IMPORTANCE Nonanatomic factors, such as histologic grade and biomarkers, can guide breast cancer management but are not included in the current TNM staging system.OBJECTIVE To use as an example the triple-negative phenotype (TNP) defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) to examine whether such inclusion improves the prognostic accuracy of TNM staging for breast cancer.DESIGN, SETTING, AND PARTICIPANTS Women diagnosed with primary invasive ductal breast cancer from January 1,1991, through December 31, 2008, were identified from a prospective institutional database. Excluded were patients who received neoadjuvant therapy, those whose staging information was incomplete, or those whose tumor lacked ER, PR, and HER2 data. Breast cancers were categorized by TNM stage and by the presence or absence of TNP.MAIN OUTCOMES AND MEASURES Overall survival at 5 years.RESULTS Database review identified 1842 consecutive eligible patients with breast cancer. When patients were stratified by TNM stage, overall survival curves for those with TNP breast cancer matched those for patients whose non-TNP breast cancer was 1 TNM stage higher. Multivariable analysis showed that TNP status was a powerful prognostic variable, and the likelihood ratio test revealed that the prognostic accuracy of the TNM staging system that incorporated TNP was superior to the current TNM staging system (P < .001). A TNM staging system that incorporated TNP reduced early-stage compression by 15%.CONCLUSIONS AND RELEVANCE The internationally recognized and easily reproducible examination of ER, PR, and HER2 status exemplifies how nonanatomic factors can improve the prognostic accuracy of breast cancer staging.

BACKGROUND: For patients with melanoma, if there has been no recurrence of disease 10 years after initial treatment, additional disease is believed to be very unlikely. However, such late recurrences are known to occur. The frequency of this phenomenon and its clinical significance are not well characterized due to the difficulty in obtaining relevant data. We examined a large, mature, institutional database to evaluate late recurrence. STUDY DESIGN: The late recurrence cohort was defined as having a disease-free interval of 10 or more years after potentially curative treatment and was compared with an early recurrence cohort recurring within 3 years. Actuarial late recurrence frequency and factors associated with late recurrence were examined. Post-recurrence overall and melanoma-specific survival and prognostic variables were analyzed. RESULTS: Among all patients, 408 exhibited late recurrence (mean disease-free interval 15.7 years). For patients who received primary treatment at our institution with 10 or more years follow-up, 327 of 4,731 (6.9%) showed late recurrence. On an actuarial basis, late recurrence rates were 6.8% and 11.3% at 15 and 20 years, respectively, for those with no recurrence at 10 years. Late recurrence was associated with both tumor (thin, non-ulcerated, non-head/neck, node negative) and patient (younger age, less male predominant) characteristics. Multivariate analysis confirmed younger age, thinner and node negative tumors in the late recurrence group. Late recurrences were more likely to be distant, but were associated with better post-recurrence survival on univariate and multivariate analyses. CONCLUSIONS: Late melanoma recurrence is not rare. It occurs more frequently in certain clinical groups and is associated with improved post-recurrence survival.

The efficacy of surgery for invasive mucinous neoplasms is unclear. We examined the natural history of invasive mucinous cystic neoplasms (MCN) and invasive intraductal papillary mucinous neoplasms (IPMN) in patients who underwent pancreatic resection.

BACKGROUND:Basal-like breast cancer (BLBC) has a poor prognosis and is often identified by the triple-negative phenotype (TNP) and/or basal cytokeratins (CKs). Overexpression of mRNA for forkhead box C1 (FOXC1) transcription factor was recently identified as a pivotal prognostic biomarker of BLBC. We investigated the prognostic value of FOXC1 protein expression in invasive breast cancer and compared its prognostic significance to that of TNP and basal CKs.METHODS:Archived TNP specimens of primary invasive ductal breast cancer from 759 patients were examined by immunohistochemical staining for FOXC1, CK5/6, and CK14; prognostic significance was assessed using multivariate analyses. In addition, the impact of adding FOXC1 versus basal CKs to TNP-based BLBC assessment was assessed.RESULTS:FOXC1 protein expression was a significant predictor of overall survival on univariate (hazard ratio [HR] 3.364 95% confidence interval [CI] 1.758-6.438, P = 0.0002) and multivariate (HR 3.389 95% CI 1.928-7.645, P = 0.0001) analyses, despite its correlation with younger age (P = 0.0003). Interestingly, nodal status was not significant on multivariate analysis when FOXC1 expression status was included in the analysis. BLBC defined by TNP plus FOXC1 demonstrated superior prognostic relevance compared to BLBC defined by TNP or TNP plus basal CKs.CONCLUSIONS:Immunohistochemical detection of FOXC1 expression in TNP invasive breast cancer is an independent prognostic indicator that is superior to conventional immunohistochemical surrogates of BLBC. Prospective validation is warranted to further define the diagnostic, prognostic, and predictive utility of FOXC1 in breast cancer management and clinical trial design.

