STAT3 is over-activated within CD163pos bone marrow macrophages in both Multiple Myeloma and the benign pre-condition MGUS.

Tumour-associated macrophages (TAMs) support cancer cell survival and suppress anti-tumour immunity. Tumour infiltration by CD163pos TAMs is associated with poor outcome in several human malignancies, including multiple myeloma (MM). Signal transducer and activator of transcription 3 (STAT3) is over-activated in human cancers, and specifically within TAMs activation of STAT3 may induce an immunosuppressive (M2-like) phenotype. Therefore, STAT3-inhibition in TAMs may be a future therapeutic strategy.We investigated TAM markers CD163, CD206, and activated STAT3 (pSTAT3) in patients with MGUS (n = 32) and MM (n = 45), as well as healthy controls (HCs, n = 13).Blood levels of the macrophage biomarkers sCD163 and sCD206, and circulating cytokines, as well as bone marrow mRNA expression of CD163 and CD206, were generally increased in MGUS and MM patients, compared to HCs, but to highly similar levels. By immunohistochemistry, bone marrow levels of pSTAT3 were increased specifically within CD163pos cells in both MGUS and MM patients.In conclusion, macrophage-related inflammatory changes, including activation of STAT3, were present already at the MGUS stage, at similar levels as in MM. Specific increase in pSTAT3 levels within CD163pos cells supports that the CD163 scavenger receptor may be a useful target for future delivery of STAT3-inhibitory drugs to TAMs in MM patients.

Validation of the UK myeloma research alliance risk profile, a new clinical prediction model for outcome in patients with newly diagnosed multiple myeloma not eligible for autologous stem cell transplantation; a population-based study from the Danish national multiple myeloma registry.

In 2019 the UK Myeloma Research Alliance introduced the Myeloma Risk Profile (MRP) for prediction of outcome in patients with newly diagnosed multiple myeloma (MM), ineligible for autologous stem cell transplantation. To validate the MRP in a population-based setting we performed a study of the entire cohort of transplant ineligible MM patients above 65 years in the Danish National MM Registry. Our data confirmed the value of the MRP. In a cohort of 1,377 patients, the MRP score separated patients into three distinct risk-groups with an observed hazard ratio of 2.91 for early death in high-risk versus low-risk patients.

Circulating extracellular vesicle-associated CD163 and CD206 in multiple myeloma.

OBJECTIVES: Extracellular vesicles (EVs) are important for intercellular signalling in cancer. Tumour-associated macrophages, expressing the haemoglobin-haptoglobin and mannose receptors CD163 and CD206, are crucial for cancer progression. We recently identified CD163 on EVs in the circulation as a fraction of total soluble CD163 (sCD163). Here, we investigated the presence of CD163 and CD206-positive EVs (EV-CD163, EV-CD206) in patients with multiple myeloma (MM). METHODS: We enrolled patients with MM (n = 32), monoclonal gammopathy of undetermined significance (MGUS) (n = 8) and healthy donors (n = 16). Plasma protein levels were determined by ELISA before and after vesicle precipitation. Monocytes were examined by flow cytometry, and leucocyte CD163 mRNA by qPCR. RESULTS: Fractions of EV-CD163 and EV-CD206 were significantly elevated in patients with newly diagnosed MM (median = 39.8%, 76.5%, respectively) compared to patients with relapse (15.6%, P = .02, 42.5%, P = .003), remission (16.9%, P < .0001, 25.2%, P < .0001), MGUS (17.8%, P < .01, 33.1%, P = .0005) and healthy donors (14.8%, P < .0001, 35.5%, P < .0001). Whole blood CD163 mRNA did not vary between the groups. The intermediate monocyte subset showed a higher CD163 expression in newly diagnosed patients. CONCLUSIONS: Our results indicate that macrophage-derived EVs may play a role in the late phase of malignant progression of MM, and encourage further EV investigations in functional experiments.

Serum galectin-1 in patients with multiple myeloma: associations with survival, angiogenesis, and biomarkers of macrophage activation.

