Association between hypoxic volume and underlying hypoxia-induced gene expression in oropharyngeal squamous cell carcinoma.

BACKGROUND: Hypoxia imaging is a promising tool for targeted therapy but the links between imaging features and underlying molecular characteristics of the tumour have not been investigated. The aim of this study was to compare hypoxia biomarkers and gene expression in oropharyngeal squamous cell carcinoma (OPSCC) diagnostic biopsies with hypoxia imaged with 64Cu-ATSM PET/CT. METHODS: 64Cu-ATSM imaging, molecular and clinical data were obtained for 15 patients. Primary tumour SUVmax, tumour to muscle ratio (TMR) and hypoxic volume were tested for association with reported hypoxia gene signatures in diagnostic biopsies. A putative gene signature for hypoxia in OPSCCs (hypoxic volume-associated gene signature (HVS)) was derived. RESULTS: Hypoxic volume was significantly associated with a reported hypoxia gene signature (rho=0.57, P=0.045), but SUVmax and TMR were not. Immunohistochemical staining with the hypoxia marker carbonic anhydrase 9 (CA9) was associated with a gene expression hypoxia response (rho=0.63, P=0.01). Sixteen genes were positively and five genes negatively associated with hypoxic volume (adjusted P<0.1; eight genes had adjusted P<0.05; HVS). This signature was associated with inferior 3-year progression-free survival (HR=1.5 (1.0-2.2), P=0.047) in an independent patient cohort. CONCLUSIONS: 64Cu-ATSM-defined hypoxic volume was associated with underlying hypoxia gene expression response. A 21-gene signature derived from hypoxic volume from patients with OPSCCs in our study may be linked to progression-free survival.

Methods: Using Three-Dimensional Culture (Spheroids) as an In Vitro Model of Tumour Hypoxia.

Regions of hypoxia in tumours can be modelled in vitro in 2D cell cultures with a hypoxic chamber or incubator in which oxygen levels can be regulated. Although this system is useful in many respects, it disregards the additional physiological gradients of the hypoxic microenvironment, which result in reduced nutrients and more acidic pH. Another approach to hypoxia modelling is to use three-dimensional spheroid cultures. In spheroids, the physiological gradients of the hypoxic tumour microenvironment can be inexpensively modelled and explored. In addition, spheroids offer the advantage of more representative modelling of tumour therapy responses compared with 2D culture. Here, we review the use of spheroids in hypoxia tumour biology research and highlight the different methodologies for spheroid formation and how to obtain uniformity. We explore the challenge of spheroid analyses and how to determine the effect on the hypoxic versus normoxic components of spheroids. We discuss the use of high-throughput analyses in hypoxia screening of spheroids. Furthermore, we examine the use of mathematical modelling of spheroids to understand more fully the hypoxic tumour microenvironment.

CD31 angiogenesis and combined expression of HIF-1α and HIF-2α are prognostic in primary clear-cell renal cell carcinoma (CC-RCC), but HIFα transcriptional products are not: implications for antiangiogenic trials and HIFα biomarker studies in primary CC-RCC.

Hypoxia-inducible factors, HIF-1α and HIF-2α, are expressed in the majority of clear-cell renal cell carcinoma (CC-RCC). In vitro, HIFα isoforms regulate a differential set of genes, and their effects in vivo within CC-RCC tumours may affect outcome. The role of angiogenesis and HIFα transcriptional products, including those involved in cell metabolism and morphological dedifferentiation have not been extensively investigated and might have relevance to the development of antiangiogenic or anti-HIFα trials in primary CC-RCC, either before or after radical nephrectomy. We analysed 168 consecutive clear-cell renal tumours from 1983 to 1999 within tissue microarrays and assessed expression of HIF-1α and HIF-2α together with the protein expression of seven of their target genes (BNIP3, CA9, Cyclin D1, GLUT-1, LDH5, Oct-4 and VEGF). The expression of these factors was compared with patient overall survival and CD31 angiogenesis. We found that HIFα antigenicity deteriorated with the age of the paraffin block (P < 0.0001) and in tumours from 1983 to 1992 was deemed not to be reliable. Similar findings were found in aged archival osteosarcoma samples. This might have important implications for retrospective biomarker studies that rely on archival tissue material. HIF-1α(HIGH)/HIF-2α(LOW) tumours had a worse overall survival compared with HIF-1α(LOW)/HIF-2α(LOW) tumours (P = 0.04). Surprisingly, on multivariate analysis, high levels of CD31(+) angiogenesis was shown to be an independent prognostic marker of increased overall survival (P = 0.003). We propose that better differentiation of vascular endothelium may be a reflection of a greater production of vessel stabilization factors versus pro-angiogenic factors, and therefore a less aggressive phenotype.

