Protein-Based Oncopanel as Addition to Target Sequencing in Head and Neck Squamous Cell Carcinoma to Individualize Treatment Decisions.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of cancers and patients have limited therapy options if primary treatment fails. Therefore, additional information about the biology of the tumor is essential. Here we performed a feasibility study of concurrently applying two precision diagnostic tools in a consecutive series of HNSCC patients. We analyzed tumor samples of 31 patients using a genomic (oncomine) and a proteomic, immunohistochemical approach (oncopanel) and compared the result, also in the focus on their overlapping therapeutical targets. We found no strong correlation between the two approaches and observed a higher proportion of marker expression for the immunohistochemical panel. However, both panels show in our HNSCC cohort distinct patterns with druggable targets. The data suggest that both approaches complement one another and can be applied side-by-side to identify the best targets for the development of individual treatment options for HNSCC patients.

GRP78-directed immunotherapy in relapsed or refractory multiple myeloma - results from a phase 1 trial with the monoclonal immunoglobulin M antibody PAT-SM6.

UNLABELLED: The primary objective of this phase 1 study was to evaluate the safety and tolerability of the anti-glucose regulated protein 78 monoclonal immunoglobulin M antibody PAT-SM6 in subjects with relapsed or refractory multiple myeloma. Twelve heavily pretreated patients received four intravenous infusions of PAT-SM6 at doses of 0.3, 1, 3, and 6 mg/kg within 2 weeks. Efficacy, pharmacokinetics and immunogenicity were followed up until the end of the trial (day 36). In addition, immune cell patterns in peripheral blood were assessed by flow cytometry and glucose regulated protein 78 expression status was evaluated in bone marrow specimens by immunohistochemistry and flow cytometry at screening. All doses administered were found to be safe and well tolerated; the maximum tolerated dose was not reached. The most common treatment emergent adverse event was leukopenia (grades 1 and 2) in eight out of the 12 multiple myeloma patients. Pharmacokinetic analysis demonstrated dose-proportional increases in drug serum concentration. The terminal half-life ranged from 5.86 to 8.41 h, the apparent volume of distribution ranged from 101 to 150 mL/kg, and clearance ranged from 8.11 to 16.1 mL/h/kg. All patients showed glucose regulated protein 78 surface expression on multiple myeloma cells. Four out of the 12 patients (33.3 %) had stable disease, according to the International Myeloma Working Group criteria, after PAT-SM6 treatment across the doses 1, 3 and 6 mg/kg. In summary, single-agent PAT-SM6 was well tolerated with modest clinical activity in relapsed or refractory multiple myeloma. Further trials exploring the combination of PAT-SM6 with existing myeloma therapies are planned. TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT01727778.

Noninvasive visualization of tumor growth in a human colorectal liver metastases xenograft model using bioluminescence in vivo imaging.

BACKGROUND: Bioluminescence imaging (BLI) is an ideal tool for noninvasive, quantitative monitoring of tumor progression/regression in animal models. The effectiveness of different treatment strategies is displayed by an altered intensity of bioluminescence, demonstrating a change of the tumor burden. The aim of this study was to establish a reliable, reproducible colorectal hepatic metastases cancer animal model. METHODS: Cells of the human colon carcinoma cell line HCT-116 Luc(pos) expressing the firefly luciferase enzyme gene were used. HCT-116 Luc(pos) cells (2.5 × 10(6)) were injected through the portal vein into the liver of immunoincompetent nude mice. BLI was used to analyze intrahepatic tumor burden and growth kinetic. RESULTS: HCT-116 Luc(pos) cells demonstrated a progressive and reproducible growth in the liver after intraportal injection. Four days after injection, the animals were analyzed for tumor growth by BLI, and mice without or too low bioluminescence signals were excluded (between 10% and 20% animals). HCT-116 Luc(pos) intrahepatic tumors responded successfully to different dosages (5 and 10 mg/kg) of 5-fluorouracil. CONCLUSIONS: BLI is an important tool with many potential advantages for investigators. The measurement of intrahepatic tumor growth by imaging luciferase activity noninvasively provides valuable information on tumor burden and effectiveness of therapy. Thus, the presented intrahepatic metastases model based on the growth of HCT-116 Luc(pos) cells is suitable for in vivo testing of different cancer therapy strategies.