Galectin-1 (Gal-1) is known to regulate cell signaling within the immune system and may be a target for new anticancer immune therapy. In patients with chronic lymphocytic leukemia (CLL) and classical Hodgkin lymphoma (cHL), high levels of Gal-1 within the tumor microenvironment were associated with worse disease state or poor outcome. Gal-1 can be secreted from cells by an unknown mechanism, and levels in blood samples were associated with high tumor burden and worse disease state in cHL and CLL patients. However, serum levels of Gal-1 have never been investigated in patients with multiple myeloma (MM). We measured serum Gal-1 levels in samples from patients with treatment demanding MM at the time of diagnosis (n=102) and after treatment (n=24) and examined associations of serum Gal-1 with clinicopathological information obtained from patient medical records, as well as data on bone marrow angiogenesis and the macrophage activation biomarkers soluble CD163 (sCD163) and soluble mannose receptor. Serum Gal-1 levels were not elevated in patients with MM at diagnosis compared with healthy donors (median values 8.48 vs 11.93 ng/mL, P=0.05), which is in contrast to results in cHL and CLL. Furthermore, Gal-1 levels did not show association with bone marrow angiogenesis, clinicopathological parameters, overall survival, or response to treatment. There was a statically significant association between Gal-1 and sCD163 levels (R=0.24, P=0.02), but not with soluble mannose receptor (P=0.92). In conclusion, our results indicate that Gal-1 is not an important serum biomarker in MM, which is in contrast to data from patients with cHL and CLL. However, the association with sCD163 is in line with previous data showing that Gal-1 may be involved in alternative (M2-like) activation of macrophages.

A rare presentation of cardiac amyloid deposits isolated to intramural vessels.

The present case illustrates the diagnostic challenges in symptomatic patients with heart failure of unknown etiology. The patients were previously diagnosed with κ-light chain amyloidosis without cardiac involvement. Echocardiography showed heart failure with mildly reduced ejection fraction but no signs of amyloidosis. Coronary angiogram showed normal arteries and 11C-PIB positron emission tomography was negative for amyloid deposits. Exercise testing revealed severe heart failure and reduced coronary flow velocity reserve. Endomyocardial biopsies showed amyloid in the intramural coronary arteries without interstitial amyloid deposits. Hence, the patient was diagnosed with microvascular dysfunction-induced heart failure due to vessel wall amyloidosis.

Smoldering multiple myeloma risk factors for progression: a Danish population-based cohort study.

Several risk scores for disease progression in patients with smoldering multiple myeloma (SMM) have been proposed; however, all have been developed using single-center registries. To examine risk factors for time to progression (TTP) to multiple myeloma (MM) for SMM, we analyzed a nationwide population-based cohort of 321 patients with newly diagnosed SMM registered within the Danish Multiple Myeloma Registry between 2005 and 2014. Significant univariable risk factors for TTP were selected for multivariable Cox regression analyses. We found that both an M-protein ≥30 g/L and immunoparesis significantly influenced TTP (HR 2.7, 95%CI (1.5;4.7), P = 0.001, and HR 3.3, 95%CI (1.4;7.8), P = 0.002, respectively). High free light chain (FLC) ratio did not significantly influence TTP in our cohort. Therefore, our data do not support recent IMWG proposal of identifying patients with FLC ratio above 100 as having ultra high-risk of transformation to MM. Using only immunoparesis and M-protein ≥30 g/L, we created a scoring system to identify low-, intermediate-, and high-risk SMM. This first population-based study of patients with SMM confirms that an M-protein ≥30 g/L and immunoparesis remain important risk factors for progression to MM.

The novel biomarker of alternative macrophage activation, soluble mannose receptor (sMR/sCD206): Implications in multiple myeloma.

Tumor-associated macrophages (TAMs) play an important role in the pathophysiology of human malignancies. They support growth of cancer cells by promoting angiogenesis, and by inhibiting tumour cell apoptosis and anti-tumor immune reactions. Several membrane proteins are well-described markers of human TAMs, including the haemoglobin scavenger receptor CD163 and the macrophage mannose receptor (MR/CD206). Interestingly, both CD163 and MR exist as soluble serum proteins (sCD163 and sMR) that may reflect the activation state of tissue macrophages, including TAMs. Here, we report the first data on sMR as a biomarker in patients with a malignant disease. We have measured concentrations of sMR in peripheral blood serum (n=104) from patients with newly diagnosed multiple myeloma (MM) by an enzyme-linked immunosorbent assay, and examined associations with data from medical records. At diagnosis, sMR levels were elevated in 27% of patients, and decreased after treatment. Further, sMR levels were associated with prognostic markers in MM, and elevated sMR (>0.43mg/L) was an independent marker of overall survival in a multivariate analysis (HR=2.20, P=0.006). Levels of sMR in blood samples showed significant association with sCD163, which may indicate common origin from CD163+MR+TAMs.