Abstract Although basal-like breast cancer (BLBC) is a distinct molecular subtype, it has no defining gene or protein marker that can be used as the basis for therapy. Estrogen receptor (ER) and HER2 receptor guide treatment of luminal and HER2 breast cancer subtypes, respectively, but chemotherapy is still the only systemic treatment available for BLBC. In view of its poor clinical outcomes, high proliferation rates and preferential metastasis to the brain, there is an urgent need for effective targeted therapy of BLBC. In a previous study using cDNA microarray analyses and immunohistochemistry of archival breast cancer tissue specimens, we showed consistent and exclusive overexpression of FOXC1 in BLBC versus other molecular subtypes. FOXC1 overexpression was significantly associated with poor overall survival and brain metastasis. In the present study, we show that overexpression of FOXC1 in breast cancer cells with low endogenous FOXC1 levels increased cellular proliferation, migration, and invasion, and induced epithelial-mesenchymal transition. Knockdown of FOXC1 by shRNA in breast cancer cells with high endogenous FOXC1 expression had the opposite effect. Cell signaling studies showed that FOXC1 activated the NF-κB pathway by upregulating the peptidyl-prolyl isomerase Pin1 and downregulating the ubiquitin ligase SOCS-1. Pin1 binds to p65, inhibits the p65 interaction with IκBα, and thus enhances p65 nuclear localization and protein stability. SOCS-1 directly interacts with p65 and promotes its degradation. FOXC1 knockdown reduced p65 protein levels and NF-κB activity. The importance of the NF-κB pathway for FOXC1 function was confirmed by the increased sensitivity to NF-κB inhibitors in cells that overexpressed FOXC1. Because BLBC under-expresses ER, FOXC1 was inversely associated with ER, as expected. Interestingly, FOXC1 repressed ER transcription and activity via NF-κB signaling, and FOXC1 overexpression switched MCF-7 breast cancer cell growth from estrogen-dependent to estrogen-independent. This may explain why ER is not normally detected in BLBC. These results suggest that FOXC1 may be a specific diagnostic and prognostic biomarker for BLBC and may play an important role in regulating aggressive traits associated with BLBC. It might also serve as a potential molecular therapeutic target for BLBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3742.

Fatty acid-binding protein-7 (FABP7) has been shown to be expressed in cutaneous melanoma; however, its role in tumor progression is unclear. Expression of FABP7 was assessed during melanoma progression through assessment of various clinicopathology stages of primary tumor progression and metastasis. FABP7 mRNA was highly expressed in 60 of 87 (69%) primary melanomas, compared with significant (P<0.0001) reduction in 13 of 68 (19%) metastatic melanomas. Analysis of 37 paired primary and metastatic melanomas by immunohistochemistry with anti-FABP7 Ab showed 73 and 27% positivity, respectively (P<0.001). FABP7 detection of metastatic tissues was inversely correlated with relapse-free (P<0.0001) and overall (P<0.0001) survival. To examine FABP7 expression loss in advanced melanomas, loss of heterozygosity (LOH) was assessed using microsatellite markers encompassing the FABP7 gene. LOH was identified in 10 of 20 (50%) metastatic melanomas at 6q22.31, compared with 0 of 14 primary melanomas (P=0.0017). FABP7 as a surrogate biomarker for circulating tumor cells (CTCs) in the blood was assessed by quantitative real-time (qRT)-PCR from melanoma patients' blood (n=134). Assessment of patients' blood showed that FABP7(+) CTC decreased with disease progression. FABP7 may function as a tumor progression gene and can be used as a potential diagnostic biomarker of early-stage melanoma systemic spreading in blood.

