Genetic polymorphisms of platelet adhesive molecules: association with breast cancer risk and clinical presentation

The main platelet adhesive receptors integrin 21, integrin IIb3 and glycoprotein (GP) Ib are also expressed in breast carcinoma cells. They play a key role in tumor cell-induced platelet aggregation and in adhesive interactions necessary for tumoral invasion and metastasis. Several polymorphisms affecting these molecules, two in integrin 2 (C807T and G1648A), one in integrin 3 (T1565C) and one in GP Ib (VNTR), influencing their levels, structure, and possibly their function, have been previously described and associated with cardiovascular diseases. In this study, we investigated the association of these polymorphisms with breast cancer risk or clinical presentation. We studied 101 patients with invasive breast cancer. The main prognostic variables were recorded, and genomic PCR analysis of these polymorphisms was performed. A group of 101 control subjects matched on age and sex was studied and compared with patients. No association was found between VNTR (GP Ib) polymorphism and breast cancer risk or presentation. Genotype and allele frequencies of C807T and G1648A polymorphisms of integrin 2 were not statistically different in breast cancer patients and controls, although we found an association between the 1648G/G genotype and higher disease stages (III and IV) (p = 0.02). Breast cancer risk was higher in carriers of 3 integrin T/T genotype (OR = 2.08, 95% CI = 1.04–4.16, p = 0.04). Furthermore, genotype 1565T/T was also associated with axillary nodal metastasis (p = 0.017) and with tumoral diameter greater than 2 cm (p = 0.02). Although confirmatory studies are needed, our results suggest that polymorphic genetic variation of integrins expressed in platelets and epithelial breast cells could modify the risk and the biological aggressiveness of breast carcinomas.     

Vitamin E concentration in breast adipose tissue of breast cancer patients (Kuopio, Finland)

Previous data on animals and humans suggest that vitamin E may be a protective factor against cancer. A low dietary vitamin E intake has been suggested to increase the risk of breast cancer. We examined the dietary intake and the concentration of vitamin E in breast adipose tissue of women in Kuopio, Finland, diagnosed between 1990 and 1992 with benign breast disease (n=34) and with breast cancer (n=32). In postmenopausal women, lower dietary intake (P=0.006) and a smaller concentration of vitamin E in breast adipose tissue (P=0.024) were observed in breast cancer patients than in subjects with benign breast disease. Partial correlation showed that the vitamin E concentration in the breast adipose tissue correlated positively with the dietary intake of vitamin E (r=0.25, P=0.023), indicating that the vitamin E concentration in breast adipose tissue reflects the dietary intake of vitamin E.Drs Zhu and Uusitupa are with the Department of Clinical Nutrition, University of Kuopio, Finland, Dr Parviainen is with the Laboratory of Helsinki University, Central Hospital, Helsinki, Finland. Drs Männistö and Pietinen are with the Department of Nutrition, National Public Health Institute, Helsinki, Finland Dr Eskelinen is with the Department of Surgery, and Dr Syrjänen is with the Department of Pathology, University of Kuopio. Address correspondence to Professor Uusitupa, Department of Clinical Nutrition, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. This study was supported by research grants from the Finnish Cancer Society, and by EVO funding for the Breast Cancer Project of Kuopio Cancer Research Center from the Kuopio University Hospital, Finland.     

Carotenoids,retinol, and vitamin E and risk of proliferative benign breast disease and breast cancer

We investigated the relationship between serum levels of retinol, -carotene, -carotene, lycopene, -tocopherol, and -tocopherol as well as intakes of retinol, carotene, and vitamin E and the risks of breast cancer and proliferative benign breast disease (BBD) in a case-control study of postmenopausal women in the Boston, MA (United States) area. Serum nutrient data were available for 377 women with newly diagnosed stage I or II breast cancer and 173 women with proliferative BBD. Controls were 403 women who were evaluated at the same institutions but did not require a breast biopsy or whose biopsy revealed nonproliferative BBD. We observed no significant associations between serum levels of these micronutrients and risk of proliferative BBD or breast cancer. The risk of breast cancer was decreased among women in the highest quintile of intake of vitamin E from food sources only (odds ratio [OR] for the highest quintile = 0.4,95 percent confidence interval [CI]=0.2–0.9; P, trend across quintiles = 0.02) but less so for total vitamin E intake including supplements (OR=0.7, CI=0.4–1.3; P, trend = 0.07).This project was supported by research grant CA 40429 from the National Cancer Institute. Dr London was supported in part by an Institutional National Research Service Award (CA 09001) from the National Cancer Institute.     