Upregulation of Syndecan-1 in the bone marrow microenvironment in multiple myeloma is associated with angiogenesis.

OBJECTIVES: Syndecan-1 (SDC1), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and interleukin-6 (IL6) are expressed by malignant plasma cells and cells in the bone marrow microenvironment and may be involved in the angiogenic process in multiple myeloma (MM). METHODS: In this study, we examined the association between bone marrow angiogenesis estimated as micro-vessel density (MVD) and gene expression of SDC1, HGF, VEGF and IL6 in whole bone marrow biopsies from healthy volunteers (n = 10), patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 35) and MM (n = 65). RESULTS: MVD was significantly higher in patients with MM than MGUS (P = 0.03) and was positively correlated with plasma cell percentage (P = 0.002). SDC1 gene expression increased with increasing MVD in patients with MGUS and MM (P < 0.001). A positive correlation between bone marrow plasma cell percentage and SDC1 gene expression was detected in patients with MM (P < 0.001). Importantly, after adjustment for plasma cell percentage, the association between MVD and SDC1 gene expression remained significant (P = 0.026). No association between bone marrow angiogenesis and gene expression of HGF, VEGF and IL6 was seen. CONCLUSION: Our study indicates that SDC1 expressed by the bone marrow microenvironment is involved in angiogenesis in MM.

Polymorphisms in the heparanase gene in multiple myeloma association with bone morbidity and survival.

OBJECTIVES: In multiple myeloma, heparanase (HSPE) is involved in myeloma cell growth, angiogenesis, osteoclastogenesis and shedding of syndecan-1, a key player in myeloma pathophysiology. Different single nucleotide polymorphisms (SNPs) in the HSPE gene with effect on gene function have been described, and some are associated with haematological malignancies. METHODS: In this study, we evaluated four SNPs rs11099592, rs4364254, rs4693608 and rs6535455 in the HSPE gene in 348 newly diagnosed multiple myeloma patients with focus on bone morbidity (lytic bone disease and vertebral fractures) and outcome after high-dose chemotherapy with stem cell support (HDT). RESULTS: We observed that homozygous carriers of the rs4693608 wild-type A-allele had a higher frequency of vertebral fractures compared to carriers of the variant G-allele, P = 0.02. In multivariate analysis, homozygous carriers of the rs6535455 variant T-allele had a longer survival than homo- and heterozygous carriers of the wild-type C-allele, hazard ratio 0.3 (95% CI 0.1-0.7, P = 0.002). CONCLUSION: The SNPs rs4693608 and rs6535455 in the HSPE gene may influence bone morbidity and outcome in multiple myeloma. Our results are an interesting observation but can be chance findings and need confirmation in studies exploring the functional role of SNPs in the HSPE gene in multiple myeloma.

Monocyte/macrophage-derived soluble CD163: a novel biomarker in multiple myeloma.

OBJECTIVES: Macrophages play an important role in cancer by suppression of adaptive immunity and promotion of angiogenesis and metastasis. Tumor-associated macrophages strongly express the hemoglobin scavenger receptor CD163, which can also be found as a soluble protein in serum and other body fluids (soluble CD163, sCD163). In this study, we examined serum sCD163 as a biomarker in patients with newly diagnosed multiple myeloma. METHODS: Peripheral blood (n = 104) and bone marrow (n = 17) levels of sCD163 were measured using an enzyme-linked immunosorbent assay. RESULTS: At diagnosis, high sCD163 was associated with higher stage according to the International Staging System (ISS) and with other known prognostic factors in multiple myeloma (creatinine, C-reactive protein, and beta-2 microglobulin). Soluble CD163 decreased upon high-dose treatment, and in a multivariate survival analysis including the covariates treatment modality and age at diagnosis, higher levels of sCD163 were associated with poor outcome (HR = 1.82; P = 0.010). The prognostic significance of sCD163 was lost when including ISS stage in the model (HR = 1.51; P = 0.085). Soluble CD163 values were significantly higher in bone marrow samples than in the matched blood samples, which indicate a localized production of sCD163 within the bone marrow microenvironment. CONCLUSIONS: Soluble CD163 was found to be a prognostic marker in patients with multiple myeloma. This may indicate that macrophages and/or monocytes have an important role in the bone marrow microenvironment of myeloma patients, supporting myeloma cell proliferation and survival. We propose the serum sCD163 value 1.8 mg/L as a cutoff concentration for survival analysis in patients with multiple myeloma, which should be validated in future studies.