Abstract Gene expression signatures for a basal-like breast cancer (BLBC) subtype have been associated with poor clinical outcomes, but a molecular basis for this disease remains unclear. Here, we report overexpression of the transcription factor FOXC1 as a consistent feature of BLBC compared with other molecular subtypes of breast cancer. Elevated FOXC1 expression predicted poor overall survival in BLBC (P = 0.0001), independently of other clinicopathologic prognostic factors including lymph node status, along with a higher incidence of brain metastasis (P = 0.02) and a shorter brain metastasis–free survival in lymph node–negative patients (P < 0.0001). Ectopic overexpression of FOXC1 in breast cancer cells increased cell proliferation, migration, and invasion, whereas shRNA-mediated FOXC1 knockdown yielded opposite effects. Our findings identify FOXC1 as a theranostic biomarker that is specific for BLBC, offering not only a potential prognostic candidate but also a potential molecular therapeutic target in this breast cancer subtype. Cancer Res; 70(10); 3870–6. ©2010 AACR.

OBJECTIVE:: B7 ligand family members have been shown to be important immunoregulatory factors in host tumor immune responses. We hypothesized that B7-H3, a coinhibitory factor, is expressed by primary breast cancer cells and associated with metastasis to regional tumor-draining lymph nodes.EXPERIMENTAL DESIGN:: American Joint Committee on Cancer stage I to III primary breast cancers (n = 82) and normal breast specimens (n = 17) were assessed for B7-H3 expression using paraffin-embedded archival tissues. B7-H3 expression by breast cancer cells was assessed by a quantitative real-time reverse transcription-polymerase chain reaction, and B7-H3 protein expression was evaluated using immunohistochemistry.RESULTS:: B7-H3 mRNA expression was detected in 32 of 82 (39%) primary breast tumors but not in normal breast tissues (P = 0.0029). B7-H3 expression in primary tumors significantly correlated with increasing tumor size, American Joint Committee on Cancer stage, and lymphovascular invasion (P < 0.0001, P < 0.0001, P = 0.0071). B7-H3 expression was highly correlated to sentinel lymph node and overall number of lymph nodes with metastasis P = 0.003, and P = 0.004, respectively). In a multivariate analysis, B7-H3 mRNA expression of the primary tumor significantly predicted metastasis to regional lymph nodes (P = 0.021, and P = 0.023, respectively). Antibody staining analysis of paraffin-embedded archival tissue breast tumors and flow cytometry of breast cancer cell lines demonstrated B7-H3 protein expression.CONCLUSIONS:: B7-H3 protein expressed by primary breast cancer cells is a tumor progression factor and is associated with extent of regional nodal metastasis.

9603 Background: Emotional state has been linked to cancer survival, but its influence on the outcome of early melanoma is unclear. The Multicenter Selective Lymphadenectomy Trial (MSLT-I) randomized patients with clinically localized cutaneous melanoma to wide local excision (WEX) plus observation or to WEX plus sentinel lymph node biopsy (SNB). Clinical endpoints included disease-specific and disease-free survival. A substudy of this phase III trial evaluated the impact of mood state on survival, and the impact of recurrence on mood state. Methods: Patients were asked to complete a 65-question form within 6 months of enrollment (baseline) and every 12 months thereafter. This questionnaire measured 6 identifiable mood states (vigor-activity, tension-anxiety, depression, anger-hostility, fatigue-inertia, confusion-bewilderment) of the Profile of Mood States (POMS), a validated mood scale for assessing responses to therapy. Self-reported data from the questionnaires were linked to demographic and clinical variables. Results: Of 2,001 patients accrued to MSLT-I, 1,620 completed the questionnaire at baseline. The baseline distribution of POMS variables was similar in the two treatment arms (data not shown). Patients with more vigor at baseline had a significantly longer disease-free and overall survival ( Table ), even after adjusting for age, tumor thickness, site, and ulceration status (p <0.001). Among 136 patients who completed a questionnaire within 6 months after recurrence, comparison of baseline and post-recurrence responses revealed significant changes in mood state: tension, fatigue and confusion increased, whereas vigor decreased (p = 0.0004, 0.0171, 0.0089, and 0.0028, respectively). Conclusions: Vigor, a measure of energy and optimism, is directly correlated with disease-free and overall survival in early melanoma. The negative impact of recurrence on mood state suggests that SNB as a tool for preventing recurrence might also improve mood state and psychological well-being. Supported by NIH CA29605. [Table: see text] No significant financial relationships to disclose.

