Next Generation Immunohistochemistry A Window Onto The Molecular Biology of Tumors Allen M. Gown, M.D. Medical Director and Chief Pathologist PhenoPath Laboratories Seattle, Washington Clinical Professor of Pathology, University of British Columbia
Hematoxylin & Eosin
Immunohistochemistry
Albert Coons American pathologist and immunologist 1912-78
Villin Tumor Diagnostics: General Trends Immunohistochemistry Companion diagnostics to new drug development, especially targeted therapies bcl-6 New technologies impacting pathology New applications of existing technology Keratins Cost/reimbursement issues TTF-1
Cell Type Analysis Has Driven IHC Development Immunohistochemistry can identify cell type with greater certainty than H&E-based morphologic patterns Most of tumor classification based upon cell type (e.g., squamous cell carcinoma, neuroendocrine carcinoma, acinar cell tumor, etc.) Cell (tumor) type is a surrogate for predicting the behavior of tumor
Cell Type Analysis Has Driven IHC Development Marker Normal Tissues Tumor CDX-2 Colorectal epithelium Colorectal adenocarcinoma SALL4 Germ cells Germ cell tumor kit Interstitial cells Cajal GISTs CD20 B cells B cell lymphoma Villin GI tract epithelium GI tract adenocas Insulin Synaptophysin Beta cells of pancreatic islet Neuroendocrine cells of intestine Insulinoma Carcinoid tumor
And now for something completely different.
Next Generation Immunohistochemistry PROVIDING A WINDOW ONTO THE MOLECULAR ALTERATIONS UNDERLYING CANCERS AND THUS IDENTIFYING APPROPRIATE THERAPIES
SCIENCE VOL 339 29 MARCH 2013
Major Genetic Alterations in Cancer Mutation Translocation Deletion Amplification Methylation
Major Genetic Alterations in Cancer Mutation Translocation Deletion Amplification Methylation Mutant Protein Loss of Expression Abnormal Localization Overexpression Expression of Fusion Proteins
Major Genetic Alterations in Cancer Mutation Translocation Deletion Amplification Methylation Mutant Protein Loss of Expression Abnormal Localization Overexpression Expression of Fusion Proteins
Examples of Gene Mutations Identifiable by Immunohistochemistry Mutant protein (e.g.,idh1) Loss of expresssion (e.g, MMR) Abnormal localization (e.g., ß-catenin) Overexpression (e.g., p53)
IDH1 and Cancer Warburg hypothesis that altered cellular respiration is origin of cancer IDH1 is enzyme integral to cell respiration Mutation causes decrease in alphaketoglutarate (KG) and increase in 2- hydroxyglutarate (2- HG) Oncometabolite driven epigenetic aberrations
Brain Pathol 20:245-54, 2010 Generated to 13 amino acid peptide coupled to KLH (containing R132H; arginine to histidine substitution) R132H mutation constitutes >90% of IDH1 mutations seen in gliomas
Mutant Specific IDH1 Antibody Capper D et al, Brain Pathol 20:245-54, 2010
IDH1 with R132H mutation Clone H09 (mouse IgG) Generated to synthetic peptide Does not cross react with wild type IDH1
Acta Neuropathol 120:707-18, 2010 IDH status is more prognostic for overall survival than standard histologic criteria By sequencing as well as IHC, with IHC identifying cases initially missed by sequencing IHC missed two cases positive by sequencing
IDH1 Mutation in Gliomas Diffuse Astrocytoma Grade II Anaplastic Astrocytoma Grade III Glioblastoma Multiforme Grade IV IDH1 Mutant 12 (100%) 13 (92.9%) 6 (12.5%) IDH1 Wild type 0 (0%) 1 (7.1%) 42 (87.5%) Am J Clin Pathol 12=38:177-84, 2012
Hartmann C et al., Acta Neuropathol 120:707-18, 2010 Grade v. IDH1 mutation status
(Adv Anat Pathol 2012;19:239 249) Obvious glioma Not so obvious glioma
Other Examples of Gene Mutations Identifiable by Immunohistochemistry EGFR mutations in lung cancer V600E BRAF in melanoma and other tumors
J Thorac Oncol 5:1551-58, 2010 Activating mutations in exons 18 through 21 predict clinical response to gefitinib Present in 10-15% of NSCLC in US and Europe All NSCLCs now tested for EGFR mutations