Vascular endothelial growth factor (VEGF) gene polymorphisms may influence the efficacy of thalidomide in multiple myeloma.

Vascular endothelial growth factor (VEGF) is a potent proangiogenic factor. Several single nucleotide polymorphisms (SNPs) in the VEGF gene with influence on VEGF expression have been described. In multiple myeloma, VEGF stimulates angiogenesis which is correlated with disease progression and prognosis. In this study, we evaluated the association between genetic variations in the VEGF gene in patients with multiple myeloma and time to treatment failure (TTF) after high-dose melphalan and stem cell support (HDT), overall survival (OS) and efficacy of the anti-angiogenic drug thalidomide. Retrospectively, the SNPs -2,578C>A (rs699947), -460C>T (rs833061), +405G>C (rs2010963) and +936C>T (rs3025039) in the VEGF gene were examined in 348 patients with newly diagnosed multiple myeloma initially treated with HDT, where 176 patients were treated with thalidomide at relapse. None of the examined geno- or haplotypes was associated with differences in TTF after initial therapy or OS. A possible relation between the haplotype -2,578A/-460C/+405G (ACG) and effect of thalidomide was seen. Patients with no copies of the haplotype ACG had a longer time to next treatment than patients with one or two copies of the haplotype ACG, median 13.7 months vs. 9.2 months, p=0.007. In conclusion, the haplotype ACG in the VEGF gene may influence the efficacy of thalidomide in multiple myeloma. Further analyses are needed to confirm these findings and get insight into the functional effect of these polymorphisms, so in the future we may be able to select multiple myeloma patients who especially will benefit from treatment with thalidomide.

Single nucleotide polymorphisms in the promoter region of the IL1B gene influence outcome in multiple myeloma patients treated with high-dose chemotherapy independently of relapse treatment with thalidomide and bortezomib.

Little is known on the impact of polymorphisms in the IL1B gene on outcome in multiple myeloma. In a population-based study of 348 Danish myeloma patients treated with high-dose treatment (HDT), 146 patients treated with INF-α maintenance treatment, and in 243 patients with relapse, we analysed the impact on outcome of HDT, INF-α maintenance treatment, and treatment with thalidomide and bortezomib at relapse, in relation to the major identified functional polymorphisms in the promoter region of IL1B. The wild-type C-allele of IL1B C-3737T and non-carriage of the IL1B promoter haplotype TGT (-3737(T), -1464(G) and -31(T)), giving high IL1B promoter activity, were associated with longer time-to-treatment failure (TTF) (HR, 1.4 (1.0-1.9) and 1.5 (1.1-2.0)) and overall survival (HR, 1.8 (1.2-2.6) and 1.6 (1.1-2.3)) after HDT. Among INF-α treated patients, a trend towards better TTF was found in patients carrying the wild-type C-allele of IL1B C-3737T (HR, 1.6 (1.1-2.4)). Furthermore, among INF-α treated patients, gene-gene interaction studies on IL1B C-3737T and NFКB1-94ins/del ATTG revealed a fourfold increase in TTF for homozygous carriers of wild-type alleles at both loci as compared to variant allele carriers at both loci. No relation to genotype and outcome was found for relapse patients treated with thalidomide or bortezomib. Our results indicate that a subpopulation of myeloma patients carrying the wild-type C-allele of IL1B C-3737T and non-carriers of the promoter haplotype TGT (-3737(T), -1464(G) and -31(T)) benefit from a better outcome of HDT and INF-α treatment, an effect that may be related to the NF-κB pathway.

The importance of a sub-region on chromosome 19q13.3 for prognosis of multiple myeloma patients after high-dose treatment and stem cell support: a linkage disequilibrium mapping in RAI and CD3EAP.