New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes.

Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.

Roles of tetrahydrobiopterin in promoting tumor angiogenesis.

Nitric oxide (NO), which is derived from endothelial NO synthase (eNOS), provides crucial signals for angiogenesis in the tumor microenvironment. Tetrahydrobiopterin (BH4) is an absolute requirement for eNOS activity. In this study, we investigated whether this activation is both maintained by a wild-type Ras/phosphatidylinositol 3-kinase (PI3K)/Akt-positive feedback loop in endothelial cells and affects tumor angiogenesis. We found that supplementation of BH4 (via the pterin salvage pathway with Sep) increased Akt/eNOS phosphorylation in both human eNOS-transfected COS-7 cells and endothelial cells concomitant with increases in NO production, cell proliferation, migration, and tube formation. This augmentation was abrogated by a PI3K inhibitor. Sepiapterin (Sep) also increased GTP-bound wild-type Ras and PI3K/Akt/eNOS activation, which was prevented by the eNOS inhibitor, Nω-Nitro-L-arginine methyl ester (L-NAME). Furthermore, expression of GTP cyclohydrolase I (the rate-limiting enzyme in de novo BH4 synthesis) under doxycycline control potentiated in vivo tumorigenesis, tumor cell proliferation, as well as angiogenesis. Conversely, both switching off GTP cyclohydrolase I expression as well as inhibiting its enzymatic activity significantly decreased eNOS expression and tumor vascularization. This study demonstrates an important role for BH4 synthesis in angiogenesis by the activation of eNOS for NO production, which is maintained by a PI3K/Akt-positive feedback loop through effects on wild-type Ras in endothelial cells. Our findings suggest that BH4 synthesis may be a rational target for antiangiogenesis therapy for tumors.

Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer.

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.

ADGRL4/ELTD1 Expression in Breast Cancer Cells Induces Vascular Normalization and Immune Suppression.

ELTD1/ADGRL4 expression is increased in the vasculature of a number of tumor types and this correlates with a good prognosis. Expression has also been reported in some tumor cells with high expression correlating with a good prognosis in hepatocellular carcinoma (HCC) and a poor prognosis in glioblastoma. Here we show that 35% of primary human breast tumors stain positively for ELTD1, with 9% having high expression that correlates with improved relapse-free survival. Using immunocompetent, syngeneic mouse breast cancer models we found that tumors expressing recombinant murine Eltd1 grew faster than controls, with an enhanced ability to metastasize and promote systemic immune effects. The Eltd1-expressing tumors had larger and better perfused vessels and tumor-endothelial cell interaction led to the release of proangiogenic and immune-modulating factors. M2-like macrophages increased in the stroma along with expression of programmed death-ligand 1 (PD-L1) on tumor and immune cells, to create an immunosuppressive microenvironment that allowed Eltd1-regulated tumor growth in the presence of an NY-ESO-1-specific immune response. Eltd1-positive tumors also responded better to chemotherapy which could explain the relationship to a good prognosis observed in primary human cases. Thus, ELTD1 expression may enhance delivery of therapeutic antibodies to reverse the immunosuppression and increase response to chemotherapy and radiotherapy in this subset of tumors. ELTD1 may be useful as a selection marker for such therapies. IMPLICATIONS: ELTD1 expression in mouse breast tumors creates an immunosuppressive microenvironment and increases vessel size and perfusion. Its expression may enhance the delivery of therapies targeting the immune system.

Automated uro-oncology data collection: the Cancer Research Uro-Oncology Database.

OBJECTIVE: To develop a relational database (Cancer Research Uro-Oncology Database, CRUD) to enable automatic data collection on all urological malignancies within our region, as there is increasing emphasis on good data collection for surgical patients with cancer, and numerous overlapping systems that are amassing data on the same patients. METHODS: Links have been established between pathological databases, multidisciplinary team data-collection systems and patient-survival monitoring facilities, providing accurate pathology, treatment and survival data on all of uro-oncology patients. We are also developing individual modules for the oncological surgeons within our unit that are compatible with the British Association of Urological Surgeons (Section of Oncology), and have plans to connect to the Medical and Clinical Oncology data systems in the future. RESULTS: Pre-existing protocols for fresh tissue, plasma and urine collection have been incorporated within CRUD via a tissue-tracking system, to comply with the Human Tissue Act 2004, and link these samples to accurate clinical, pathological and survival data. Many research and audit projects have already used these data, including the construction of a 274-case tissue microarray for renal cell carcinoma, microRNA hybridization arrays and analysis of 900 nephrectomy cases from the past three decades. CONCLUSIONS: Our work over the past 3 years in Oxford has established numerous links with organizations collecting data on our uro-oncological patients, and we are now able to collect this excellent combined data on all of these patients, in an automated manner.