EGFR Mutations in NSCLC Exon 19 deletions account for ~50% of total mutations 11 different mutations resulting in deletions of 3 to 7 amino acids; all result in deletions of codons for amino acids 747-749 Second major mutation group are missense mutations in exon 21, with L858R accounting for 39% of exon 21 mutations
EGFR Mutations in NSCLC
Kato Y et al., J Thorac Oncol 5:1551-58, 2010 Antibody EGFR E746- A750del Antibody EGFR L858R
Kato Y et al., J Thorac Oncol 5:1551-58, 2010 90 Sensitivity 81.8% 75% N = 70 67,5 E746-A750 L858R
Kato Y et al., J Thorac Oncol 5:1551-58, 2010 125 Specificity 100% 96.6% 100 N = 70 75 E746-A750 L858R
J Thorac Oncol 5:1551-58, 2010 Owing to very high specificity, may be used as screening device to identify positives, but all negatives would need to be tested by molecular methods
Other EGFR IHC Studies Study N Target Sensitivity Specificity Yu et al. Clin Cancer Res 15:3023-8, 2009 340 exon 19 del L858R 92 99 Brevet et al J Mol Diagn 12:169-76, 2010 218 exon 19 del L858R 85% 95% - FAn X et al., Hum Pathol 44:1499-507, 2013 169 exon 19 del L858R 87.7% (combined 98.3% (combined) Bondgaard AL et al, Modern Pathol 27:1590-98,2014 210 exon 19 del L858R 63.2% 80.0% 98.8% 97.8%
Other EGFR IHC Studies Potential Problems Low sensitivity Does not identify all mutations clinically actionable Sensitivity and specificity will depend upon cutoff for positivity (1+? 2+)
Other Examples of Gene Mutations Identifiable by Immunohistochemistry EGFR mutations in lung cancer V600E BRAF in melanoma and other tumors
Examples of Mutations Leading to Loss of Protein Expression INI-1/SMARCB1 Mismatch Repair (MLH1, MSH2, MSH6, PMS2) Rhabdoid tumors (and others) Coloretal adenocarcinoma E-cadherin Lobular breast cancer Succinic dehydrogenase PTEN Subset of gastrointestinal stromal tumors Endometrial, breast cancer
Examples of Gene Mutations Identifiable by Immunohistochemistry Mutant protein (e.g.,idh1) Loss of expresssion (e.g, MMR, SDH) Abnormal localization (e.g., ß-catenin) Overexpression (e.g., p53)
Reasons for MMR IHC Identifying Lynch Syndrome patients Identifying patients with sporadic MSI tumors (who may not require FU-based chemotherapy) Identifying carcinomas of unknown primary that are minimally differentiated colorectal adenocarcinoma
HNPCC (Lynch Syndrome) Hereditary Non-polyposis Colorectal Cancer Autosomal dominant Mutation in MLH1 (~60%), MSH2 (~30%), or MSH6 (~10%) Accounts for 2-5% of colorectal adenocarcinoma Tumors develop at early age, usually found on right side Also develop endometrial adenocarcinoma Synchronous and metachronous colorectal cancers: 40% develop within 10 years without total colonic resection
DNA Mismatch Repair System MLH1 PMS2 MLH2 MSH6
DNA Mismatch Repair DNA mismatch repair promotes genomic stability by correcting base-base and small insertion/deletion mispairs that arise during DNA replication and recombination http://www.helsinki.fi/bioscience/mmrandcancer/mmrgenetics.html
MLH1
MSH2
MSH6
PMS2
New Nomenclature d dmmr pmmr
Are MSI-H tumors distinct? MSI-H tumors more likely arise on the right side MSI-H tumors more likely to occur in people with positive family history of colorectal cancer MSI-H tumors more likely to be cribriform, solid, signet ring, high grade ( medullary ), mucinous Lymphocytic infiltration most important feature for predicting MSI-H (nodular Crohn-like peritumoral or TIL)
Histologic Patterns of MSI Adenocarcinomas Mucinous Signet Ring Medullary Medullary TIL Pushing Border from Bellizzi AM and Frankel WL, Adv Anat Pathol 16:405-17, 2009
IHC vs. MSI Testing IHC MSI Cost $$ $$$ Analyte Protein DNA How much tumor required Very little Very little Requirements Tumor only Tumor + normal Possibility of contamination by normal No Yes Turnaround Next day 2-7 days Identifies involved gene Yes No Assay sensitive to fixation Yes No adapted from Bellizzi AM and Frankel WL, Adv Anat Pathol 16:405-17, 2009