The timing of treatment in breast cancer: gaps and delays in treatment can be harmful

Timing of treatment in breast cancer may refer to intervals within a single management or between different managements. Rates of shrinkage of breast cancers in response to treatment are related to histological grade and may be used as surrogates for growth rates. Histological grade should predict appropriate timing of treatment. Four cases of locally advanced breast cancer that illustrate a number of different types of interval are presented. Two tumours of differing histological grade (II and III) had been managed by historical split-course radiotherapy and two similar grade III tumours were managed by primary medical treatment, followed at different intervals by radiotherapy. In the grade III tumours different radiotherapy fractionation régimes and effects of varying intervals between mangements are compared. The theoretical advantage of shrinkage (leading to reoxygenation) during the gap in split-course radiotherapy is realized only in relatively slowly growing and shrinking tumours. Grade III tumours grow rapidly. They have the potential to shrink rapidly in response to appropriate treatment, namely intensive chemotherapy or radiotherapy but not hormones. Inadequate treatment leads to growth in intervals between individual doses, whether of drugs or radiation, and to failure of local control. The advantage of surgery or primary medical treatment will be lost if the interval between managements is too long in relation to the volume doubling time. Histological grade is a good guide of this parameter; the grade III tumours are particularly vulnerable to gaps in treatment.     

Carotenoids, alpha-tocopherols, and retinol in plasma and breast cancer risk in northern Sweden

Objective: Using a nested case–referent design we evaluated the relationship between plasma levels of six carotenoids, alpha-tocopherol, and retinol, sampled before diagnosis, and later breast cancer risk. Methods: In total, 201 cases and 290 referents were selected from three population-based cohorts in northern Sweden, where all subjects donated blood samples at enrolment. All blood samples were stored at –80 °C. Cases and referents were matched for age, age of blood sample, and sampling centre. Breast cancer cases were identified through the regional and national cancer registries. Results: Plasma concentrations of carotenoids were positively intercorrelated. In analysis of three cohorts as a group none of the carotenoids was found to be significantly related to the risk of developing breast cancer. Similarly, no significant associations between breast cancer risk and plasma levels of -tocopherol or retinol were found. However, in postmenopausal women from a mammography cohort with a high number of prevalent cases, lycopene was significantly associated with a decreased risk of breast cancer. A significant trend of an inverse association between lutein and breast cancer risk was seen in premenopausal women from two combined population-based cohorts with only incident cases. A non-significant reduced risk with higher plasma -carotene was apparent throughout all the sub-analyses. Conclusion: In conclusion, no significant associations were found between plasma levels of carotenoids, -tocopherol or retinol and breast cancer risk in analysis of three combined cohorts. However, results from stratified analysis by cohort membership and menopausal status suggest that lycopene and other plasma-carotenoids may reduce the risk of developing breast cancer and that menopausal status has an impact on the mechanisms involved.     

The T61 human breast cancer xenograft: An experimental model of estrogen therapy of breast cancer

Summary Endocrine therapy is one of the principal treatment modalities of breast cancer, both in an adjuvant setting and in advanced disease. The T61 breast cancer xenograft described here provides an experimental model of the effects of estrogen treatment at a molecular level. T61 is an estrogen receptor positive tumor which was originally derived from a T1N0M0 invasive ductal cancer and has been carried as a serially transplanted xenograft in nude mice. T61 is a hormone sensitive tumor whose growth is suppressed by both estrogen and tamoxifen, in contrast to other estrogen receptor positive tumors such as MCF-7 which are stimulated by estrogen.Molecular studies have demonstrated that T61 expresses easily detectable levels of mRNA for a number of peptide growth factors, including transforming growth factor alpha (TGF-) and insulin-like growth factors I and II (IGF-I and IGF-II), but not transforming growth factor beta-1 (TGF-1). Of these, IGF-II is the only peptide whose expression is altered by endocrine therapy. Treatment of T61-bearing nude mice with physiologic doses of estrogen is accompanied by loss of IGF-II mRNA expression within 24 hours, and rapid regression of tumor. T61 tumor growth is also inhibited in animals treated with a monoclonal antibody which blocks binding of ligand to the IGF-I receptor, which mediates the mitogenic signal of bound IGF-II through autophosphorylation of its intracellular tyrosine kinase domain.These results demonstrate the utility of the T61 model in the study of the molecular mechanism of estrogen therapy in breast cancer, and suggest that in this system, modulation of a specific growth factor (IGF-II) by endocrine therapy can have profound effects on tumor growth.     