Targeted panel sequencing in the routine diagnosis of mature T- and NK-cell lymphomas: report of 128 cases from two German reference centers.

Diagnosing any of the more than 30 types of T-cell lymphomas is considered a challenging task for many pathologists and currently requires morphological expertise as well as the integration of clinical data, immunophenotype, flow cytometry and clonality analyses. Even considering all available information, some margin of doubt might remain using the current diagnostic procedures. In recent times, the genetic landscape of most T-cell lymphomas has been elucidated, showing a number of diagnostically relevant mutations. In addition, recent data indicate that some of these genetic alterations might bear prognostic and predictive value. Extensive genetic analyses, such as whole exome or large panel sequencing are still expensive and time consuming, therefore limiting their application in routine diagnostic. We therefore devoted our effort to develop a lean approach for genetic analysis of T-cell lymphomas, focusing on maximum efficiency rather than exhaustively covering all possible targets. Here we report the results generated with our small amplicon-based panel that could be used routinely on paraffin-embedded and even decalcified samples, on a single sample basis in parallel with other NGS-panels used in our routine diagnostic lab, in a relatively short time and with limited costs. We tested 128 available samples from two German reference centers as part of our routine work up (among which 116 T-cell lymphomas), which is the largest routine diagnostic series reported to date. Our results showed that this assay had a very high rate of technical success (97%) and could detect mutations in the majority (79%) of tested T-cell lymphoma samples.

Identification of Disparities in Personalized Cancer Care-A Joint Approach of the German WERA Consortium.

(1) Background: molecular tumor boards (MTBs) are crucial instruments for discussing and allocating targeted therapies to suitable cancer patients based on genetic findings. Currently, limited evidence is available regarding the regional impact and the outreach component of MTBs; (2) Methods: we analyzed MTB patient data from four neighboring Bavarian tertiary care oncology centers in Würzburg, Erlangen, Regensburg, and Augsburg, together constituting the WERA Alliance. Absolute patient numbers and regional distribution across the WERA-wide catchment area were weighted with local population densities; (3) Results: the highest MTB patient numbers were found close to the four cancer centers. However, peaks in absolute patient numbers were also detected in more distant and rural areas. Moreover, weighting absolute numbers with local population density allowed for identifying so-called white spots-regions within our catchment that were relatively underrepresented in WERA MTBs; (4) Conclusions: investigating patient data from four neighboring cancer centers, we comprehensively assessed the regional impact of our MTBs. The results confirmed the success of existing collaborative structures with our regional partners. Additionally, our results help identifying potential white spots in providing precision oncology and help establishing a joint WERA-wide outreach strategy.

Recurrent Oncogenic JAK and STAT Alterations in Cutaneous CD30-Positive Lymphoproliferative Disorders.

The group of cutaneous CD30-positive lymphoproliferative disorders (LPD) comprises two different entities, namely lymphomatoid papulosis (LyP) and cutaneous anaplastic large T-cell lymphoma (cALCL). LyP constitutes a benign lymphoproliferation with spontaneously regressing papules, whereas cALCL presents with solitary or multiple skin tumors with a low propensity to disseminate. To elucidate the hitherto largely unknown molecular pathogenesis of these entities, we performed comprehensive next-generation sequencing in a well-characterized cohort of 12 patients. Considering the low tumor cell content of LyP, we applied targeted sequencing technologies with a hybrid capture-based DNA library preparation approach and for the identification of fusion transcripts an anchored multiplex PCR enrichment kit. As the major finding, we detected, in 50% of LPD, genetic events that implied a constitutively activated Janus kinase-signal transducer and activator of transcription signaling (JAK-STAT) pathway in these entities. The identified molecular aberrations comprised either pathogenic STAT mutations or oncogenic fusion transcripts comprising effector domains of JAK. With respect to LyP, we report to our knowledge such previously unreported genetic aberrations in this specific entity. The detection of these convergent aberrations within the JAK-STAT signaling pathway deciphers common potential driving mechanisms of lymphomagenesis within LPD being shared between LyP and cALCL. Moreover, the presence of these oncogenic alterations paves the way to develop novel personalized treatment strategies.

Prognostic Value of O-(2-[18F]Fluoroethyl)-L-Tyrosine PET/CT in Newly Diagnosed WHO 2016 Grade II and III Glioma.