IHC v. MSI Testing Concordance very high in most studies High concordance possible even with just two antibodies (e.g., MLH1, MSH2) but even higher with four (MLH1, MSH2, PMS2, MSH6) Potential shortcoming if IHC is inability to detect missense mutations that nevertheless result in immunoreactive but nonfunctional protein
MMR IHC and Colorectal Adenocarcinoma Immunohistochemical localization integrates what happens at the genomic level to MMR genes Identifies genotypically distinct variants of colorectal adenocarcinoma with important clinical implications
NO ADJUVANT CHEMOTHERAPY N=570 Ribic CM et al. NEJM 349:247-57, 2003
ADJUVANT CHEMOTHERAPY N=570 Ribic CM et al. NEJM 349:247-57, 2003
J Clin Oncol 28:3219-26, 2010 Add Sergeant paper JCO here n = 457 Outcome of Patients with Stage III Colorectal Adenocarcinoma Treated with Adjuvant 5-FU dmmr pmmr
Conflicting Data on Predictive Role of MSI in 5-FU Response in Colorectal Adenocarcinoma Confl5-FU icting data with respect to prediction of response to 5-FU Vilar E and Tabernero J, Cancer Discovery May 2013 502-11
Reasons for MMR IHC Identifying Lynch Syndrome patients Identifying patients with sporadic MSI tumors (who may not require FU-based chemotherapy) Identifying carcinomas of unknown primary that are minimally differentiated colorectal adenocarcinoma
MMR IHC Interpretation Caveats There must be complete loss of MMR expression in the tumor cell population There can be variegated and incomplete immunostaining owing to fixation issues as well as intrinsic variation (e.g., MSH6) Don t overcall dmmr if there is no staining within the non-neoplastic elements
Gastrointestinal Stromal Tumors (GISTs) Originally thought to be smooth muscle tumors ( leiomyoblastoma ) or autonomic nerve tumor ( GANT ) Related to interstitial cells of Cajal (ICC) Both GISTs and ICCs express KIT, CD34, and DOG1
KIT and PDGFRA Mutations in GISTs from Marrari A et al, Arch Pathol Lab Med 136:483-9, 2012
c-kit IHC is a Cell Type Specific Marker! Is marker both of normal interstitial cells of Cajal as well counterpart tumor, gastrointestinal stromal tumor Presence of c-kit expression in GIST is not evidence of presence of activating mutation and hence eligibility for imatinib
SDH Mutations and GISTs PNAS 108:314-8, 2011 Carney-Stratakis syndrome caused by germ line mutations in SDH subunits B, C, or D Predisposes to GISTs and paragangliomas Investigated sporadic GISTs in patients lacking kit or PDGFRA mutations (N =34)
SDH-Deficient GISTs Feature SDH Deficient SDH Proficient Age Children and young adults Sex distribution F > M F = M Older adults Location Stomach Entire GI tract Multinodular Almost always Rare Multifocality Common Rare Histology Epithelioid or mixed Spindle common Lymph node mets Common Rare Course of mets Indolent Aggressive Imatinib sensitivity No Most cases c-kit positive IHC Yes Yes c-kit mutations None ~95% SDH mutations ~50% None adapted from Doyle LA and Hornick JL, Histopathol 64:53-67, 2014
SDH-Deficient GISTs 2.6% of GISTs negative for both DOG1 and kit, and 50% of these are PDGRA-mutant types IHC for SDHB or SDHA is helpful in evaluation of SDH deficient GISTs ~30% of SDH deficient GISTs have mutations in SDHA; only SDHA mutant tumors show loss of SDHA adapted from Doyle LA and Hornick JL, Histopathol 64:53-67, 2014
SDH Mutations and GISTs Janeway KA et al., PNAS 108:314-8, 2011
SDH Mutation GISTs Multinodular architecture Loss of Expression of SDH-B Mixed spindle and epithelioid morphology Doyle LA, Histopathol 61:801-9, 2012
http://upload.wikimedia.org/wikipedia/commons/5/5a/succdeh.svg Succinate dehydrogenase is an enzyme complex, bound to the inner mitochondrial membrane of mammalian mitochondria and many bacterial cells. It is the only enzyme that participates in both the citric acid cycle and the electron transport chain.
