Comparison of trastuzumab emtansine, trastuzumab deruxtecan, and disitamab vedotin in a multiresistant HER2-positive breast cancer lung metastasis model.

Human epidermal growth factor 2 (HER2)-positive breast cancer with lung metastases resistant to targeted agents is a common therapeutic challenge. Absence of preclinical lung metastasis models that are resistant to multiple anti-HER2 targeted drugs hampers the development of novel therapies. We established a novel HER2-positive breast cancer cell line (L-JIMT-1) with a high propensity to form lung metastases from the parenteral JIMT-1 cell line by injecting JIMT-1 cells into immunodeficient SCID mice. Lung metastases developed in all mice injected with L-JIMT-1 cells, and more rapidly and in greater numbers compared with the parental JIMT-1 cells. L-JIMT-1 cells expressed more epidermal growth factor receptor and HER2 than JIMT-1 cells. L-JIMT-1 cells were resistant to all five tyrosine kinase inhibitors tested in vitro (afatinib, erlotinib, lapatinib, sapitinib, and tucatinib). When we compared JIMT-1 and L-JIMT-1 sensitivity to three HER2-targeting antibody-drug conjugates (ADCs) trastuzumab emtansine (T-DM1), trastuzumab deruxtecan (T-DXd), and disitamab vedotin (DV) in vitro, JIMT-1 cells were resistant T-DXd, partially sensitive to T-DM1, and sensitive to DV, while L-JIMT-1 cells were resistant to both T-DM1 and T-DXd, but moderately sensitive to DV. In a mouse model, all three ADCs inhibited the growth of L-JIMT-1 lung metastases compared to a vehicle, but DV and T-DXd more strongly than T-DM1, and DV treatment led to the smallest tumor burden. The L-JIMT breast cancer lung metastasis model developed may be useful in the evaluation of anti-cancer agents for multiresistant HER2-positive advanced breast cancer.

HER2Δ16 Engages ENPP1 to Promote an Immune-Cold Microenvironment in Breast Cancer.

The tumor-immune microenvironment (TIME) is a critical determinant of therapeutic response. However, the mechanisms regulating its modulation are not fully understood. HER2Δ16, an oncogenic splice variant of the HER2, has been implicated in breast cancer and other tumor types as a driver of tumorigenesis and metastasis. Nevertheless, the underlying mechanisms of HER2Δ16-mediated oncogenicity remain poorly understood. Here, we show that HER2∆16 expression is not exclusive to the clinically HER2+ subtype and associates with a poor clinical outcome in breast cancer. To understand how HER2 variants modulated the tumor microenvironment, we generated transgenic mouse models expressing either proto-oncogenic HER2 or HER2Δ16 in the mammary epithelium. We found that HER2∆16 tumors were immune cold, characterized by low immune infiltrate and an altered cytokine profile. Using an epithelial cell surface proteomic approach, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as a functional regulator of the immune cold microenvironment. We generated a knock-in model of HER2Δ16 under the endogenous promoter to understand the role of Enpp1 in aggressive HER2+ breast cancer. Knockdown of Enpp1 in HER2Δ16-derived tumor cells resulted in decreased tumor growth, which correlated with increased T-cell infiltration. These findings suggest that HER2Δ16-dependent Enpp1 activation associates with aggressive HER2+ breast cancer through its immune modulatory function. Our study provides a better understanding of the mechanisms underlying HER2Δ16-mediated oncogenicity and highlights ENPP1 as a potential therapeutic target in aggressive HER2+ breast cancer.

Extracellular vesicles as modifiers of antibody-drug conjugate efficacy.

Antibody-drug conjugates (ADCs) are a new class of anti-cancer drugs that consist of a monoclonal antibody, a highly potent small-molecule cytotoxic drug, and a chemical linker between the two. ADCs can selectively deliver cytotoxic drugs to cancer cells leading to a reduced systemic exposure and a wider therapeutic window. To date, nine ADCs have received marketing approval, and over 100 are being investigated in nearly 600 clinical trials. The target antigens of at least eight out of the nine approved anti-cancer ADCs and of 69 investigational ADCs are present on extracellular vesicles (EVs) (tiny particles produced by almost all types of cells) that may carry their contents into local and distant cells. Therefore, the EVs have a potential to mediate both the anti-cancer effects and the adverse effects of ADCs. In this overview, we discuss the mechanisms of action of ADCs and the resistance mechanisms to them, the EV-mediated resistance mechanisms to small molecule anti-cancer drugs and anti-cancer monoclonal antibodies, and the EVs as modifiers of ADC efficacy and safety.