Glioblastoma Multiforme in an Asian Population: Evidence for a Distinct Genetic Pathway

In Singapore astrocytic tumours occur in only 25% of patients with primary brain tumours compared to 40–60% in other series. Glioblastoma multiforme arises either de novo as a primary glioblastomas associated with epidermal growth factor receptor (EGFR) and mdm2 over-expression or as a secondary glioblastomas, through malignant progression from low-grade astrocytomas, associated with p53 mutations and PDGFR- over-expression. Using immunohistochemical methods and DNA sequencing, we studied our population of glioblastomas for over-expression of EGFR, mdm2, p53, and PDGFR- as well directly for mutations of the p53 gene. While levels of over-expression of EGFR and mdm2 were consistent with levels expected for primary glioblastomas, levels of p53 and PDGFR- were consistent with levels documented for secondary glioblastomas. Notably 96% of the samples over-expressed p53 as detected with monoclonal antibody pAb 240. Of the 39 samples available for DNA sequencing 18% (7/39) had p53 mutations, including three mutations previously undocumented in glioblastomas. These results provide strong evidence that glioblastomas in Asian patients do not conform to currently accepted models of glioblastoma development, and that clinically defined glioblastomas in these patients show genetic changes consistent with both primary and 'secondary glioblastomas.     

Clinical significance of prostacyclin and thromboxane in cancer of the female breast and genital tract

Studies investigating the role of thromboxane A₂ and prostacyclin in cancer of the female breast and genital tract are reviewed. Whereas thromboxane A₂ was found to promote the tumour growth and metastasis, prostacyclin exerted a protective effect in maintaining vascular and platelet homeostasis. Thus, monitoring of prostacyclin and thromboxane levels in plasma and urine of cancer patients may be essential for the evaluation of tumour growth and metastasis. Of all modulators of thromboxane and prostacyclin biosynthesis, nafazatrom was found to exhibit promising results for the treatment of advanced breast cancer, although its use in the routine therapy is questionable at this stage.     

Comparison of topoisomerase-IIalpha gene status between primary breast cancer and corresponding distant metastatic sites

Background. Topoisomerase-II (topo-II) is a key enzyme in DNA replication and a molecular target for anti-cancer drugs called topoisomerase-II inhibitors, such as anthracyclines. Its value as a predictive marker of responsiveness to these cytotoxic drugs is currently being evaluated with promising results. However, even in the metastatic setting, the choice of treatment is based on the biologic characteristics of the primary tumor. Few data are available regarding the expression of biological markers between the primary tumor and the corresponding distant metastases. Methods. Topo-II gene status was evaluated in 29 breast cancer patients in which a primary tumor sample and a corresponding metastatic sample were both available. Fluorescent in situ hybridization (FISH) with the Vysis triple probe (Vysis multi-color topo-II spectrum orange, Her-2 spectrum green and CEP17 spectrum aqua probe) was used, which allowed the concomitant evaluation of HER-2 gene status. Results. As previously reported, topo-II gene aberrations are always associated with HER-2 gene amplification; indeed no topo-II gene aberrations have been observed in the HER-2 negative tumors. Conversely, 38.5% (five patients) of the HER-2 positive primary breast tumors (13 patients) were topo-II amplified, while 61.5% (eight patients) had a normal topo-II gene. No topo-II gene deletion was found in our series. Topo-II gene amplification in the primary tumor was always associated with amplification in the corresponding metastases, and no metastases with topo-II gene amplification were seen without amplification in the primary tumor. Furthermore, the amplification level of topo-II (i.e., ratio topo-II: CEP17) remained unchanged in primary and metastatic sites. Conclusion. Despite the low number of patients, our results seem to indicate that topo-II gene status evaluation in the primary breast tumor accurately reflects its status in the corresponding distant metastases.     