PURPOSE: The use of [18F]fluoroethyl)-L-tyrosine ([18F]FET) positron emission tomography/computed tomography (PET/CT) has proven valuable in brain tumor management. This study aimed to investigate the prognostic value of radiotracer uptake in newly diagnosed grade II or III gliomas according to the current 2016 World Health Organization (WHO) classification. PROCEDURES: A total of 35 treatment-naive patients (mean age, 48 ± 17 years) with histologically proven WHO grade II or III gliomas as defined by the current 2016 WHO classification were included. Static PET/CT imaging was performed 20 min after intravenous [18F]FET injection. Images were assessed visually and semi-quantitatively using regions of interest for both tumor (SUVmax, SUVmean) and background (BKGmean) to calculate tumor-to-background (TBR) ratios. The association among histological results, molecular markers (including isocitrate dehydrogenase enzyme and methylguanine-DNA methyltransferase status), clinical features (age), and PET findings was tested and compared with outcome (progression-free [PFS] and overall survival [OS]). RESULTS: Fourteen patients presented with grade II (diffuse astrocytoma n = 10, oligodendroglioma n = 4) and 21 patients with grade III glioma (anaplastic astrocytoma n = 15, anaplastic oligodendroglioma n = 6). Twenty-seven out of the 35 patients were PET-positive (grade II n = 8/14, grade III n = 19/21), with grade III tumors exhibiting significantly higher amino acid uptake (TBRmean and TBRmax; p = 0.03 and p = 0.02, respectively). PET-negative lesions demonstrated significantly prolonged PFS (p = 0.003) as compared to PET-positive gliomas. PET-positive disease had a complementary value in prognostication in addition to patient age, glioma grade, and molecular markers. CONCLUSIONS: Amino acid uptake as assessed by [18F]FET-PET/CT imaging is useful as non-invasive read-out for tumor biology and prognosis in newly diagnosed, treatment-naive gliomas according to the 2016 WHO classification.

Establishing Pure Cancer Organoid Cultures: Identification, Selection and Verification of Cancer Phenotypes and Genotypes.

Precision medicine requires in vitro models which will both faithfully recapitulate the features of an individual's disease and enable drug testing on a wide variety of samples covering the greatest range of phenotypes possible for a particular disease. Organoid technology has immense potential to fulfill this demand, but it will be necessary to develop robust protocols that enable the generation of organoids in a dependable manner from nearly every patient. Here we provide a user's guide, including detailed step-by-step protocols, to the establishment, isolation and verification of gastric cancer organoids. Selection strategies include omission of growth factors, addition of drugs, isolation of distinct phenotypes and generation of monoclonal lines. For confirmation of cancer identity, we use sequencing, drug selection, karyotyping and histology. While we specify these protocols for human gastric cancer organoids here, the methods described are applicable to organoids derived from other tissues as well.

A new tumor-specific variant of GRP78 as target for antibody-based therapy.

The chaperone GRP78 is a member of the heat-shock protein 70 (HSP70) family and is responsible for cellular homeostasis by preventing stress-induced apoptosis. GRP78 is expressed in all cells of the body. In malignant cells, which are permanently exposed to environmental stress, GRP78 is overexpressed and increased levels can be found in the cytoplasm and on the cell membrane. Thus, GRP78 promotes tumor proliferation, survival, metastases and resistance to a wide variety of therapies. Like other tumor-specific membrane molecules, GRP78 can also be present on cancer cells in a variant form. This modification qualifies it as a target for immune surveillance and antibody responses. The fully human monoclonal IgM antibody, SAM-6, was isolated from a gastric cancer patient and it binds to a new variant of GRP78 with a molecular weight of 82 kDa. The epitope is an O-linked carbohydrate moiety and is specific for malignant cells. These data show that cancer-specific modifications of cell-surface protection molecules are (a) subject of an immune response and (b) ideal targets for new therapeutical approaches.

The human IgM antibody SAM-6 induces tumor-specific apoptosis with oxidized low-density lipoprotein.

