CRIPTO promotes extracellular vesicle uptake and activation of cancer associated fibroblasts.

Expression of CRIPTO, a factor involved in embryonic stem cells, fetal development, and wound healing, is tied to poor prognosis in multiple cancers. Prior studies in triple negative breast cancer (TNBC) models showed CRIPTO blockade inhibits tumor growth and dissemination. Here, we uncover a previously unidentified role for CRIPTO in orchestrating tumor-derived extracellular vesicle (TEV) uptake and fibroblast activation through discrete mechanisms. We found a novel mechanism by which CRIPTO drives aggressive TNBC phenotypes, involving CRIPTO-laden TEVs that program stromal fibroblasts, toward cancer associated fibroblast cell states, which in turn prompt tumor cell invasion. CRIPTO-bearing TEVs exhibited markedly elevated uptake in target fibroblasts and activated SMAD2/3 through NODAL-independent and - dependent mechanisms, respectively. Engineered expression of CRIPTO on EVs enhanced the delivery of bioactive molecules. In vivo , CRIPTO levels dictated TEV uptake in mouse lungs, a site of EV-regulated premetastatic niches important for breast cancer dissemination. These discoveries reveal a novel role for CRIPTO in coordinating heterotypic cellular crosstalk which offers novel insights into breast cancer progression, delivery of therapeutic molecules, and new, potentially targetable mechanisms of heterotypic cellular communication between tumor cells and the TME.

Clinicopathologic Features of Breast Carcinoma With 2018 American Society of Clinical Oncology/College of American Pathologists Fluorescence In Situ Hybridization Group 3 (Human Epidermal Growth Factor Receptor 2 Chromosome 17 Centromere Ratio <2.0 and Average Human Epidermal Growth Factor Receptor 2 Copy Number ≥6.0) Breast Cancers.

CONTEXT.­: The American Society of Clinical Oncology/College of American Pathologists 2018 update of the human epidermal growth factor receptor 2 (HER2) testing guideline includes a fluorescence in situ hybridization (FISH) group with a HER2 to chromosome 17 centromere (CEP17) ratio less than 2.0 and HER2 copy number 6.0 or greater (group 3), which requires integrated review of HER2 immunohistochemistry (IHC). OBJECTIVE.­: To assess the clinicopathologic features of group 3 patients and determine features associated with HER2-positive status after workup. DESIGN.­: Cases submitted for HER2 FISH between January 2019 and June 2022 were identified, and relevant clinicopathologic information was obtained. RESULTS.­: One hundred forty-two HER2 FISH cases (1.6%) were group 3. In 52 cases (36.6%) IHC was negative (0/1+), in 3 (2.8%) IHC was positive (3+), and in 86 (60.6%) IHC was 2+. Annotated IHC 2+ slides were recounted by a second reviewer in targeted areas, where 16 of 86 (18.6%) had a HER2:CEP17 ratio less than 2.0 and a HER2 copy number of 4.0 or greater to less than 6.0 (HER2 negative). After combined IHC/FISH review, 74 of 142 (52.1%) were classified as HER2 positive. HER2 copy number/cell was higher in HER2-positive compared with HER2-negative cases after the workup. The extent and intensity of staining in IHC 2+ cases did not correlate with the level of gene amplification. Twenty percent of HER2-positive patients achieved pathologic complete response. CONCLUSIONS.­: About half of group 3 cases were classified as HER2 positive after additional workup. Pathologic complete response rates in HER2-positive cases were lower than expected for group 1 (HER2:CEP17 ratio ≥2.0; HER2 copy number ≥4.0) patients. IHC targeted FISH recounts may be redundant and may potentially lead to classification of some patients as HER2 negative, resulting in withholding of targeted therapy.

Metaplastic Breast Carcinoma: Clinicopathologic Features and Recurrence Score Results From a Population-based Database.

OBJECTIVES: Metaplastic breast carcinoma (MBC) is a rare, aggressive form of cancer comprising epithelial and mesenchymal elements. The purpose of this study was to use population-based data to review the clinicopathologic, molecular features, and outcomes of MBC. METHODS: Surveillance, Epidemiology, and End Results Program (SEER) data were used to identify MBC and invasive ductal carcinoma (IDC), no special type (NOS) between 2004 and 2015. Results from Oncotype DX's 21-gene assay linked to SEER registries were included for hormone receptor (HR)-positive tumors. χ 2 analysis was performed to determine the differences between MBC and IDC. Kaplan-Meier curves and multivariate Cox proportional hazards models were used for breast cancer specific death (BCSD). RESULTS: Compared with IDC, NOS (n=509,864), MBC (n=3876) were more likely to present at an older age, be black, have negative lymph nodes, be >2 cm, grade 3, and triple negative (TN). All subtypes [HR-positive/human epidermal growth receptor 2 (HER2)-negative, HR-positive/HER2-positive, HR-negative/HER2-positive, and TN] had higher BCSD than IDC, NOS. 22.3% of MBC cases were HR-positive. HR-positive MBCs tested for a recurrence score (RS) 65% were high-risk compared with 16.8% of IDC, NOS. Within the MBC cohort, no significant differences in BCSD were identified with respect to different molecular subtypes. In a fully adjusted model, TN or HER2-positive status did not adversely affect BCSD compared with HR-positive MBC. CONCLUSIONS: All molecular subtypes of MBC had a poorer prognosis compared with IDC, NOS. The different molecular subtypes of MBC did not affect the BCSD. HR-positive MBC patients had a significantly higher high-risk RS than IDC, NOS patients.