ARX788, a novel anti-HER2 antibody-drug conjugate, shows anti-tumor effects in preclinical models of trastuzumab emtansine-resistant HER2-positive breast cancer and gastric cancer.

The majority of HER2-positive breast or gastric cancers treated with T-DM1 eventually show resistance to this agent. We compared the effects of T-DM1 and ARX788, a novel anti-HER2 antibody-drug conjugate, on cell growth and apoptosis in HER2-positive breast cancer and gastric cancer cell lines sensitive to T-DM1, gastric cancer cell lines resistant to T-DM1, HER2-negative breast cancer cell lines, and T-DM1-resistant xenograft models. ARX788 was effective in T-DM1-resistant in vitro and in vivo models of HER2-positive breast cancer and gastric cancer. ARX788 showed a pronounced growth inhibitory effect on all five HER2-positive cell lines tested, of which two gastric cancer cell lines had acquired resistance to T-DM1. ARX788 evoked more apoptotic events compared to T-DM1. While JIMT-1 and RN-87 xenograft tumors progressed on T-DM1 treatment, all such tumors responded to ARX788, and four out of the six JIMT-1 tumors and nine out of the twelve RN-87 tumors disappeared during the ARX788 treatment. Mice treated with ARX788 survived longer than those treated with T-DM1. The data support evaluation of ARX788 in patients with HER2-positive breast cancer or gastric cancer including cancers that progress during T-DM1 therapy.

A Novel Anti-HER2 Antibody-Drug Conjugate XMT-1522 for HER2-Positive Breast and Gastric Cancers Resistant to Trastuzumab Emtansine.

Most patients with HER2-positive breast or gastric cancer exhibit primary or acquired resistance to trastuzumab emtansine (T-DM1), and such patients may have limited therapeutic options. XMT-1522 is a novel anti-HER2 antibody-drug conjugate. We compared XMT-1522 to T-DM1 in preclinical models. The effects of XMT-1522 and T-DM1 on cell survival and apoptosis were compared in six HER2-positive breast cancer or gastric cancer cell lines, of which three lines were T-DM1-sensitive (N-87, OE-19, JIMT-1) and three T-DM1-resistant (RN-87, ROE-19, SNU-216). We compared these agents also in the HER2-negative breast cancer cell line MCF-7, and in mouse RN-87 and JIMT-1 xenograft models. Cell survival was assessed using the AlamarBlue method and apoptosis with the Caspase-Glo 3/7 method. XMT-1522 inhibited the growth of all six HER2-positive cell lines. The proportions of cells that survived XMT-1522 treatment were smaller as compared with T-DM1, particularly in the T-DM1-resistant cell lines. XMT-1522 induced more cell apoptosis compared with T-DM1. While RN-87 and JIMT-1 xenograft tumors progressed on T-DM1 treatment, all tumors responded to XMT-1522, and all but one tumor disappeared during the XMT-1522 treatment. XMT-1522 had a strong antitumor effect on RN-87 and JIMT-1 xenografts that progressed on T-DM1. We conclude that XMT-1522 was effective in HER2-positive breast cancer and gastric cancer cell lines resistant to T-DM1, and in xenograft models resistant to T-DM1. The results support the testing of XMT-1522 in clinical trials in patients with HER2-positive cancer.

Cancer-derived exosomes from HER2-positive cancer cells carry trastuzumab-emtansine into cancer cells leading to growth inhibition and caspase activation.

