; 21(20); 4686–97. ©2015 AACR.

Supplementary Figure 1 Legend - PDF file 52K, Effects on CALU-3, CALU-3 GEF-R, H1299, H1975 and GLC82 cell growth of the combination of metformin and gefitinib. Cell viability was determined using a 3-(4,5 methylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay. Treatment combinations and sequences are described in Materials and Methods. CI values were calculated according to the Chou and Talalay mathematical model for drug interactions using the Calcusyn software for different fractions affected (fa). CI is a quantitative measure of the degree of interaction between different drugs. If CI = 1, it denotes additivity; if CI > 1, it denotes antagonism; if 1 < CI > 0.7, it denotes slight synergism; if CI = 0.7-0.3, it denotes synergism; if CI < 0.3, it denotes strong synergism. Results are the median of three independent experiments, each done in eight replicate wells for experimental point

Graphycal representation of the interplay between Hedgehog and MET pathway.

PDF file - 116KB

supplementary figure 1. Effects of cetuximab in combination with regorafenib in a panel of CRC cell lines with primary and acquired resistance to anti-EGFR inhibitor in vitro.

PDF file 145K, genes down regulated in GEO-CR versus GEO

PDF file 84K, Supplementary figure legend

PDF file - 1325KB, Caption Supplementary Figure 1 A and 1B. The combined treatment of cetuximab and the selective MEK1/2 inhibitor BAY 86-9766 induce a synergistic growth inhibitory effects in CRC cell lines with primary and acquired resistance to cetuximab and an antagonistic effects in cetuximab-sensitive CRC cell lines. Caption Supplementary Figure 2. The BAY 86-9766 treatment, in CRC cancer cell lines with primary and acquired resistance to cetuximab, induce a dose dependent reduction of MAPK and MEK phosphorylation. Caption Supplementary Figure 3. The combined treatment of cetuximab and the selective MEK1/2 inhibitor BAY 86-9766 was well tolerated by mice, with no weight loss or other signs of acute or delayed toxicity. Caption Supplementary Table 1A and 1B. The panel of CRC cell lines have been treated with several concentrations of cetuximab and selective MEK1/2 inhibitors (BAY 86-9766, Selumetinib and Pimasertib), showing differential sensitivity to the drugs. Caption Supplementary Table 2. We have performed the experiments on a panel of eight human CRC cell lines having different mutation profiles in KRAS, NRAS, BRAF, PIK3CA and EGFR genes. Caption Supplementary Table 3. In the CRC cell lines with acquired and primary resistance to cetuximab, the treatment with the selective MEK1/2 inhibitor BAY 86-9766 determined synergistic growth inhibitory effects in combination with cetuximab.

Purpose: The EGFR-independent activation of the RAS/RAF/MEK/MAPK pathway is one of the resistance mechanisms to cetuximab.Experimental Design: We have evaluated, in vitro and in vivo, the effects of BAY 86-9766, a selective MEK1/2 inhibitor, in a panel of human colorectal cancer cell lines with primary or acquired resistance to cetuximab.Results: Among the colorectal cancer cell lines, five with a KRAS mutation (LOVO, HCT116, HCT15, SW620, and SW480) and one with a BRAF mutation (HT29) were resistant to the antiproliferative effects of cetuximab, whereas two cells (GEO and SW48) were highly sensitive. Treatment with BAY 86-9766 determined dose-dependent growth inhibition in all cancer cells, including two human colorectal cancer cells with acquired resistance to cetuximab (GEO-CR and SW48-CR), with the exception of HCT15 cells. Combined treatment with cetuximab and BAY 86-9766 induced a synergistic antiproliferative and apoptotic effects with blockade in the MAPK and AKT pathway in cells with either primary or acquired resistance to cetuximab. The synergistic antiproliferative effects were confirmed using other two selective MEK1/2 inhibitors, selumetinib and pimasertib, in combination with cetuximab. Moreover, inhibition of MEK expression by siRNA restored cetuximab sensitivity in resistant cells. In nude mice bearing established human HCT15, HCT116, SW48-CR, and GEO-CR xenografts, the combined treatment with cetuximab and BAY 86-9766 caused significant tumor growth inhibition and increased mice survival.Conclusion: These results suggest that activation of MEK is involved in both primary and acquired resistance to cetuximab and the inhibition of EGFR and MEK could be a strategy for overcoming anti-EGFR resistance in patients with colorectal cancer. Clin Cancer Res; 20(14); 3775–86. ©2014 AACR.