Prognostic value of an activation state marker for epidermal growth factor receptor in tissue microarrays of head and neck cancer.

Overexpression and mutation of epidermal growth factor receptors (EGFR) have been shown to be important in the prognosis of several cancers, including head and neck cancers. However, our inability to define the activation status of these and other receptors limits our ability to assess the importance of these pathways and to exploit effectively new molecularly targeted treatments directed at their catalytic activities. Here we describe the use of automated, high-throughput fluorescence lifetime imaging microscopy to measure EGFR autophosphorylation status by fluorescence resonance energy transfer (FRET) in head and neck tumors. We have correlated FRET efficiency with the clinical and survival data. The results from head and neck arrays show that high FRET efficiency is correlated with worsening disease-free survival but not with overall survival. This powerful tool could be exploited as a new independent quantitative prognostic factor in clinical decisions and cancer management.

Expression of terminally glycosylated calcitonin receptor-like receptor in uterine leiomyoma: endothelial phenotype and association with microvascular density.

PURPOSE: The role for the hypoxia-inducible angiogenic factor adrenomedullin (AM) in tumor growth and progression has been suggested. Calcitonin receptor-like receptor (CL) is a G protein-coupled receptor (GPCR) that mediates effects of AM, but little information is available on its expression and functional state in human tumors. The present study attempted to determine CL potential for antiangiogenic therapy of uterine leiomyoma. EXPERIMENTAL DESIGN AND RESULTS: GPCR CL is transported to the cell surface and recognized by AM only when terminally/mature glycosylated. The presence and localization of this form of the receptor in tumor and surrounding myometrial tissues obtained from leiomyoma-bearing uteri were examined using deglycosylation, immunoblotting, and immunofluorescence analysis. The mature CL glycoprotein was expressed in both tissues and localized exclusively in normal and tumor endothelium within leiomyoma-bearing uteri. The functionality of the receptor expressed in myometrial microvascular endothelial cells (MMVEC) was examined in vitro using receptor internalization and angiogenic assays. The mature CL glycoprotein expressed by primary MMVECs was functional because AM interacted with this GPCR and induced its internalization as well as angiogenic effects (proliferation and migration) in MMVECs in vitro. Finally, the levels of tissue-expressed mature CL glycoprotein as a functional form of this GPCR were analyzed by immunoblotting. The expression of this functional form of the receptor in vivo was significantly decreased (P = 0.01) in leiomyoma tissue, and this was concurrent with the decrease in microvascular density (measured by Chalkley counting) in tumor compared with surrounding myometrium (P = 0.031). CONCLUSIONS: Our findings suggest that GPCR CL mediates angiogenic effects of AM in myometrium and that further evaluation of the properties of the CL expressed in both normal and tumor endothelium in vivo may be essential before targeting this endothelial GPCR for antiangiogenic therapies.

The role of hypoxia-inducible factor-1 in three-dimensional tumor growth, apoptosis, and regulation by the insulin-signaling pathway.

The purpose of this study was to establish the effect of hypoxia-inducible factor-1 (HIF-1) directly on tumor growth, independently of angiogenesis. This was done by growing wild-type mouse hepatoma cells (HEPA-1) and their HIF-1-deficient counterpart C4 as multicellular tumor spheroids and quantifying differences in growth rates and proliferative and apoptotic indices. Insulin and insulin-like growth factor-I are key growth factors, also able to regulate hypoxia-responsive genes via HIF-1; thus, the effects of insulin on this model were also investigated. Two-dimensional growth was serum dependent and no difference was seen between wild-type HEPA-1 and C4 cell growth profiles, but major differences were seen in three-dimensional growth. HIF-1 supported spheroid growth under hypoxia as the HEPA-1 spheroids grew faster than the C4. Surprisingly, the HIF-1-deficient cells had a higher proliferation rate in three-dimensional growth (C4 mean S-phase index, 13.6%; HEPA-1 mean S-phase index, 9%; P = 0.009) that was associated with an inhibition of apoptosis. However, the apoptosis rate was much greater in these spheroids (C4 mean apoptotic index, 6.4; HEPA-1 mean apoptotic index, 0.78%; P = 0.0006). Addition of insulin increased proliferation and apoptosis in both HEPA-1 and C4 spheroids, demonstrating an HIF-1-independent effect of insulin signaling in three-dimensional growth. These results indicate that the enhancing effect of HIF-1 in three-dimensional tumor growth is a balance of both reduced proliferation and enhanced survival, the latter being proportionally greater.