Background Solitary dermal melanoma (SDM) is confined to the dermal and/or subcutaneous tissue without an epidermal component. It is unclear whether this lesion is a subtype of primary melanoma or distant cutaneous metastasis from an unknown primary. We evaluated our large experience to determine the prognosis and optimal management of SDM. Methods Our melanoma referral center’s database of prospectively acquired records was used for identification and clinicopathologic analysis of patients presenting with SDM between 1971 and 2005. Results Of 12,817 database patients seen during a 34-year period, 101 (0.8%) had SDM. Of 92 patients free of distant metastasis on initial presentation, 55 (60%) were observed and 37 (40%) underwent surgical nodal staging: regional metastases were identified in 7 (19%). Nodal recurrence occurred in 1 of 30 patients (3.3%) with histopathology-negative nodes compared with 13 of 55 patients (24%) who underwent nodal observation instead of nodal staging. Thus, 21 of 92 patients (23%) had nodal metastasis identified during surgical nodal staging or postoperative nodal observation. At a median follow-up of 68 months, estimated 5-year overall survival rate was 73% for 71 patients with localized disease versus 67% for 21 patients with regional disease ( P = 0.25) versus 22% for 9 patients with distant disease ( P = 0.009, regional versus distant disease). Conclusions SDM resembles intermediate-thickness primary cutaneous melanoma with respect to prognostic characteristics and clinical evolution, but its rate of distant metastasis justifies radiographic staging and its high rate of regional node metastasis justifies wide excision and sentinel node biopsy.

PURPOSE:Lymph node metastases are the most significant prognostic indicator for patients with breast cancer. Sentinel node biopsy (SNB) has led to an increase in the detection of micrometastases in the sentinel node (SN). This prospective study was designed to determine the survival impact of micrometastases in SNs of patients with invasive breast cancer. This study is based on the new sixth edition of the American Joint Committee on Cancer (AJCC) staging criteria.PATIENTS AND METHODS:Between January 1, 1992 and April 30, 1999, 790 patients entered this prospective study at the John Wayne Cancer Institute. The SN was examined first by hematoxylin and eosin (HE), and if the SN was negative with HE, then immunohistochemical staining was performed. The patients were then divided into four groups based on AJCC nodal staging: pN0(i-), no evidence of tumor (n = 486); pN0(i+), tumor deposit < or = 0.2 mm (n = 84); pN1mi, tumor deposit more than 0.2 mm but < or = 2 mm (n = 54), and pN1, tumor deposit more than 2 mm (n = 166). Disease-free survival (DFS) and overall survival (OS) were estimated using the Kaplan-Meier method. The log-rank test was used to determine differences in DFS and OS of patients from different groups.RESULTS:At a median follow-up of 72.5 months, the size of SN metastases was a significant predictor of DFS and OS.CONCLUSION:Patients with micrometastatic tumor deposits, pN0(i+) or pN1mi, do not seem to have a worse 8-year DFS or OS compared with SN-negative patients. As expected, there was a significant decrease in 8-year DFS and OS in patients with pN1 disease in the SN.

AbstractPurpose: The availability of a variety of immune response modifiers creates an opportunity for improved efficacy of immunotherapy, but it also leads to uncertainty in how to combine agents and how to assess those combinations. We sought to assess the effect of the addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) to vaccination with a melanoma vaccine.Experimental Design: Ninety-seven patients with resected melanoma (stage II-IV) were enrolled, stratified by stage, and randomized to receive a cellular melanoma vaccine with or without GM-CSF. The primary endpoint was delayed-type hypersensitivity (DTH) response to melanoma cells. Antibody responses, peripheral leukocyte counts, and survival were also examined.Results: The GM-CSF arm showed enhanced antibody responses with an increase in IgM titer against the TA90 antigen and increased TA90 immune complexes. This arm also had diminished antimelanoma cell delayed-type hypersensitivity response. Peripheral blood leukocyte profiles showed increases in eosinophils and basophils with decreased monocytes in the GM-CSF arm. These immune changes were accompanied by an increase in early melanoma deaths and a trend toward worse survival with GM-CSF.Conclusion: These data suggest that GM-CSF is not helpful as an immune adjuvant in this dose and schedule and raise concern that it may be harmful. Based on the discordant findings of an immune endpoint and clinical outcome, the use of such surrogate endpoints in selecting treatments for further evaluation must be done with a great deal of caution. (Clin Cancer Res 2009;15(22):702935)