BACKGROUND: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that carries a cytotoxic drug (DM1) to HER2-positive cancer. The target of T-DM1 (HER2) is present also on cancer-derived exosomes. We hypothesized that exosome-bound T-DM1 may contribute to the activity of T-DM1. METHODS: Exosomes were isolated from the cell culture medium of HER2-positive SKBR-3 and EFM-192A breast cancer cells, HER2-positive SNU-216 gastric cancer cells, and HER2-negative MCF-7 breast cancer cells by serial centrifugations including two ultracentrifugations, and treated with T-DM1. T-DM1 not bound to exosomes was removed using HER2-coated magnetic beads. Exosome samples were analyzed by electron microscopy, flow cytometry and Western blotting. Binding of T-DM1-containing exosomes to cancer cells and T-DM1 internalization were investigated with confocal microscopy. Effects of T-DM1-containg exosomes on cancer cells were investigated with the AlamarBlue cell proliferation assay and the Caspase-Glo 3/7 caspase activation assay. RESULTS: T-DM1 binds to exosomes derived from HER2-positive cancer cells, but not to exosomes derived from HER2-negative MCF-7 cells. HER2-positive SKBR-3 cells accumulated T-DM1 after being treated with T-DM1-containg exosomes, and treatment of SKBR-3 and EFM-192A cells with T-DM1-containing exosomes resulted in growth inhibition and activation of caspases 3 and/or 7. CONCLUSION: T-DM1 binds to exosomes derived from HER2-positive cancer cells, and T-DM1 may be carried to other cancer cells via exosomes leading to reduced viability of the recipient cells. The results suggest a new mechanism of action for T-DM1, mediated by exosomes derived from HER2-positive cancer.

Comparison of Antibodies for Immunohistochemistry-based Detection of HER3 in Breast Cancer.

BACKGROUND: Growth factor receptor HER3 (ErbB3) lacks standardized immunohistochemistry (IHC)-based methods for formalin-fixed paraffin-embedded (FFPE) tissue samples. We compared 4 different anti-HER3 antibodies to explain the differences found in the staining results reported in the literature. MATERIALS AND METHODS: Four commercial HER3 antibodies were tested on FFPE samples including mouse monoclonal antibody clones, DAK-H3-IC and RTJ1, rabbit monoclonal antibody clone SP71, and rabbit polyclonal antibody (SAB4500793). Membranous and cytoplasmic staining patterns were analyzed and scored as 0, 1+, or 2+ according to the intensity of the staining and completeness of membranous and cytoplasmic staining. A large collection of HER2-amplified breast cancers (n=177) was stained with the best performing HER3 antibody. The breast cancer cell line, MDA-453, and human prostate tissue were used as positive controls. IHC results were confirmed by analysis of flow cytometry performed on breast cancer cell lines. Staining results of FFPE samples were compared with samples fixed with an epitope-sensitive fixative (PAXgene). RESULTS: Clear circumferential cell membrane staining was found only with the HER3 antibody clone DAK-H3-IC. Other antibodies (RTJ1, SP71, and polyclonal) yielded uncertain and nonreproducible staining results. In addition to cell membrane staining, DAK-H3-IC was also localized to the cytoplasm, but no nuclear staining was observed. In HER2-amplified breast cancers, 80% of samples were classified as 1+ or 2+ according to the HER3 staining on the cell membrane. The results from FFPE cell line samples were comparable to those obtained from unfixed cells in flow cytometry. IHC conducted on FFPE samples and on PAXgene-fixed samples showed equivalent results. CONCLUSIONS: We conclude that IHC with the monoclonal antibody, DAK-H3-IC, on FFPE samples is a reliable staining method for use in translational research. Assessment of membranous HER3 expression may be clinically relevant in selecting patients who may most benefit from pertuzumab or other novel anti-HER3 therapies.