Purpose: Although cetuximab, an anti-EGF receptor (EGFR) monoclonal antibody, is an effective treatment for patients with KRAS wild-type metastatic colorectal cancer (mCRC), its clinical use is limited by onset of resistance.Experimental Design: We characterized two colorectal cancer models to study the mechanisms of acquired resistance to cetuximab.Results: Following chronic treatment of nude mice bearing cetuximab-sensitive human GEO colon xenografts, cetuximab-resistant GEO (GEO-CR) cells were obtained. In GEO-CR cells, proliferation and survival signals were constitutively active despite EGFR inhibition by cetuximab treatment. Whole gene expression profiling identified a series of genes involved in the hepatocyte growth factor (HGF)-MET–dependent pathways, which were upregulated in GEO-CR cells. Furthermore, activated, phosphorylated MET was detected in GEO-CR cells. A second colorectal cancer cell line with acquired resistance to cetuximab was obtained (SW48-CR). Inhibition of MET expression by siRNA restored cetuximab sensitivity in GEO-CR and SW48-CR cells, whereas exogenous activation of MET by HGF stimulation in cetuximab-sensitive GEO and SW48 cells induced resistance to cetuximab. Treatment of GEO-CR and SW48-CR cells with PHA665752, a selective MET inhibitor, inhibited cell growth, proliferation, and survival signals and impaired cancer cell migration. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. In fact, TGF-α overexpression induced the EGFR–MET interaction, with subsequent MET phosphorylation and activation of MET downstream effectors in GEO-CR and SW48-CR cells.Conclusions: These results suggest that overexpression of TGF-α through induction of EGFR–MET interaction contributes to cetuximab resistance in colorectal cancer cells. The combined inhibition of EGFR and MET receptor could represent a strategy for preventing and/or overcoming cetuximab resistance in patients with colorectal cancer. Clin Cancer Res; 19(24); 6751–65. ©2013 AACR.

Purpose: EGF receptor (EGFR) tyrosine kinase inhibitors (TKI) have been found to be effective against lung cancer, but clinical resistance to these agents has developed as their usage has increased. Metformin is a widely used antidiabetic drug and also displays significant growth-inhibitory and proapoptotic effects in several cancer models, alone or in combination with chemotherapeutic drugs.Experimental Design: The effects of gefitinib, a selective EGFR-TKI, and metformin on a panel of non–small cell lung cancer (NSCLC) cell lines were assessed by using MTT, bromide assay, flow cytometry, anchorage-independent growth, coimmunoprecipitation, and Western blot analysis.Results: The combination of metformin with gefitinib induced a strong antiproliferative and proapoptotic effect in NSCLC cell lines that harbored wild-type LKB1 gene. Treatment with metformin as single agent, however, induced an activation and phosphorylation of mitogen-activated protein kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. The inhibition of EGFR phosphorylation and of downstream signaling by adding gefitinib to metformin treatment abrogated this phenomenon and induced a strong apoptotic effect in vitro and in vivo.Conclusions: Metformin and gefitinib are synergistic in LKB1 wild-type NSCLC cells. However, further studies are required to investigate better the effect of metformin action on the RAS/RAF/MAPK pathway and the best context in which to use metformin in combination with molecular targeted agents. Clin Cancer Res; 19(13); 3508–19. ©2013 AACR.

Supplemental matherial and methods

supplementary table 1. mutational status

A) GLI1 promoter activity in EGFR-WT NSCLC cell lines. Effects of Hh and EGFR inhibition in EGFR-WT NSCLC cell lines on their abilities to B) growth, C) invade , D)migrate and E) colony forming. F)In vivo efficacy of erlotinib and LDE225 on EGFR-WT NSCLC xenografts in nude mice.

EGFR mutational status, gefitinib sensitivity and HH pathway activation in our panel of NSCLC cell lines.

Supplementary Figure 2 Legend - PDF file 58K, Supplementary Figure 2 legend: Effects on the downstream pathways by metformin treatment in A549 and H460. Western blotting analysis of AMPK, ACC, MAPK, AKT, p70S6K, S6, 4EBP1, activation following treatment with the indicated concentration of metformin. beta-actin was included as a loading control

supplementary figure 3. Pro-apoptotic effects of cetuximab in combination with regorafenib in SW620 CRC cell line with primary resistance to anti-EGFR inhibitor.

Supplementary Figure 1 - PDF file 81K, Effects on CALU-3, CALU-3 GEF-R, H1299, H1975 and GLC82 cell growth of the combination of metformin and gefitinib

Western blot analysis of protein levels and phosphorylated forms of MET, EGFR, MAPK, AKT, GLI1 and beta actin in protein lysates from tumors harvested from nude mice treated with the indicated drugs.

Intensity score , percentage score and IHC score of Vimentina, MET and PTCH on tumor xenografts in nude mice collected at the end of treatment.