Role of Delta-like 4 in Jagged1-induced tumour angiogenesis and tumour growth.

Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands implicated in tumour angiogenesis. They were shown to have opposite effects on mouse retinal and adult regenerative angiogenesis. In tumours, both ligands are upregulated but their relative effects and interactions in tumour biology, particularly in tumour response to therapeutic intervention are unclear. Here we demonstrate that DLL4 and JAG1 displayed equal potency in stimulating Notch target genes in HMEC-1 endothelial cells but had opposing effects on sprouting angiogenesis in vitro. Mouse DLL4 or JAG1 expressed in glioblastoma cells decreased tumour cell proliferation in vitro but promoted tumour growth in vivo. mDLL4-expressing tumours showed fewer but larger vessels whereas mJAG1-tumours produced more vessels. In both tumour types pericyte coverage was decreased but the vessels were more perfused. Both ligands increased tumour resistance towards anti-VEGF therapy but the resistance was higher in mDLL4-tumours versus mJAG1-tumours. However, their sensitivity to the therapy was restored by blocking Notch signalling with dibenzazepine. Importantly, anti-DLL4 antibody blocked the effect of JAG1 on tumour growth and increased vessel branching in vivo. The mechanism behind the differential responsiveness was due to a positive feedback loop for DLL4-Notch signalling, rendering DLL4 more dominant in activating Notch signalling in the tumour microenvironment. We concluded that DLL4 and JAG1 promote tumour growth by modulating tumour angiogenesis via different mechanisms. JAG1 is not antagonistic but utilises DLL4 in tumour angiogenesis. The results suggest that anti-JAG1 therapy should be explored in conjunction with anti-DLL4 treatment in developing anti-Notch therapies in clinics.

Relation of erythropoietin and erythropoietin receptor expression to hypoxia and anemia in head and neck squamous cell carcinoma.

PURPOSE: The use of erythropoietin in head and neck squamous cell carcinoma (HNSCC) has been associated with poor survival. This study examines the protein and mRNA expression of erythropoietin and erythropoietin receptor in HNSCC and their relation to hypoxia, hemoglobin (Hb), and clinical outcome. EXPERIMENTAL DESIGN: The immunohistochemical expression of erythropoietin and erythropoietin receptor was assessed in 151 cases of HNSCC. Expression was compared with the hypoxia-dependent proteins hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase-9 (CA-9) and correlated with clinical outcome. The mRNA expression of erythropoietin and erythropoietin receptor was measured in paired samples of HNSCC. RESULTS: Erythropoietin and erythropoietin receptor were expressed in 95% and 99% of tumors, respectively. Using a weighed expression score, there was a positive correlation between erythropoietin and erythropoietin receptor expression (r = 0.18, P = 0.03). HIF-1alpha (r = 0.38, P < 0.01) and CA-9 (r = 0.26, P = 0.002) correlated with erythropoietin expression, but there was no correlation with erythropoietin receptor. No correlation was found between Hb and erythropoietin (r = 0.07, P = 0.36) or erythropoietin receptor (r = -0.02, P = 0.8), and no survival difference between high and low erythropoietin or erythropoietin receptor expression (P = 0.59 and P = 0.98, respectively). The mRNA expression of erythropoietin (P = 0.03) but not erythropoietin receptor (P = 0.62) was significantly increased in 11 paired samples of HNSCC. CONCLUSION: In vivo, the HIF pathway regulates erythropoietin at the mRNA level but not erythropoietin receptor expression in HNSCC. Anemia does not seem to influence the hypoxic microenvironment of tumors sufficiently to alter the expression of erythropoietin. The effects of exogenous erythropoietin may be acting via receptors expressed on tumor cells in vivo, or on vascular cells, which also express the pathway.

Frozen biopsy collection and storage: frozen biopsy samples.

This chapter describes some simple standard operating procedures for the regular collection of samples from surgical resections and their rapid preservation by freezing for long-term cryogenic storage.

Preparation of tumor homogenates for subsequent preparation of cytosols, membrane fractions, RNA, and DNA.