Trastuzumab emtansine: mechanisms of action and drug resistance.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that is effective and generally well tolerated when administered as a single agent to treat advanced breast cancer. Efficacy has now been demonstrated in randomized trials as first line, second line, and later than the second line treatment of advanced breast cancer. T-DM1 is currently being evaluated as adjuvant treatment for early breast cancer. It has several mechanisms of action consisting of the anti-tumor effects of trastuzumab and those of DM1, a cytotoxic anti-microtubule agent released within thetarget cells upon degradation of the human epidermal growth factor receptor-2 (HER2)-T-DM1 complex in lysosomes. The cytotoxic effect of T-DM1 likely varies depending on the intracellular concentration of DM1 accumulated in cancer cells, high intracellular levels resulting in rapid apoptosis, somewhat lower levels in impaired cellular trafficking and mitotic catastrophe, while the lowest levels lead to poor response to T-DM1. Primary resistance of HER2-positive metastatic breast cancer to T-DM1 appears to be relatively infrequent, but most patients treated with T-DM1 develop acquired drug resistance. The mechanisms of resistance are incompletely understood, but mechanisms limiting the binding of trastuzumab to cancer cells may be involved. The cytotoxic effect of T-DM1 may be impaired by inefficient internalization or enhanced recycling of the HER2-T-DM1 complex in cancer cells, or impaired lysosomal degradation of trastuzumab or intracellular trafficking of HER2. The effect ofT-DM1 may also be compromised by multidrug resistance proteins that pump DM1 out of cancer cells. In this review we discuss the mechanism of action of T-DM1 and the key clinical results obtained with it, the combinations ofT-DM1 with other cytotoxic agents and anti-HER drugs, and the potential resistance mechanisms and the strategies to overcome resistance to T-DM1.

Trastuzumab-DM1 is highly effective in preclinical models of HER2-positive gastric cancer.

BACKGROUND: A novel antibody-drug conjugate (trastuzumab-DM1, T-DM1) is currently in clinical trials for patients with trastuzumab resistant HER2-positive breast cancer. Since no clinical data is available from gastric cancer, we studied T-DM1 on HER2-positive human gastric cancer cells and xenograft tumors. METHODS: Effects of T-DM1 were studied in four HER2-positive gastric cancer cell lines (N-87, OE-19, SNU-216 and MKN-7) in vitro. Xenograft tumors from N-87 and OE-19 were studied to determine the effect of T-DM1 in vivo. RESULTS: T-DM1 was found more effective than trastuzumab in N-87 and OE-19, and moderately effective in MKN-7 cells. On SNU-216 cells both trastuzumab and T-DM1 showed limited efficacy. In xenograft tumor experiments, complete pathological response was observed in all OE-19 xenografted mice and in half of the N-87 xenografted mice. The results were equally good irrespective of the tumor burden at therapy initiation, or preceding trastuzumab treatment. T-DM1 treatment showed direct effects (apoptotic cell death and aberrant mitosis) as well as it mediated antibody-dependent cellular cytotoxicity (ADCC). CONCLUSIONS: T-DM1 showed a promising anti-tumor effect in HER2-positive gastric cancer cell lines in vitro and in vivo, even in tumors which had developed resistance to trastuzumab. T-DM1 therapy may warrant clinical trials for HER2-positive gastric cancer patients.

Trastuzumab-DM1 causes tumour growth inhibition by mitotic catastrophe in trastuzumab-resistant breast cancer cells in vivo.

INTRODUCTION: Trastuzumab is widely used for the treatment of HER2-positive breast cancer. Despite encouraging clinical results, a significant fraction of patients are, or become, refractory to the drug. To overcome this, trastuzumab-DM1 (T-DM1), a newer, more potent drug has been introduced. We tested the efficacy and mechanisms of action of T-DM1 in nine HER2-positive breast cancer cell lines in vitro and in vivo. The nine cell lines studied included UACC-893, MDA-453 and JIMT-1, which are resistant to both trastuzumab and lapatinib. METHODS: AlamarBlue cell-proliferation assay was used to determine the growth response of breast cancer cell lines to trastuzumab and T-DM1 in vitro. Trastuzumab- and T-DM1-mediated antibody-dependent cellular cytotoxicity (ADCC) was analysed by measuring the lactate dehydrogenase released from the cancer cells as a result of ADCC activity of peripheral blood mononuclear cells. Severe Combined Immunodeficient (SCID) mice were inoculated with trastuzumab-resistant JIMT-1 cells to investigate the tumour inhibitory effect of T-DM1 in vivo. The xenograft samples were investigated using histology and immunohistochemistry. RESULTS: T-DM1 was strongly growth inhibitory on all investigated HER2-positive breast cancer cell lines in vitro. T-DM1 also evoked antibody-dependent cellular cytotoxicity (ADCC) similar to that of trastuzumab. Outgrowth of JIMT-1 xenograft tumours in SCID mice was significantly inhibited by T-DM1. Histologically, the cellular response to T-DM1 consisted of apoptosis and mitotic catastrophe, the latter evidenced by an increased number of cells with aberrant mitotic figures and giant multinucleated cells. CONCLUSIONS: Our results suggest mitotic catastrophe as a previously undescribed mechanism of action of T-DM1. T-DM1 was found effective even on breast cancer cell lines with moderate HER2 expression levels and cross-resistance to trastuzumab and lapatinib (MDA-453 and JIMT-1).