Lipids are essential for normal and malignant cells during growth and differentiation. The turnover is strictly regulated because an uncontrolled uptake and accumulation is cytotoxic and can lead to lipoapoptosis: lipoptosis. The human monoclonal antibody SAM-6 binds to a cell surface receptor on malignant cells and to oxidized low-density lipoprotein (LDL). SAM-6 induces an excess of intracellular lipids, by overfeeding malignant cells with oxidized LDL, via a receptor-mediated endocytosis. The treated cells overaccumulate depots of cholesteryl esters and triglycerides. This lipid overaccumulation is tumor specific; nonmalignant cells neither bind the antibody nor harvest lipids after incubation. Because for both forms of apoptosis, the death domain dependent ("extrinsic") and independent ("intrinsic"), the activation of proteases is crucial, we also investigated this pathway in more detail. It was found that shortly after internalization of antibody/oxidized LDL/receptor complex and formation of lipid depots, cytochrome c is released by mitochondria. Followed by this, initiator caspase-8 and caspase-9 and effector caspase-3 and caspase-6 are activated. The mechanism of mitochondrial trigger (e.g., by free fatty acids) is under investigation. However, the present data indicate that the SAM-6 antibody induces an intrinsic-like form of apoptosis by overfeeding malignant cells with lipoproteins.

Tumors: too sweet to remember?

Immunity, based on a natural and an educated system, is responsible for recognition and elimination of infectious particles, cellular waste, modified self and transformed cells. This dual system guarantees that dangerous particles are removed immediately after appearance and that a memory with maturated weapons exists, if the organism is re-infected by the same particle. For malignant cells, however, the immune response seems to be restricted to innate immunity, because at least for the humoral response, all so far detected tumor-specific antibodies belong to the natural immunity. In this review we try to explain why malignant cells might be "too sweet" to induce a memory.

Natural IgM antibodies: the orphaned molecules in immune surveillance.

Natural IgM antibodies are typical victims of prejudices which originated in the mid 80 s. Over the years, these molecules were considered as the pariahs among the immune competent molecules and their characteristic properties, like low affinity, cross-reactivity and pentameric structure, were assessed as useless, difficult, nebulous, etc. Today, mainly based on a few scientists' persistent work and the key discoveries on innate immune recognition, natural IgM antibodies are "back on stage". Their role in the immune response against bacteria, viruses, fungi and possibly modified self-components as well as in therapy and diagnosis of malignancies is accepted. All the so far negatively judged features are seen in a different light, e.g. low affinity seems to be good for function and does not exclude specificity, and cross-reactivity is no longer judged as unspecific, but instead as a very economic way of immune recognition. And at last, with the use of natural IgM antibodies, a new field of tumor-specific targets has been encountered, the carbo-neo-epitopes. Therefore, by having learned from nature, the renaissance of natural IgM antibodies opens a new area of cancer therapeutics and diagnostics.

Cysteine-rich fibroblast growth factor receptor 1, a new marker for precancerous epithelial lesions defined by the human monoclonal antibody PAM-1.

Precancerous epithelial lesions are sites of uncontrolled cellular proliferation, generated by irreversible genetic changes. Not all of these lesions progress to invasive cancer, some may even regress, but early detection of abnormal cells can be crucial for survival of the patient. Diagnosis is mainly performed by using morphological parameters. Proliferation markers can facilitate the analysis, if they show a consistent expression, and distinguish between healthy and malignant cells. The fully human monoclonal IgM antibody PAM-1 was isolated from a patient with stomach carcinoma and binds to a new variant of cysteine-rich fibroblast growth factor receptor 1 (CFR-1). This CFR-1/PAM-1 receptor is expressed on nearly all of the epithelial cancers of every type and origin, but not on healthy tissue. It is also present on precursor lesions found in: Helicobacter pylori-induced gastritis, intestinal metaplasia and dysplasia of the stomach, ulcerative colitis-related dysplasia and adenomas of the colon, Barrett's metaplasia and dysplasia of the esophagus, squamous cell metaplasia and dysplasia of the lung, and cervical intraepithelial neoplasia. The unique, growth-dependent expression of this new CFR-1 isoform makes the PAM-1 antibody an ideal diagnostic tool for the detection of precancerous and cancerous lesions.

Lipoptosis: tumor-specific cell death by antibody-induced intracellular lipid accumulation.