Background. American Society of Clinical Oncology (ASCO) guidelines recommend axillary dissection (ALND) as the primary treatment for sentinel node micrometastases (SNMM) in breast cancer. We hypothesized that variation exists in adherence to these guidelines and management of SNMM as a result of the medical specialty of the treating physician.Methods. A questionnaire was e-mailed to 2511 active members of ASCO who specialize in breast cancer and who are board certified in surgery, medical oncology, or radiation oncology. Questions covered treatment of SNMM and factors influencing clinical decisions. Survey responses were analyzed by the Kruskal-Wallis and Fisher's exact test.Results. Of 612 questionnaires viewed by recipients, 537 (88%) were completed by 382 medical oncologists, 100 surgeons, and 55 radiation oncologists. Most (98.5%) regarded SNMM as clinically important. Unexpectedly, ALND for SNMM was recommended by only 23% of surgeons, 23% of medical oncologists, and 15% of radiation oncologists ( no statistically significant intergroup difference). Factors identified as important in selecting patients for ALND were tumor size, age, tumor grade, lymphovascular invasion, and size of metastases. Most respondents in each specialty (89% overall) would consider adjuvant chemotherapy for primary tumors <1 cm if SNMM were present. Tumor size, age, size of metastases, estrogen receptor/progesterone receptor status, HER-2 neu status, and gene expression profile would influence this decision. Radiation oncologists (76%) were more likely than medical oncologists (57%) or surgeons (47%) to consider axillary radiation instead of ALND for SNMM (P = 0.0021).Conclusions. Guidelines recommending ALND for SNMM are not being followed by most of the ASCO members we surveyed.

3013 Background: Vaccines are currently under evaluation in melanoma, and many vaccine strategies use GM-CSF as an immune adjuvant. There are few randomized data yet available to determine the clinical impact of the addition of GM-CSF to vaccination. We conducted a randomized, prospective trial of an allogeneic melanoma vaccine with or without GM-CSF to determine the immune and clinical effects. Methods: 95 patients were stratified by stage and randomized to Canvaxin vaccine (with bacille Calmette-Guerin [BCG]) or Canvaxin (+BCG) and GM-CSF (200 mcg/m2/d) injected at the vaccine sites after vaccination for 5 days. Antibody titers against a urinary tumor-associated melanoma antigen were measured by ELISA, and DTH skin testing was done using PPD and unadulterated vaccine cells. Results: There was a significant early rise in both IgG and IgM titers in the GM-CSF arm with an early high plateau at about 8 weeks for both. The rise in antibody titer was slower in the non-GM-CSF arm with IgG rising throughout the study period and IgM peaking at 20 weeks. Conversely there was a delay in PPD responsiveness in the GM-CSF arm. PPD conversion percentage surpassed 90% in the non-GM-CSF arm at 6 weeks and peaked at 100% at 12 weeks. In the GM-CSF arm, only 76% converted at 6 weeks and only 87% were ever sensitized. A trend toward diminished DTH response was noted in the GM-CSF arm as early as 4 weeks and became significant at 16 weeks (p=0.01) and when considered throughout the trial. There was a marked increase in early recurrences in the GM-CSF arm leading to a significant survival difference when examined at 2 years (96% versus 72%; log-rank p=0.0017). Conclusions: When given at the site of vaccination, GM-CSF appears to accelerate the onset of a humor immune response while delaying cellular responses. In addition there appears to be evidence of adverse clinical effects in terms of early recurrence and possibly survival. Recent reports of increased myeloid suppressor cells with GM-CSF treatment may provide an immune mechanism for our findings. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration CancerVax