Multiple molecular mechanisms underlying trastuzumab and lapatinib resistance in JIMT-1 breast cancer cells.

Trastuzumab plays an important role in breast cancer therapy. However, a significant fraction of patients do not respond to therapy or they tend to develop resistance shortly after beginning therapy. Although some resistance mechanisms have been described, it is unclear whether these mechanisms can coexist. In this study, we analyzed the resistance mechanisms in the breast cancer cell line JIMT-1, a model of intrinsic trastuzumab resistance. We compared the JIMT-1 cell line with a panel of eight HER-2 positive breast cancer cell lines. All cell lines were characterized for the phosphatidylinositol 3-kinase (PIK3CA) mutation status, expression levels of the phosphatase and tensin homolog on chromosome 10 (PTEN) and neuregulin-1 (NRG1) mRNA, HER-2 gene copy number, and protein expression. The results were correlated to the sensitivity to trastuzumab and lapatinib as well as the potency of trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) evoked by trastuzumab. JIMT-1 cells showed several co-existing drug resistance mechanisms, including an activating mutation of the PIK3CA gene, low expression of PTEN, high expression of NRG1, and relatively low expression of HER-2 receptor protein (despite gene amplification). All these features were present at variable levels in other cell lines, whereas JIMT-1 was unique in displaying all these factors at the same time. Unexpectedly, ADCC reaction by normal lymphocytes was equally strong in all HER-2 positive cell lines, without any correlation to molecular markers or direct sensitivity to the drugs. Resistance to trastuzumab and lapatinib is probably caused by several co-existing molecular mechanisms. Direct sensitivity to trastuzumab and lapatinib was not correlated with ADCC.

Characterization of a novel, trastuzumab resistant human breast cancer cell line.

HER2-positive breast cancers represent a distinct phenotype and are intrinsically more aggressive than HER2-negative tumors. Although HER2-targeted therapies have been rationally developed, resistance to these treatments represents a process understood poorly. There are few experimental models that allow studying the molecular mechanism of resistance. Our aim was to characterize a trastuzumab resistant breast cancer cell line (B585) that was established from an invasive ductal carcinoma. B585 grows only in immunodeficient mice as a xenograft. CGH and FISH were used to define cytogenetic alterations, gene-expression analysis and immunohistochemistry were applied to detect RNA and protein expression. By array-CGH focused amplifications were identified for C-MYC, EGFR, ErbB2, CCND1 and TOP2-A oncogenes. ErbB2 was co-amplified with TOP2-A. mRNA overexpression was detected for the amplified genes. ErbB2 protein was overexpressed and showed heterogeneous distribution. In summary, molecular cytogenetic analysis and expression profiling of B585 revealed several new alterations. Based on the experiments performed in SCID mice and the genotypic/phenotypic characteristics, this new in vivo breast cancer xenograft is a valuable model to investigate molecular mechanism of trastuzumab resistance.

Die hard: are cancer stem cells the Bruce Willises of tumor biology?

In recent years, an exponentially growing number of studies have focused on identifying cancer stem cells (CSC) in human malignancies. The rare CSCs could be crucial in controlling and curing cancer: through asymmetric division CSCs supposedly drive tumor growth and evade therapy with the help of traits shared with normal stem cells such as quiescence, self-renewal ability, and multidrug resistance pump activity. Here, we give a brief overview of techniques used to confirm the stem cell-like behavior of putative CSCs and discuss markers and methods for identifying, isolating, and culturing them. We touch on the limitations of each marker and why the combined use of CSC markers, in vitro and in vivo assays may still fail to identify all relevant CSC populations. Finally, the various experimental findings supporting and contradicting the CSC hypothesis are summarized. The large number of tumor types thus far with a subpopulation of uniquely tumorigenic and therapy resistant cells suggests that despite the unanswered questions and inconsistencies, the CSC hypothesis has a legitimate role to play in tumor biology. At the same time, experimental evidence supporting the established alternative theory of clonal evolution can be found as well. Therefore, a model that describes cancer initiation and progression should combine elements of clonal evolution and CSC theory.