This chapter outlines methods for the preparation of several different cellular fractions from whole samples of tumor and normal tissue.

Isolation of DNA from tumor samples.

A method for producing high-molecular-weight DNA from pulverized tissue, nuclear fractions, or cultured cells. This isolation method relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent sodium dodecyl sulfate. Ethylenediaminetetraacetic acid is included in the lysis buffer to inhibit DNases.

Isolation of RNA from tumor samples: single-step guanidinium acid-phenol method.

The guanidinium acid-phenol method of RNA extraction is relatively fast (4 h) and is useful for the processing of large numbers of samples, without the need for ultracentrifugation. This protocol produces total RNA that includes ribosomal, transfer, and messenger RNA. This high-quality RNA is suitable for Northern blot analysis, dot-blot hybridization, poly (A) RNA selection, in vitro translation, cDNA library construction, reverse transcriptase-polymerase chain reaction, ribonuclease protection assay, and primer extension experiments.

Hypoxia-inducible factors HIF-1alpha and HIF-2alpha in head and neck cancer: relationship to tumor biology and treatment outcome in surgically resected patients.

Hypoxia within head and neck squamous cell carcinoma (HNSCC) predicts a poor response to radiotherapy and poor prognosis. Hypoxia-inducible factor (HIF)-1 and HIF-2 are nuclear transcription factors that regulate the cellular response to hypoxia and are important for solid tumor growth and survival. Overexpression of HIF-1alpha and HIF-2alpha was demonstrated in three HNSCC cell lines under hypoxia and tumor tissue versus normal tissue (n = 20, HIF-1alpha, P = 0.023; HIF-2alpha, P = 0.013). On immunostaining, HIF-1alpha and HIF-2alpha expression were localized to tumor nuclei; HIF-2alpha expression was also seen in tumor-associated macrophages. Expression of HIF-1alpha in surgically treated patients with HNSCC (n = 79) was associated with improved disease-free survival (P = 0.016) and overall survival (P = 0.027).

Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer.

How tumors access and spread via the lymphatics is not understood. Although it is clear that dissemination via the blood system involves hemangiogenesis, it is uncertain whether tumors also induce lymphangiogenesis or simply invade existing peritumoral vessels. To address the issue we quantitated tumor lymph vessels in archival specimens of head and neck cancer by immunostaining for the recently described lymphatic endothelial marker LYVE-1, the vascular endothelial marker CD34, and the pKi67 proliferation marker, correlating lymph vessel density and proliferation index with clinical and pathological variables. Discrete "hotspots" of intratumoral small proliferating lymphatics were observed in all carcinomas, and a high intratumoral lymph vessel density was associated with neck node metastases (n = 23; P = 0.027) and an infiltrating margin of tumor invasion (P = 0.046) in the oropharyngeal subgroup. Quantitation of the lymphangiogenic growth factor vascular endothelial growth factor C by real-time PCR and immunohistochemistry revealed higher levels of mRNA in tumor tissue than in normal samples (n = 8; P = 0.017), but no obvious correlation with intratumoral lymphatics. Our results provide new evidence that proliferating lymphatics can occur in human cancers and may in some cases contribute to lymph node metastasis.

Relation of hypoxia-inducible factor-2 alpha (HIF-2 alpha) expression in tumor-infiltrative macrophages to tumor angiogenesis and the oxidative thymidine phosphorylase pathway in Human breast cancer.

Tumor-associated macrophages (TAMs) produce angiogenic factors and in breast cancer are associated with high vascular grade and poor survival. TAMs preferentially migrate to hypoxic areas within tumors and strongly express hypoxia-inducible factor (HIF)-2 alpha. This study examined whether HIF-2 alpha was involved in TAM angiogenic activation by correlating its expression with tumor microvessel density as a marker of angiogenesis, and other tumor variables, in a series of human primary invasive breast carcinomas. A correlation was found between high TAM HIF-2 alpha and high tumor vascularity (P < 0.0001), as well as high tumor grade (P = 0.007). The relation of HIF-2 alpha expression to a recently described oxygen-dependent pathway of angiogenesis was also studied, and an inverse relationship was found between TAM HIF-2 alpha and tumor thymidine phosphorylase expression (P = 0.02). These results suggest that TAM HIF-2 signaling may be a useful target for future antiangiogenic strategies but show that tumors use both oxygen-dependent and oxygen deficiency-regulated pathways for angiogenesis. Thus, combined blockade of pathways and careful assessment of these pathways in trials are necessary.