Courtesy of Jason L. Hornick, MD PhD
Courtesy of Jason L. Hornick, MD PhD KIT
Courtesy of Jason L. Hornick, MD PhD SDHB
Courtesy of Jason L. Hornick, MD PhD KIT
Courtesy of Jason L. Hornick, MD PhD SDHB
Courtesy of Jason L. Hornick, MD PhD
Courtesy of Jason L. Hornick, MD PhD
Examples of Gene Mutations Identifiable by Immunohistochemistry Mutant protein (e.g.,idh1) Loss of expression (e.g, MMR, SDH) Abnormal localization (e.g., ß-catenin) Overexpression (e.g., p53)
ß-Catenin: Role in Cell Adhesion and Signaling Axin and APC are negative regulators of Wnt signalling cascade. Axin and APC phosphorylate ß- catenin on APC binding sites, thereby degrading and inactivating the protein. Regulation of ß-catenin critical to APC s tumor suppressor effect
ß-Catenin
Nuclear beta catenin The presence of beta catenin in the nucleus indicates a disruption of the WNT pathway In nucleus, beta catenin interacts with transcription factors of TCF/LEF family and thus takes part in alteration of gene expression ß-catenin in nucleus continuously drives transcription of target genes Lead to increased cell proliferation and/or inhibition of apoptosis
Mesenteric Fibromatosis Aggressive fibromatosis, desmoid tumor All ages Associated with Gardner syndrome Abdominal and extra-abdominal (shoulder, chest wall, back) Deep-seated, poorly circumscribed Most present with asymptomatic abdominal mass
ß-Catenin and Fibromatoses Montgomery et al, AJSP, 2002 Fibromatoses have mutation in APC/ ß-catenin pathway Abnormal nuclear accumulation of ß-catenin protein Studied expression by IHC in mesenteric fibromatosis, GIST, and sclerosing mesenteritis
Mesenteric Fibromatosis ß-Catenin
` GIST ß-Catenin
ß-Catenin Immunohistochemistry can be employed to see the nuclear ß-catenin protein abnormally located in the nucleus Nuclear ß-catenin is a surrogate marker for the presence of APC or ß-catenin mutations
Is Nuclear ß-catenin Expression Found in Other Tumors? Modern Pathology 18:68-74, 2005
Ng TL et al., Modern Pathology 18:68-74, 2005 Tumors POSITIVE for high level nuclear ß-catenin expression Desmoid type fibromatosis (71%) Solitary fibrous tumor (40%) Endometrial stromal sarcoma (40%) Synovial sarcoma (28%)
Abnormal localization of ß-catenin to nucleus May be mutation of ß-catenin or adenosis polyposis coli (APC) genes APC mutations more common in setting of familial adenomatous polyposis ß-catenin mutations more common in sporadic aggressive fibromatosis Demonstrates that mutation of one gene may result in abnormal localization of another gene product
Examples of Gene Mutations Identifiable by Immunohistochemistry Mutant protein (e.g.,idh1) Loss of expresssion (e.g, MMR) Abnormal localization (e.g., ß-catenin) Overexpression (e.g., p53)
p53 Most (but not all) inactivating mutations result in conformational change of p53 molecule that results in prolonged half-life Half-life 20 minutes for wild type, hours for mutant proteins Wild type protein detectable by immunohistochemistry but at low levels that seem to correlate with cell proliferation Large deletions or truncating mutations may result in apparent loss of p53 expression
J Pathol 222:191-8, 2010 DO-7 anti-p53 monoclonal antibody (cross reacts with wild type and mutant) Scored in three bins: complete absence of expression, focal expression, overexpression (>50%) Outcome in two different cohorts
p53 and Ovarian Cancer Kobel M et al., J Pathol 222:191-8, 2010
p53 and Ovarian Cancer Kobel M et al., J Pathol 222:191-8, 2010 Pelvic high grade serous ovarian cancers show either complete loss or overexpression in 88% of cases p53 overexpression associated with reduced risk of recurrence Complete absence of expression associated with unfavorable outcome Suggests functional differences underlying overexpression v. absence of expression
p53
p53 Immunohistochemistry Three unique immunostaining patterns Mutated Wild type Mutated
p53 and Barrett s Kaye PV, et al. Histopathol 54:699-712, 2009 N = 154 biopsy specimens with Barrett s, 32 specimens without dysplasia p53 immunohistochemistry assists in diagnosis in difficult cases and predicts progression
Suggested Algorithm Kaye PV, et al. Histopathol 54:699-712, 2009
Gut 62:1676-83, 2013 Low grade dysplasia currently only accepted predictor for neoplastic progression in Barrett s esophagus Can alterations in p53 improve risk stratification?