Hereditary persistence of {alpha}-fetoprotein: Case report and review of the literature

Persistently elevated -fetoprotein (AFP) levels of 24 to 30µg/ml (normal <10 µg/ml) were found in a 38-year-old healthyman. Subsequently, AFP was found to be elevated in another five out of 13family members within three generations. The pedigree is consistent with anautosomal dominant inheritance pattern. No discernible disease and nofunctional abnormality appears to be associated with this clinically benigndisorder which has been recorded in the literature on four occasions todate. The reported AFP levels in these other cases ranged from 18 to 198µg/ml.Physiologically, AFP is mainly produced in the liver and the yolk sac ofhuman fetuses more than four weeks old, with peak values of up to 4 mg/ml at12 to 16 weeks of gestation. After birth, AFP levels usually fall, withineight to 12 months, to a very low concentration of <10 µg/ml andpersist at low levels throughout life. However, AFP levels can rise abovenormal in both children and adults in distinct conditions and diseases whichwill be discussed.Hereditary persistence of -fetoprotein (HPAFP) should be consideredin both children and adults with unexplained and persistent elevation of AFPe.g., those screened for hepatocellular carcinoma or diagnosed for germ celltumor. It should also be recognized in AFP screening for neural tube defectsor Down's syndrome during pregnancy. Hereditary persistence of AFP can beeasily confirmed by analyzing AFP levels in family members.     

Prognostic relevance of transforming growth factor alpha (TGF-α) and tumor necrosis factor alpha (TNF-α) detected in breast cancer tissues by immunohistochemistry

Summary The function of different growth factors in the development and progression of malignant tumors and the role of cytotoxic cytokines in the host response generated against neoplasms have been recently studied. Anti-TGF- and anti-TNF- monoclonal antibody families have been developed and characterized previously by our laboratory. Libraries of anti-TGF- and anti-TNF- monoclonal antibodies were selected for equal immunoreactivity both in native (frozen) and in formaldehyde fixed, paraffin embedded histological sections. No differences were found between native and fixed samples demonstrated in 10 cases in the present prospective study. Retrospective investigation was performed in 35 histopathological specimens of breast cancer patients detailed clinically and observed during 5 years after the surgical treatment. Correlation between TGF- and/or TNF- expression and clinical staging - TNM score, lymph node metastasis, tumor recurrence and survival time - was analyzed. According to our present study, the TGF- positive patients had worse clinical prognosis than the TNF- positive and double positive cases during long term observation.     

Urinary 2-hydroxyestrone/16alpha-hydroxyestrone ratio and family history of breast cancer in premenopausal women

It has been hypothesized that the ratio of urinary 2-hydroxyestrone to 16-hydroxyestrone (2-OHE₁/16-OHE₁ represents a biomarker for breast cancer risk; the lower the ratio the higher the risk. We obtained early morning urine samples from 70 'high risk' premenopausal women who had a first degree family history of breast cancer and 27 'low risk' women with no such history. Five estrogen metabolites in urine were determined: 2-OHE₁, 16-OHE₁, estrone (E₁), estradiol (E₂), and estriol (E₃) conjugates. We compared geometric mean levels of each metabolite adjusted for age and weight. 'High risk' women did not have elevated levels of any of these metabolites. Instead, we observed decrements of 3–27% in women with a family history of breast cancer compared with women without such history, this difference was statistically significant for E₂, 2- OHE₁, and 16-OHE₁. The ratio of 2-OHE₁/16-OHE₁ was identical in women with and without a family history of breast cancer. These results were unchanged, when additionally adjusted for recent intake of alcohol or cruciferous vegetables. Our data suggest that among premenopausal women, family history is not associated with higher urinary estrogen levels or a lower ratio of urinary 2-OHE₁/16-OHE₁.     