EGFR and ErbB2 are functionally coupled to CD44 and regulate shedding, internalization and motogenic effect of CD44.

Activation of the ErbB family of receptor tyrosine kinases is involved in a range of human cancers. Transmembrane signaling mediated by ErbB proteins is stimulated by peptide growth factors and is blocked by monoclonal antibodies such as trastuzumab and pertuzumab. ErbB receptors exert their function in conjunction with non-ErbB proteins, e.g. CD44. Here we show that epidermal growth factor (EGF) and heregulin induce CD44 shedding in JIMT-1, an ErbB2-overexpressing cell line resistant to trastuzumab, accompanied by internalization and intramembrane proteolysis of CD44 and enhanced cellular motility. These effects of EGF and heregulin are blocked by pertuzumab. Trastuzumab inhibits the heregulin- and hyaluronan oligosaccharide-induced shedding and internalization of CD44 and their motogenic effect. Trastuzumab also blocks CD44 shedding from JIMT-1 xenograft tumors in vivo. At the same time the internalization rate of trastuzumab is increased by hyaluronan oligosaccharide treatment in vitro. Our experiments point to an unexpected, but potentially important mechanism of action of ErbB receptor-targeted monoclonal antibodies used in the treatment of cancer.

ErbB-directed immunotherapy: antibodies in current practice and promising new agents.

The ErbB family is well known for its significance in oncogenesis. As bad prognostic markers, overexpressed or mutated ErbB1 and ErbB2 have an important role in the molecular diagnosis of various cancers, but as membrane proteins, expressed often selectively in tumor tissues, they can be targeted with kinase inhibitors or therapeutic antibodies. In addition to trastuzumab, the first humanized antibody that was approved for the therapy of solid tumors by the FDA, now many humanized antibodies are in late clinical trials, or already approved. Conjugation of antibodies with radioactive isotopes, cytotoxic agents or liposomes loaded with chemotherapeutic drugs led to improved therapeutic efficiency over their parent "unarmed" antibodies. Novel, engineered antibody derivates with better pharmacodynamic properties are also available and allow the targeting of ErbB1 or ErbB2 positive cancers in a wider patient population. In this review, we discuss the biological and clinical background of ErbB targeting, and describe the most successful antibodies against ErbB1 (cetuximab, panitumumab, matuzumab, nimotuzumab, ICR62, mAb 528, ch806 and MDX-447) and ErbB2 (trastuzumab, pertuzumab, MDX-H210, 2B1, C6.5xscFv(NM3E2), ertumaxomab and FRP-5 derivates) that are in clinical trials or already approved, along with the various relevant conjugation and engineering strategies. Recent data pertinent to the prevalent problem of clinical resistance to treatment with trastuzumab are also discussed.

Trastuzumab decreases the number of circulating and disseminated tumor cells despite trastuzumab resistance of the primary tumor.

We have recently shown that despite of the fact that the ErbB2-positive JIMT-1 human breast cancer cells intrinsically resistant to trastuzumab in vitro, trastuzumab inhibited the outgrowth of early phase JIMT-1 xenografts in SCID mice via antibody-dependent cellular cytotoxicity (ADCC). Here we show that trastuzumab significantly reduces the number of circulating and disseminated tumor cells (CTCs and DTCs) in this xenograft model system at a time when the primary tumor is already unresponsive to trastuzumab. This observation suggests that ErbB2 positive CTCs and DTCs might be sensitive to trastuzumab-mediated ADCC even if when the primary tumor is already non-responsive. Thus, trastuzumab treatment might also be beneficial in the case of patients with breast cancer that is already trastuzumab resistant.

Hyaluronan-induced masking of ErbB2 and CD44-enhanced trastuzumab internalisation in trastuzumab resistant breast cancer.