A balanced lipid metabolism is crucial for all cells. Disturbance of this homeostasis by nonphysiological intracellular accumulation of fatty acids can result in apoptosis. This was proven in animal studies and was correlated to some human diseases, like lipotoxic cardiomyopathy. Some metabolic mechanisms of lipo-apoptosis were described, and some causes were discussed, but reagents, which directly induce lipo-apoptosis, have thus far not been identified. The human monoclonal IgM antibody SAM-6 was isolated from a stomach cancer patient by using the conventional human hybridoma technology (trioma technique). The addition of SAM-6 to tumor cells leads to an increase in the intracellular accumulation of neutral lipids, followed by tumor cell apoptosis. The antibody SAM-6 does not react with noncancerous human epithelial and fibroblastic cells, because the M(r) 140000 membrane molecule, recognized by the antibody, is specifically expressed on human malignant cells. The antibody is coded by the germ-line genes IgHV3-30.3*01 and IgLV3-1*01 and is a component of the innate immunity to cancer. In this article, we describe an antibody-induced tumor-specific cell death, named lipoptosis. This is, to our knowledge, the first description of this specific form of lipo-apoptosis as an antibody-mediated mechanism of tumor cell killing.

Natural IgM antibodies and immunosurveillance mechanisms against epithelial cancer cells in humans.

Malignancy is like a chronic disease, and the immune system is permanently involved in recognizing and eliminating the transformed cells. The human hybridoma technology offers the unique opportunity to study the mechanisms, structures, and targets involved in recognition and elimination of aberrant cells. Thousands of tumor-reactive human monoclonal antibodies were isolated by this technique from cancer patients and from healthy donors, and all of these antibodies were IgM antibodies; no IgG and IgA antibodies were found. Fourteen of these antibodies were selected for DNA sequence analysis, characterization of their binding patterns, and determination of their origin and genetics. All of the IgM antibodies studied expressed only few or no mutations at all (germ-line coded), bound to carbohydrates on modified tumor-specific receptors and induced apoptosis. The degree of cross-reactivity to other tumors correlated reciprocally with the number of mutations in coding regions. By using an anti-idiotypic antibody we were able to show that the IgM-producing cells were of CD5+ B-cell origin. The data presented here indicate that the innate immunity and natural IgM antibodies play an important role in immunosurveillance mechanisms against epithelial tumors in humans.

A GRP78-Directed Monoclonal Antibody Recaptures Response in Refractory Multiple Myeloma with Extramedullary Involvement.

PURPOSE: Glucose-regulated protein (GRP) 78 is overexpressed in multiple myeloma, and both its surface expression and its biologic significance as key sensor of the unfolded protein response make GRP78 an ideal candidate for immunotherapeutic intervention. The monoclonal antibody PAT-SM6 targets surface GRP78 and leads to disease stabilization when used as single agent in a clinical trial. In this article, we evaluated expression of GRP78 in relapsed-refractory disease and explored PAT-SM6 therapy in combination regimens. EXPERIMENTAL DESIGN: GRP78 expression was immunohistochemically analyzed during disease progression and development of drug resistance throughout different stages of multiple myeloma. Activity of PAT-SM6 was evaluated in combination with anti-multiple myeloma agents lenalidomide, bortezomib, and dexamethasone in vitro Finally, we report on a multiple myeloma patient with relapsed-refractory disease treated with PAT-SM6 in combination with bortezomib and lenalidomide. RESULTS: Although sGRP78 expression was present at all stages, it increased with disease progression and was even strongly elevated in patients with drug-resistant and extramedullary disease. Pretreatment with dexamethasone as well as dual combination of PAT-SM6/lenalidomide further increased sGRP78 expression and consecutively showed synergistic anti-multiple myeloma effects with PAT-SM6 in proliferation assays. As proof of concept, a 62-year-old male with triple resistant multiple myeloma treated with PAT-SM6, bortezomib, and lenalidomide experienced partial remission of both intra- and extramedullary lesions. CONCLUSIONS: PAT-SM6 therapy in combination regimens showed efficacy in relapsed-refractory multiple myeloma. Clin Cancer Res; 22(17); 4341-9. ©2016 AACR.

CFR-1 receptor as target for tumor-specific apoptosis induced by the natural human monoclonal antibody PAM-1.