Barrett s esophagus with low grade dysplasia Barrett s esophagus with low grade dysplasia Esophageal adenoca with complete loss of p53
Kastelein F et al., 2013 N = 635 patients Retrospective study of p53 protein expression as determined by IHC 8% of patients developed high grade dysplasia or adenocarcinoma More powerful predictor than histologic diagnosis of LGD Strongly associated with overexpression and especially loss of p53 expression
Incidence of p53 Overexpression and Absence of Expression Kastelein F et al., 2013
Gut 63:7-42, 2014 Marker with greatest body of evidence which can also be applied routinely is p53 50-89% positive in Barrett s dysplasia Can improve inter observer variability for reporting dysplasia (especially LGD v. atypical reactive [ID]) Powerful predictor of progression (OR 3-8)
Major Genetic Alterations in Cancer Mutation Translocation Deletion Amplification Methylation Loss of Expression Abnormal Localization Overexpression Expression of Fusion Proteins
Examples of Chromosomal Translocations Identifiable by NG-IHC Tumor Translocation Fusion Generated NG-IHC Target PNET/ES t(11;22)(q24;q12) EWSR1-FLI1 FLI1 ALCL t(2;5)(p23;q35) NPM-ALK ALK ASPS Synovial sarcoma der(17)t(x;17)(p11;q25 ) ASPL-TFE3 TFE3 t(x;18)(p11.2;q11.2) SYT-SSX1 TLE-1 DSRCT t(11;22)(q11;q12) EWSR1-WT1 WT-1 AML t(8;21)(q22;q22) AML1-ETO AML1-ETO Chromosme 2
Alveolar Soft Part Sarcoma Rare tumor in young patients with poor prognosis First coined and described by Christopherson, 1952 Characteristic histology: organoid nests, pseudoalveolar pattern Can show clear cell change, PAS+ crystalline inclusions
der(17)t(x;17)(p11;q25) of ASPS and generation of ASPL-TFE3 fusion protein N ASPL (Chr 17) C N TFE3 (X Chr) C
der(17)t(x;17)(p11;q25) of ASPS and generation of ASPL-TFE3 fusion protein N ASPL (Chr 17) C N TFE3 (X Chr) C
der(17)t(x;17)(p11;q25) of ASPS and generation of ASPL-TFE3 fusion protein ASPL (Chr 17) C N C N TFE3 (X Chr)
der(17)t(x;17)(p11;q25) of ASPS and generation of ASPL-TFE3 fusion protein C N ASPL - TFE3 C N
Composite ASPL-TFE3 Protein ASPL promotor constitutively expressed and fusion protein highly expressed TFE3 (in family of basic helix loop helix leucine zipper nuclear transcription factors) binds to DNA
TFE3
TFE3 Expression Is Also Found in Some RCCs Group of renal cell carcinomas distinguished by chromosomal translocations with breakpoints involving the TFE3 gene on Xp11.2 Ross H and Argani P, Pathology 42:369-73, 2010
ASPL-TFE3 Renal Cell Carcinomas Have Distinctive Morphologic Features Voluminous clear cytoplasm Discrete cell borders Alveolar or papillary growth pattern Psammoma bodies
ASPL-TFE3 Renal Cell Carcinoma
ASPL-TFE3 Renal Cell Carcinoma
Am J Surg Pathol 27:760-61, 2003
Problems with Identification of TFE3 by IHC TFE3 detection highly fixation dependent Epitope retrieval Specificity of antibody employed Cutoff point for positivity
Am J Surg Pathol 37:804-15, 2013
TFE3 Expression Is Also Found in Other Tumors Epithelioid hemangioendothelioma variant with YAP1-TFE3 gene fusion (v. WWTR1-CAMTA1 fusion in ~90% of these tumors) TFE3 translocation associated PEComas of gynecologic tract
Anaplastic Large Cell Lymphoma and ALK
CD20
CD3
CD30
Ki67
ALK
The Many Histologic Faces of ALCL Common Lymphohistiocytic variant Hodgkins-like variant Sarcomatoid variant Monomorphic variant Small cell variant