Although trastuzumab, a recombinant humanised anti-ErbB2 antibody, is widely used in the treatment of breast cancer, neither its mechanism of action, nor the factors leading to resistance are fully understood. We have previously shown that antibody-dependent cellular cytotoxicity is pivotal in the in vivo effect of trastuzumab against JIMT-1, a cell line showing in vitro resistance to the antibody, and suggested that masking of the trastuzumab-binding epitope by MUC-4, a cell surface mucin, took place. Here, we further explored the role of masking of ErbB2 in connection with CD44 expression and synthesis of its ligand, hyaluronan. We show that high expression of CD44 observed in JIMT-1 cells correlates with ErbB2 downregulation in vivo, while siRNA-mediated inhibition of CD44 expression leads to decreased rate of trastuzumab internalisation and low cell proliferation in vitro. An inhibitor of hyaluronan synthesis, 4-methylumbelliferon (4-MU) significantly reduced the hyaluronan level of JIMT-1 cells both in vivo and in vitro leading to enhanced binding of trastuzumab to ErbB2 and increased ErbB2 down-regulation. Furthermore, the inhibitory effect of trastuzumab on the growth of JIMT-1 xenografts was significantly increased by 4-MU treatment. Our results point to the importance of the CD44-hyaluronan pathway in the escape of tumour cells from receptor-oriented therapy.

Trastuzumab causes antibody-dependent cellular cytotoxicity-mediated growth inhibition of submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug resistance.

Trastuzumab is a recombinant antibody drug that is widely used for the treatment of breast cancer. Despite encouraging clinical results, some cancers are primarily resistant to trastuzumab, and a majority of those initially responding become resistant during prolonged treatment. The mechanisms of trastuzumab resistance have not been fully understood. We examined the role of antibody-dependent cellular cytotoxicity (ADCC) using JIMT-1 cells that are ErbB2 positive but intrinsically resistant to trastuzumab in vitro. Unexpectedly, in experiments mimicking adjuvant therapy of submacroscopic disease in vivo (JIMT-1 cells inoculated s.c. in severe combined immunodeficiency mice), trastuzumab was able to inhibit the outgrowth of macroscopically detectable xenograft tumors for up to 5-7 weeks. The effect is likely to be mediated via ADCC because trastuzumab-F(ab')(2) was ineffective in this model. Moreover, in vitro ADCC reaction of human leukocytes was equally strong against breast cancer cells intrinsically sensitive (SKBR-3) or resistant (JIMT-1) to trastuzumab or even against a subline of JIMT-1 that was established from xenograft tumors growing despite trastuzumab treatment. These results suggest that ADCC may be the predominant mechanism of trastuzumab action on submacroscopic tumor spread. Thus, measuring the ADCC activity of patient's leukocytes against the tumor cells may be a relevant predictor of clinical trastuzumab responsiveness in vivo.

EGFR gene copy number alterations in primary cutaneous malignant melanomas are associated with poor prognosis.

Copy number alterations of the epidermal growth factor receptor (EGFR) gene have been extensively analyzed in different cancers, but no data are available for primary malignant melanoma. The aim of the present study was to simultaneously investigate the EGFR gene and chromosome 7 copy number alterations in 81 cutaneous malignant melanomas by interphase FISH and correlate the data with clinicopathological parameters of patients. EGFR mRNA levels were detected by Affymetrix GeneChip Human Genome U133 Plus 2.0 expression arrays for 16 lesions. Both increased gene dosage and chromosome 7 alterations were found in 70% of tumors. Extra EGFR copies were detected in an additional 10% of samples. Polysomy 7 was associated with EGFR gene amplification. Significant correlation was found between EGFR alterations and histological subtypes, tumor thickness, ulceration and metastases formation. Amplification was significantly higher in lesions that developed metastases within 2 years after surgical excision of the primary tumor. Gene copy alterations were associated with elevated mRNA expression in 77% of lesions when compared to tumors with disomic EGFR status, the correlation was not directly proportional to gene copy number. Associations between protein expression and mRNA levels were even less prominent. In conclusion, our study indicates that amplification of the EGFR gene and polysomy 7 are frequent alterations in primary melanomas and are associated with bad prognosis. Further studies are required to clarify whether melanoma patients with EGFR alterations can benefit from anti-EGFR therapy.