The gene RAI was originally described as an inhibitor of RelA/p65 subunit of nuclear factor κB (NF-κB). Here, we analyse the association between genetic variation in the genes RAI and CD3EAP and outcome of 348 myeloma patients treated with high-dose treatment (HDT), 146 patients treated with interferon-α (INF-α) as maintenance treatment, 177 patients treated with thalidomide, and 74 patients treated with bortezomib at relapse and address if the effects of polymorphisms in CD3EAP and RAI are modified by a functional polymorphism in NFКB1. By linkage disequilibrium mapping, we found that variant alleles of several polymorphisms in a sub-region of 19q13.3 spanning the regions RAI-intron1-1 to RAI intron1-3 and the region exon1 to exon3­6 in CD3EAP were associated with prolonged time-to-treatment failure (TTF; p = 0.003) and overall survival (OS; p = 0.02). Haplotype analyses revealed that none of the haplotypes were more strongly associated to TTF or OS than the two strongly linked SNPs, RAI-intron1-1 (rs4572514) and CD3EAP G-21A (rs967591). The association of RAI-intron1-1 and CD3EAP G-21A with TTF was independent of NFKB1-94 ins/del, but homozygous ins-allele carriers which were also variant allele carriers of RAI-intron1-1 or CD3EAP G-21A had the longest OS. Among patients treated with INF-α or thalidomide, no effect was seen in relation to genotype. Our results indicate that polymorphism in RAI and CD3EAP are associated with outcome of myeloma patients treated with HDT. Combination analyses with the functional polymorphism in NFKB1 suggest that a possibly functional effect of RAI or CD3EAP could be related to NF-κB availability.

No influence of the polymorphisms CYP2C19 and CYP2D6 on the efficacy of cyclophosphamide, thalidomide, and bortezomib in patients with Multiple Myeloma.

BACKGROUND: The response to treatment varies among patients with multiple myeloma and markers for prediction of treatment outcome are highly needed. Bioactivation of cyclophosphamide and thalidomide, and biodegradation of bortezomib, is dependent on cytochrome P450 metabolism. We explored the potential influence of different polymorphisms in the CYP enzymes on the outcome of treatment. METHODS: Data was analyzed from 348 patients undergoing high-dose treatment and stem cell support in Denmark in 1994 to 2004. Clinical information on relapse treatment in 243 individual patients was collected. The patients were genotyped for the non-functional alleles CYP2C19*2 and CYP2D6*3, *4, *5 (gene deletion), *6, and CYP2D6 gene duplication. RESULTS: In patients who were treated with bortezomib and were carriers of one or two defective CYP2D6 alleles there was a trend towards a better time-to-next treatment. We found no association between the number of functional CYP2C19 and CYP2D6 alleles and outcome of treatment with cyclophosphamide or thalidomide. Neither was the number of functional CYP2C19 and CYP2D6 alleles associated with neurological adverse reactions to thalidomide and bortezomib. CONCLUSION: There was no association between functional CYP2C19 and CYP2D6 alleles and treatment outcome in multiple myeloma patients treated with cyclophosphamide, thalidomide or bortezomib. A larger number of patients treated with bortezomib are needed to determine the role of CYP2D6 alleles in treatment outcome.

Improved survival of multiple myeloma patients with late relapse after high-dose treatment and stem cell support, a population-based study of 348 patients in Denmark in 1994-2004.

OBJECTIVE: To analyse if patients with early relapse after high-dose chemotherapy with stem cell support (HDT) benefit from new treatment strategies in a population-based setting. METHODS: We conducted a retrospective study of relapse treatment and survival in 348 patients undergoing HDT in Denmark in 1994-2004. Patients were divided into two groups according to time-to-treatment failure (i) within 18 months after HDT and (ii) later than 18 months after HDT. The fraction of patients surviving 3 yr after first relapse was evaluated in relation to calendar periods for introduction of new drugs: before the introduction of thalidomide (1995-1999), before the introduction of bortezomib and lenalidomide (2000-2002) and when patients had access to all treatment modalities (2003-2008). RESULTS: Two hundred and forty-three patients suffered from relapse which required treatment. The median follow-up time was 91.4 months (60-158.8 months) and overall survival was 56.3 months after HDT. The fraction of patients alive 3 yr after first relapse increased in the periods after the year 2000 for patients with late relapse: 1995-1999, 36%; 2000-2002, 57%; and 2003-2008, 72% (P = 0.03), in contrast to patients with early relapse: 1995-1999, 25%; 2000-2002, 33%; and 2003-2008, 31% (P = 0.7). CONCLUSION: Improved survival was only observed among patients with late relapse after HDT and this may be because of increased use of salvage HDT, improved supportive care and introduction of new drugs.