4502 Introduction: Forkhead box protein 3 (Foxp3) controls the development and function of CD4+/CD25+ T-regulatory cells (Tregs). The role of Tregs in tolerance or suppression of malignancy is not well understood. Intratumoral accumulation of Tregs was associated with poor survival in ovarian cancer but improved survival in patients with hematologic malignancy. In this study we examined Foxp3 expression levels in tumor-positive and -negative sentinel nodes (SNs) and in non-SNs (NSNs) from patients with early-stage melanoma. Methods: RNA was extracted from freshly dissected and preserved tangential cut peripheral tissue sections of 21 tumor-negative SNs, 18 tumor-positive SNs (micrometastases

There are no standard methods to predict response to treatment or outcome of stage IV melanoma. Our previous assessment of peripheral blood mononuclear cells (PBMC) from immunized patients demonstrated that interleukin (IL)-10 expression might be associated with prognosis. However, PBMC are a mixture of CD4+ cells, CD8+ cells, and monocytes. This study identified the subset of PBMC responsible for IL-10 expression and evaluated the prognostic value of IL-10 expression in immunized stage IV patients. Eighty-seven patients with stage IV melanoma were randomly selected from our database. All patients had received an allogeneic melanoma whole-cell vaccine (Canvaxin) after complete resection of clinical disease. Blood samples had been collected serially during Canvaxin administration and cryopreserved. Intracellular IL-10 expression was assessed by double staining fluorescence-activated cell sorter. CD14+ monocytes are the predominant PBMC producing IL-10. Sixteen weeks after treatment (week 16), IL-10 levels were significantly (P=0.02) higher in poor-survival patients than those with favorable outcomes. Patients were separated into 2 groups on the basis of the CD14+ monocyte IL-10 response: either increasing or decreasing IL-10 expression from preimmunization (week 0) to week 16 blood draws. Patients with increasing IL-10 levels had significantly shorter survival than those whose IL-10 levels decreased at week 16 (P<0.0001). Multivariate analysis demonstrated that trends in IL-10 levels inversely correlated with survival (P<0.0001). We conclude that CD14+ monocytes are the dominant cellular source of IL-10 among PBMC and that changes in IL-10 expression may serve as an immunologic-based surrogate for predicting outcome for stage IV patients after surgical resection.

Purpose: Identification of regional node metastasis is important for accurate staging and optimal treatment of early melanoma. We hypothesize that the nodal profile of immunoregulatory cytokines can confirm the identity of the first tumor-draining regional node, i.e., the sentinel node (SN) and indicate its tumor status. Experimental Design: RNA was extracted from freshly dissected and preserved nodal tissue of 13 tumor-negative SNs, 10 tumor-positive SNs (micrometastases <2 mm), and 11 tumor-negative non-SNs (NSN). RNA was converted into cDNA and then amplified by PCR. Expression of 96 cytokines and chemokines was assessed using cDNA microarray and compared by using hierarchical clustering. Results: Fifty-seven genes were expressed at significantly (P < 0.05) different levels in SNs and NSNs (4 genes had higher expression, and 53 genes had lower expression in SNs). Expression levels of interleukin-13 (IL-13), leptin, lymphotoxin β receptor (LTbR), and macrophage inflammatory protein 1b (MIP1b) were significantly higher (P < 0.04, P < 0.01, P < 0.05, and P < 0.01, respectively), and expression level of IL-11Ra was lower (P < 0.03) for tumor-positive as compared with tumor-negative SN. Receiver-operator characteristics curve analyses showed that the area under the curve (AUC) for IL-13, leptin, LTbR, MIP1b, and IL-11Ra was 0.79, 0.83, 0.75, 0.81, and 0.77, respectively. The AUC for the five genes in combination was 0.973, suggesting high concordance of gene-expression profiles with SN staging. Conclusions: SNs have a different immunoregulatory cytokine profile than NSNs. The cytokine profile of tumor-positive SNs; increased expression of IL-13, leptin, LTbR, and MIP1b and decreased expression of IL-11Ra, may provide clues to the local tumor lymph node interaction seen in the earliest steps of melanoma metastasis.