Purpose: In colorectal cancer, the activation of the intracellular RAS–RAF and PIK3CA–AKT pathways has been implicated in the resistance to anti-EGFR mAbs. We have investigated the role of regorafenib, an oral multikinase inhibitor, in combination with cetuximab, an anti-EGFR mAb, to overcome anti-EGFR resistance.Experimental Design: We have tested, in vitro and in vivo, the effects of regorafenib in a panel of human colorectal cancer cell lines with a KRAS mutation (SW480, SW620, HCT116, LOVO, and HCT15) or with a BRAF mutation (HT29), as models of intrinsic resistance to cetuximab treatment, and in two human colorectal cancer cell lines (GEO and SW48) that are cetuximab-sensitive, as well as in their derived cells with acquired resistance to cetuximab (GEO-CR and SW48-CR).Results: Treatment with regorafenib determined a dose-dependent growth inhibition in all colorectal cancer cell lines. The combined treatment with cetuximab and regorafenib induced synergistic antiproliferative and apoptotic effects in cetuximab-resistant cell lines by blocking MAPK and AKT pathways. Nude mice were injected s.c. with HCT116, HCT15, GEO-CR, and SW48-CR cells. The combined treatment caused significant tumor growth inhibition. Synergistic antitumor activity of regorafenib plus cetuximab was also observed in an orthotopic colorectal cancer model of HCT116 cells. In particular, the combined treatment induced a significant tumor growth inhibition in the primary tumor site (cecum) and completely prevented metastasis formation.Conclusions: The combined treatment with cetuximab and regorafenib could be a strategy to overcome resistance to anti-EGFR therapies in metastatic colorectal cancer patients. Clin Cancer Res; 21(13); 2975–83. ©2015 AACR.

PDF file 1516K, Inhibition of MET expression restores cetuximab sensitivity in SW48-CR cells

PDF file 184K, genes up regulated in GEO-CR versus GEO

PDF file 1732K, Development and characterization of cetuximab-resistant SW48 (SW48-CR) colon cancer cells

supplementary figure legend

supplementary figure 4. Effects of cetuximab in combination with regorafenib on mice body weight.

Supplementary Tables 1-3 from Heterodimerization of Insulin-like Growth Factor Receptor/Epidermal Growth Factor Receptor and Induction of Survivin Expression Counteract the Antitumor Action of Erlotinib

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been used to treat non–small cell lung cancer (NSCLC). However, the overall response rate to EGFR TKIs is limited, and the mechanisms mediating resistance to the drugs are poorly understood. Here, we report that insulin-like growth factor-I receptor (IGF-IR) activation interferes with the antitumor activity of erlotinib, an EGFR TKI. Treatment with erlotinib increased the levels of EGFR/IGF-IR heterodimer localized on cell membrane, activated IGF-IR and its downstream signaling mediators, and stimulated mammalian target of rapamycin (mTOR)–mediated de novo protein synthesis of EGFR and survivin in NSCLC cells. Inhibition of IGF-IR activation, suppression of mTOR-mediated protein synthesis, or knockdown of survivin expression abolished resistance to erlotinib and induced apoptosis in NSCLC cells in vitro and in vivo. Our data suggest that enhanced synthesis of survivin protein mediated by the IGFR/EGFR heterodimer counteracts the antitumor action of erlotinib, indicating the needs of integration of IGF-IR–targeted agents to the treatment regimens with EGFR TKI for patients with lung cancer. (Cancer Res 2006; 66(20): 10100-11)

Background Chemotherapy-induced neutropenia (CIN) has been demonstrated to be a prognostic factor in several cancer conditions. We previously found a significant prognostic value of CIN on overall survival (OS), in a pooled dataset of patients with advanced non-small-cell lung cancer (NSCLC) receiving first line chemotherapy from 1996 to 2001. However, the prognostic role of CIN in NSCLC is still debated. Methods We performed a post hoc analysis pooling data prospectively collected in six randomized phase 3 trials in NSCLC conducted from 2002 to 2016. Patients who never started chemotherapy and those for whom toxicity data were missing were excluded. Neutropenia was categorized on the basis of worst grade during chemotherapy: absent (grade 0), mild (grade 1–2), or severe (grade 3–4). The primary endpoint was OS. Multivariable Cox model was applied for statistical analyses. In the primary analysis, a minimum time (landmark) at 180 days from randomization was applied in order to minimize the time-dependent bias. Results Overall, 1529 patients, who received chemotherapy, were eligible; 572 of them (who received 6 cycles of treatment) represented the landmark population. Severe CIN was reported in 143 (25.0%) patients and mild CIN in 135 (23.6%). At multivariable OS analysis, CIN was significantly predictive of prognosis although its prognostic value was entirely driven by severe CIN (hazard ratio [HR] of death 0.71; 95%CI: 0.53–0.95) while it was not evident with mild CIN (HR 1.21; 95%CI: 0.92–1.58). Consistent results were observed in the out-of-landmark group (including 957 patients), where both severe and mild CIN were significantly associated with a reduced risk of death. Conclusion The pooled analysis of six large trials of NSCLC treatment shows that CIN occurrence is significantly associated with a longer overall survival, particularly in patients developing severe CIN, confirming our previous findings.