A polymorphism in NFKB1 is associated with improved effect of interferon-{alpha} maintenance treatment of patients with multiple myeloma after high-dose treatment with stem cell support.

BACKGROUND: Maintenance therapy with interferon-alpha after high-dose treatment with stem cell support in multiple myeloma has been intensively debated. In this study, we evaluated the response to treatment with interferon-alpha in relation to genetic variation in genes related to inflammation. DESIGN AND METHODS: In a retrospective study of 296 patients with multiple myeloma undergoing high-dose therapy between 1994 and 2004, 146 patients were treated with interferon-alpha as maintenance therapy. We tested the polymorphisms IL1B T-31C, IL6 G-174C, NFKB1-94ins/delATTG, CD3EAP G-21A and PPP1R13L IVS1 A4364G for associations with time to treatment failure and overall survival with and without interferon-alpha treatment. RESULTS: The wild type ins-allele of polymorphism NFKB1-94 ins/delATTG was, by multivariate Cox analysis, associated with longer time to treatment failure (p=0.01) and overall survival (p=0.0084) when tested between treatment arms and in the subgroup of patients treated with interferon-alpha the wild type ins-allele was associated with longer overall survival (p=0.002). In the absence of interferon-alpha treatment, there was no association between the polymorphisms and treatment outcome, except for patients homozygous for the wild type G allele of IL6 G-174C who survived longer (p= 0.0074) than variant allele carriers. There was no association between the polymorphisms IL1B T-31C, CD3EAP G-21A and PPP1R13L IVS1 A4364G and treatment outcome for interferon-alpha. CONCLUSIONS: Patients who are homozygous carriers of the wild type ins-allele of the NFKB1 -94ins/delATTG polymorphism may benefit from treatment with interferon-alpha, in contrast to patients carrying the variant allele. This result may indicate that the effect of interferon-alpha treatment is dependent on the availability of nuclear factor-kappaB and the polymorphism in NFKB1 may, therefore, be a good prognostic marker for multiple myeloma patients on maintenance treatment with interferon-alpha after high-dose therapy. A prospective study of interferon-alpha treatment in relation to NFKB1 -94ins/delATTG is highly warranted.

High serum YKL-40 concentration is associated with severe bone disease in newly diagnosed multiple myeloma patients.

OBJECTIVES: In multiple myeloma (MM) YKL-40 is present in the bone marrow microenvironment and is suggested to play a role in remodelling of the extracellular matrix. Here, the association between serum YKL-40 and severity of bone disease in MM is investigated. METHODS: Serum YKL-40 was measured in 34 MM patients at diagnosis. Bone disease was assessed by radiography and biochemical markers of bone metabolism. Patients were treated with conventional chemotherapy and followed for up to 30 months. RESULTS: Patients with a serum YKL-40 elevated above the age specific reference range (56%) had a higher total X-ray score (P = 0.003) and higher levels of the markers of bone resorption serum C-terminal telopeptide of collagen type I (P = 0.003), urine pyridinoline (P = 0.04) and urine deoxypyridinoline (P = 0.002), while the levels of urine N-terminal telopeptide of collagen type I (NTX-1) and the markers of bone formation equalled those seen in patients with a normal serum YKL-40. Serum YKL-40 level (beta = 7.6; P = 0.002) and urine NTX-1 (log(2)) (beta = 3.5; P = 0.006) were independent predictors of total X-ray score at diagnosis. Patients with elevated serum YKL-40 at diagnosis had shorter time to first osteolytic event compared to patients with normal serum YKL-40 (12 months vs. not reached; Log rank test: P = 0.03). CONCLUSIONS: Newly diagnosed MM patients with elevated serum concentrations of YKL-40 have more severe bone destruction including increased bone resorptive activity and shorter time to progression of bone disease. At this point, a potential role for YKL-40 in myeloma-related bone disease must be considered.