Infiltrating dendritic/Langerhans cells in primary breast cancer

It is fully anticipated that dendritic cells (DCs) will become a mainstay for inclusion in biological therapies for patients with cancer including breast cancer. To elucidate the cellular composition of DCs infiltrating human breast cancers, we investigated the correlations between the density of infiltrating DCs and some clinicopathological factors of breast cancer patients, examined cytokine expression on cancer cells and finally, assessed the numbers of CD45RO⁺ tumor infiltrating lymphocytes (TIL). Tissues adjacent to cancer nests contained significantly more S-100 protein⁺ and S-100 protein⁺ CD1a^– DCs, but less CD1a⁺ DCs, than the nests. In invasive ductal carcinomas infiltration by S-100 protein⁺ DCs within and adjacent to nests, CDla⁺ DCs within nests and S-100 protein⁺ CD1a^– DCs adjacent to nests was denser than that in non-invasive carcinomas. With respect to the histological subtypes, there were fewer DCs in scirrhous carcinomas. Patients with stage IV disease had significantly fewer DCs of primary lesions than at other clinical stages. There were good correlations between infiltration by S-100 protein⁺ DCs and expression of the cytokines GM-CSF, IL-1 and TNF- on cancer cells and between GM-CSF expression and S-100 protein⁺ CD1a^– DCs. There was a close correlation between CD45RO⁺ TIL and S-100 protein⁺ DC densities both within and adjacent to the cancer nests and the S-100 protein⁺ CD1a^– DC density adjacent to the cancer nests. Despite extensive immunoelectron microscopic observation, CD1a⁺ DCs within cancer nests contained only few Birbeck's granule-like structure. These data indicate that cancer nests are infiltrated predominantly by CD1a⁺ DCs, whereas S-100 protein⁺ CD1a^– DCs predominate in surrounding tissues, and a infiltration by DCs may require cytokine expression on cancer cells and simultaneous lymphocyte infiltration. The findings of this clinicopathological study indicate the importance of evaluating simultaneously the types and localizations of infiltrating DCs in cancer tissues.     

Tamoxifen and fenretinide in women with metastatic breast cancer

Background: Tamoxifen and fenretinide combination therapy has been shown to be an active treatment regimen in metastatic breast cancer patients. This pilot study sought to determine whether the addition of fenretinide to tamoxifen would be associated with antitumor activity in metastatic breast cancer patients who had been previously treated with tamoxifen or who had hormone receptor negative disease. The effect of this therapy on circulating plasma transforming growth factorbeta (TGF) levels and serum lipids was also examined.Patientsand Methods: Thirtyone patients were treated with tamoxifen (20mg po daily), and fenretinide (400mg po daily with a 3day drug holiday each month). Plasma TGF testing was performed using isoform specific sandwich ELISA.Results: Twenty four of the 31 patients were evaluable for an antitumor response including 14 estrogen receptor (ER) positive patients who had failed prior tamoxifen therapy, seven ERnegative patients, and three hormone therapy naive ERpositive patients. There were no objective antitumor responses; three patients had stable disease for 8, 8, and 24 months. Five patients (16%) discontinued therapy for toxicity (one for grade 3 skin rash and four for abnormal dark adaptation). There was a statistically significant decrease in total cholesterol (median change per patient of –13.5mg/dl; p=0.049, a 6.5% decrease), and an increase in HDL levels (median change per patient of +18mg/dl, p=0.0001, a 35% increase) with tamoxifen and fenretinide therapy.TGF-1 plasma levels were normal in 26 of 28 patients, and no changes in these levels post-treatment were demonstrated.Conclusions: Tamoxifen and fenretinide therapy is not an active combination in ER negative metastatic breast cancer or in patients whose disease has progressed on tamoxifen. This combination had a beneficial effect on total serum cholesterol and HDL levels with no associated rise in serum triglyceride levels. The 400mg dose of fenretinide was associated with symptomatic nyctalopia in one-third of patients making it an unsuitable dose for use in breast cancer prevention studies.     