Recurring Cytogenetic Changes in ALCL Translocation Partner Gene Frequency Location t(2;5) Nucleophosmin 75% Cytoplasmic/nu clear t(1;2) Tropomyosin 3 10-20% Cytoplasmic t(2;3) TK fused gene 2-5% Cytoplasmic Inv 2 ATIC 2-5% Cytoplasmic t(2;22) Clathrin heavy chain 2-5% Cytoplasmic/gra nular
Falini B et al., Blood 94:3509-15, 1999
Falini B et al., Blood 94:3509-15, 1999 IHC of classical ALCL shows nuclear plus cytoplasmic pattern IHC of ALCL variants with non- NPM-ALK fusions show cytoplasmic only pattern
NPM-ALK Variant ALK Falini B et al., Blood 94:3509-15, 1999
Falini B et al., Blood 94:3509-15, 1999
CONCLUSION Immunohistochemistry can be used to identify the presence of a subset of ALCL that have variant translocations but have similar clinical outcomes to (2;5) translocation ALCLS producing NPM-ALK Falini B et al., Blood 94:3509-15, 1999
The Evolution of Anaplastic Large Cell Lymphoma
Examples of Chromosomal Translocations Identifiable by NG-IHC Tumor Translocation Fusion Generated NG-IHC Target PNET/ES t(11;22)(q24;q12) EWSR1-FLI1 FLI1 ALCL t(2;5)(p23;q35) NPM-ALK ALK ASPS der(17)t(x;17)(p11;q25) ASPL-TFE3 TFE3 Synovial sarcoma t(x;18)(p11.2;q11.2) SYT-SSX1 TLE-1 DSRCT t(11;22)(q11;q12) EWSR1-WT1 WT-1 AML t(8;21)(q22;q22) AML1-ETO AML1-ETO Lung cancer Chromosme 2 inversion EML4-ALK ALK
Major Genetic Alterations in Cancer Mutation Translocation Deletion Amplification Methylation Loss of Expression Abnormal Localization Overexpression Expression of Fusion Proteins
Examples of Amplified Proteins Identifiable by NG-IHC Tumor Breast, gastric cancer Liposarcoma Lymphoma Lung cancer Protein amplified HER2 MDM-2, CDK4 bcl-2 EGFR
What does the Future of Pathology Look Like? The paradigm shift to molecular based classification of tumors will continue and will accelerate Molecular oncodiagnostics will play an increasingly large role in tumor analysis
From Morphology And Back Anaplastic large cell lymphoma Rhabdoid Tumor MSI Type Colorectal CAs Lobular Breast CA IHC-Cell type CD30 Vimentin and CK+ inclusions Maybe loss of CK20 and CDX2 Lobular cell, CK pattern Genetic t(2;5) INI1/SMARCB1 mutation Mutations of MMR genes Molecular switching off of E-cad New IHC ALK INI1/SMARCB1 loss Loss of MMRs Loss of E- cadherin New related entities Small cell monomorphic variant Chondrosarcoma, epithelioid sarcoma MSI Type Colorectal CAs with no special histologic features Pseudointraductal carcinoma
Next Generation Immunohistochemistry 1 NG-IHC can be used to identify molecular alterations that characterize selected malignancies NG-IHC acts as a surrogate for molecular studies, and is less expensive and time consuming and, in some cases, can provide more information
Next Generation Immunohistochemistry 2 NG-IHC can integrate different genotypic changes which result in the same phenotypic changes NG-IHC can thus expand and better define categories of disease
The Changing Role of the Pathologist As more drugs that target specific components of signal-transduction pathways become available and as we increase our knowledge of the complexity of these signalling networks, the burden of selecting the correct drug combinations for each individual cancer patient will ultimately shift to the pathologist, who must identify the underlying defect in each tumor. Shaw RJ and Cantley LC. Nature 441:424-30, 2006
gown@phenopath.com Thank you for your attention Photograph by Dave Morrow