The human monoclonal antibody PAM-1 was isolated from a patient with stomach cancer. The germ-line coded IgM antibody identifies a recently described 130 kDa variant of CFR-1 (cysteine-rich fibroblast growth factor receptor 1). This CFR-1/PAM-1 receptor is post-transcriptionally modified and over-expressed on human epithelial tumors and carcinoma pre-cancer lesions such as H. pylori induced gastritis, intestinal metaplasia and dysplasia of the stomach, ulcerative colitis-related dysplasia and adenomas of the colon, Barrett metaplasia and dysplasia of the esophagus, squamous cell metaplasia and dysplasia of the lung and cervical intraepithelial neoplasia. Furthermore, the expression of CFR-1/PAM-1 correlates with the proliferation rate and increases with the grade of malignancy. This study demonstrates that the human monoclonal antibody PAM-1 inhibits cell growth and induces apoptosis, in vitro and in vivo. Both, the unique tumor-specific expression of the CFR-1/PAM-1 receptor and the growth inhibitory effect of the PAM-1 antibody makes this combination a good diagnostic and therapeutic tool for all kinds of epithelial cancers and precursor lesions.

Nature's best weapons to fight cancer. Revival of human monoclonal IgM antibodies.

The unique features of monoclonal antibodies (specificity, effectiveness, purity and unlimited reproducibility) make them ideal tools for the specific treatment of all kind of diseases. The third generation of monoclonal antibodies for the treatment of human diseases will be, after murine and "humanised" murine immunoglobulins, fully human antibodies. The best source of human monoclonal antibodies are the antibody pools of cancer patients themselves with the best technique for generating them being conventional human hybridoma technology. This technique, will generate human monoclonal antibodies which will not only define important new targets on cancerous tissue, but will also provide the necessary therapeutic human antibodies in the fight against cancer.

PAM-1, a natural human IgM antibody as new tool for detection of breast and prostate precursors.

Early detection and differential analysis of premalignant lesions are very important for both prognosis and therapy of cancer patients. A good source of diagnostic tools is the natural antibody pool of humans. Tumor-specific antibodies can be established by using hybridoma technology. The fully human germline-coded monoclonal IgM antibody PAM-1 was isolated from a patient with a stomach carcinoma. PAM-1 reacts with a post-transcriptionally modified isoform of membrane receptor CFR-1 which is overexpressed on almost all epithelial cancers of all types and origins. The expression of CFR-1/PAM-1 on precancerous stages of breast and prostate cancer was analyzed by immunohistochemistry and compared with normal breast and prostate tissue as well as adenocarcinomas of both. In addition FACS analysis was performed to detect receptor expression on benign and malign prostate cells. 73 different tissue samples of prostate and breast precancerous stages and prostate and breast carcinomas were analysed for CFR-1/PAM-1 expression immunohistochemically. The CFR-1/PAM-1 receptor was expressed on nearly all precancerous stages and carcinomas while normal breast and prostate tissue showed negative results. These results were confirmed by FACS analysis showing a CFR-1/PAM-1 expression only on prostate carcinoma cells but not on benign prostate hyperplasia cells. The unique expression of this new CFR-1/PAM-1 receptor makes the PAM-1 antibody an ideal diagnostic and even therapeutic tool for precancerous and cancerous epithelial lesions of the breast and the prostate.

Human monoclonal IgM antibodies with apoptotic activity isolated from cancer patients.

Monoclonal antibodies are accepted as ideal adjuvant therapeutic reagents for all kinds of diseases. Polyvalent (cross-linking) and low-mutated IgM antibodies (less immunogenic) are believed to be the most effective weapons against cancer. The best sources for these types of antibodies are the cancer patients themselves. Using conventional hybridoma technology, not only are fully human monoclonal IgM antibodies isolated, but also new tumor-related targets can be identified using the same experimental approach. The resulting antibodies can be used directly for therapeutic purposes without further modulation and manipulation. This report describes five newly established human monoclonal IgM antibodies; antibody LM-1 that was isolated from a patient with lung cancer, antibodies PM-1 und PM-2 that were isolated from a patient with pancreatic cancer, and antibodies CM-1 and CM-2 which were isolated from a patient with colon carcinoma. The mainly germ-line encoded antibodies are specific for malignant tissues and show only restricted reactivity with healthy cells. When tested for in vitro functional activity, all five antibodies inhibit tumor cell proliferation of carcinoma cells by inducing apoptosis.