HER-2/neu and topoisomerase IIalpha in breast cancer

In breast cancer, the predominant genetic mechanism for oncogene activation is through an amplification of a gene. The HER-2 (also known as ErbB2/c-erbB2/HER-2/neu) oncogene is the most frequently amplified oncogene in breast cancer, and its overexpression is associated with poor clinical outcome. In addition to its important role in breast cancer growth and progression, HER-2 is also a target for a new form of chemotherapy. Breast cancer patients have been treated with considerable success since 1998 with trastuzumab, a recombinant antibody designed to block signaling through HER-2 receptor. HER-2 has also been implicated in altering the chemosensitivity of breast cancer cells to different forms of conventional cytotoxic chemotherapy, particularly of topoII-inhibitors (e.g., anthracyclines). Topoisomerase II gene is located just by the HER-2 oncogene at the chromosome 17q12–q21 and is amplified or deleted in almost 90% of the HER-2 amplified primary breast tumors. Recent data suggests that amplification and deletion of topoisomerase II may account for both relative chemosensitivity and resistance to anthracycline therapy, depending on the specific genetic defect at the topoII locus. Expanding our understanding of HER-2 amplification also changes its role in the pathogenesis of breast cancer. HER-2 is an oncogene that clearly can drive tumor induction and growth and is also a target for a new kind of chemotherapy, but its function as a marker for chemoselection may be due to associated genetic changes, of which topoisomerase II is a good example. Moreover, despite potential evidence that genes other than HER-2, such as topoisomerase II, may be more important predictors of therapeutic response in breast cancer, HER-2 status still has a very significant role in therapeutic selection, mainly as the major criterion for administering trastuzumab in treating breast cancer. Thus, the clinical and therapeutic importance of the HER-2 and topoisomerase II status to breast cancer management should only increase in the next few years.     

Growth factor involvement in the multihormonal regulation of MCF-7 breast cancer cell growth in soft agar

Summary The hormone dependency of the MCF-7 breast cancer cell line, while extensively tested in liquid culture, has not been previously evaluated under conditions of anchorage-independent growth in serum-free media. Using the soft agar clonogenic assay, we demonstrate that physiologically relevant concentrations of estradiol (E₂), progesterone (Pg), and prolactin (PRL) similarly stimulated MCF-7 cell colony formation in the absence of serum. Addition of an anti-insulin-like growth factor-I (IGF-I) antibody inhibited E₂- and Pg-stimulated growth, while PRL action was not affected. Similar results were obtained with an anti-IGF-I receptor antibody, except that its inhibitory effect on Pg-induced colony formation was modest and not statistically significant. Administration of either an anti-transforming growth factor- (TGF-) antibody or an anti-epidermal growth factor (EGF) receptor antibody similarly inhibited E₂-stimulated MCF-7 cell growth in soft agar, while neither antibody influenced Pg or PRL effects. Addition of TGF-₁, -₂, -₃ similarly suppressed MCF-7 cell colony formation in a dose dependent manner to a degree comparable to that observed with 4-OH-tamoxifen (4-OH-T). Furthermore, the growth inhibitory effect of 4-OH-T was completely reversed by an anti-TGF- antibody. We conclude that IGFs and TGF- are important mediators of E₂-stimulated MCF-7 cell growth in soft agar. IGFs may also be playing a role in Pg action, while neither growth factor is involved in PRL-stimulated colony formation. Finally, TGF- appears to be an important mediator of antiestrogen-induced inhibition of tumor growth.Abbreviations IGF insulin-like growth factor - TGF transforming growth factor - E₂ estradiol-17 - Pg progesterone - PRL prolactin - 4-OH-T 4-hydroxy-tamoxifen - IMEM improved minimal essential medium     

Evaluation of response to postradiation eight in one chemotherapy in childhood brain tumors

Ten children, 3 to 15 years of age with high risk primary brain tumors were treated with postradiation eight in one chemotherapy; vincristine, lomustine, procarbazine, hydroxyurea, cisplatin, cytosine arabinoside, cyclophosphamide and methylprednisolone. The tumors comprised of three medulloblastomas, two primitive neuroectodermal tumors, one ependymoblastoma and four anaplastic ependymomas. Treatment involved surgery (two total resection, six subtotal and two biopsy only) followed by conventional radiotherapy (primary tumor: 50–54 Gy, whole brain: 30–45 Gy, and spinal axis: 25–36 Gy). Objective tumor response with radiotherapy was achieved in 7 of 9 patients (78%) (6/8 patients with residual tumor and one patient with complete resection but positive cerebrospinal fluid cytology). Complete response was attained in 4 of 9 patients (44%). Eight in one chemotherapy was initiated four weeks after radiation and repeated at 4 weekly intervals for 5–8 courses. Postradiation eight in one failed to show any additional effect on tumor responses. Median survival was 34 months (range 9–48 months) with five of ten patients alive: four in complete and one in partial remission. All the five survivors were among the patients who had achieved response to initial treatment. This result suggested that degree of response to initial treatment might determine subsequent outcome and thus the choice of modality for initial therapy might be important.