Digital imaging correlation of immunohistochemistry and fluorescence in situ hybridization in breast carcinoma cases with HER2 genetic heterogeneity.

Genetic heterogeneity (GH) is a rare but important event in the evaluation of HER2 amplification status. We investigated whether HER2 fluorescence in situ hybridization (FISH) GH correlated with increased protein expression by immunohistochemistry (IHC) and/or morphologic features using image analyses. Retrospective search of HER2 FISH GH cases 2016-2020 was performed. Cases with both FISH and IHC slides available were considered eligible and were digitally imaged. Additional demographic, histological, and treatment information was compiled from pathology and medical records when available. Overall, 11 of 15 cases (73.3%) had HER2 FISH GH that matched to areas of HER2 overexpression or focally different morphology. Nine cases with areas of gene amplification overlapped with <10% of intense circumferential protein expression ("Mini 3+"), and 1 case with focal micropapillary features. Clinical information was available on 6 patients (40%), all were alive with no evidence of disease (mean follow-up 30.5 months; range, 12-65 months). One patient with GH and a lymph node metastasis showed nonamplified population in the nodal tumor. GH, when defined as discrete clusters of amplified cells following 2013 ASCO/CAP guidelines, even when less than 10% of the tumor cells, frequently has morphologic correlates such as focal intense protein overexpression or micropapillary morphology. Clinical significance of these focal gene amplification and protein overexpression needs to be further investigated.

RAI1 alternate probe identifies additional gastroesophageal adenocarcinoma cases as amplified following equivocal HER2 fluorescence in situ hybridization testing: experience from a national reference laboratory.

The College of American Pathologists/American Society of Clinical Oncology recommends HER2 testing prior to initiation of targeted therapy for patients with advanced Gastroesophageal adenocarcinoma (GEA), using immunohistochemistry (IHC) followed by fluorescence in situ hybridization (FISH) in cases with an equivocal (score 2 + ) result on IHC. The FISH results are considered indeterminate if the HER2/CEP17 ratio is <2.0 with an average CEP17 copy number of ≥3.0 and a HER2 copy number ≥4.0 and ≤6.0 after counting additional tumor cells. Indeterminate results may be resolved by using an alternative chromosome 17 probe such as RAI1. The purpose of this study is to review our experience with RAI1 alternate probe in HER2 FISH testing of GEA in a large reference laboratory setting. Esophageal, gastroesophageal, and gastric adenocarcinomas received for HER2 FISH testing in our lab between 9/2018 and 1/2020 were included. HER2/CEP17 and HER2/ RAI1 ratios, and the average HER2, CEP17, RAI1 signals per cell were recorded. 328 GEA had HER2 testing performed in our lab during the study period. 101 (30.8%) were amplified, 169 (51.5%) were non-amplified and 58 (17.7%) were indeterminate. Following RAI1 testing, 42 (72.4%) of 58 indeterminate cases were reclassified as non-amplified and 16 (27.6%) were reclassified as amplified, increasing the total amplified cases to 117 (35.7%). The correlation between the average CEP17 and RAI1 copy number for all cases was weak (R2 = 0.095). In summary, using the alternate probe RAI1 reclassifies 27.6% of original HER2 FISH indeterminate gastroesophageal carcinomas as amplified, which makes them eligible for targeted therapies.

The 21-Gene Recurrence Score in Special Histologic Subtypes of Breast Cancer.

CONTEXT.­: Recurrence score (RS) testing was developed and validated in invasive ductal and rare lobular carcinomas, although it is used for all special types of breast cancers. OBJECTIVE.­: To determine association of histologic type (HT) and RS, specifically high-risk RS. DESIGN.­: We used RSs linked to Surveillance, Epidemiology, and End Results Program registries of invasive breast cancers diagnosed in 2004 through 2015. Multivariable logistic regression was used to evaluate association between HT and high-risk RS. Relationships between HT and low-, intermediate-, and high-risk RS were compared with χ2 test. Kaplan-Meier curves were compared using log-rank test. RESULTS.­: A total of 110 318 patients had RS testing. Of these, 23 220 (21%) had low, 70 822 (64.2%) intermediate, and 16 276 (14.8%) high RS. Histologic types were 80 476 (73%) ductal, 12 713 (11.5%) lobular, 12 449 (11.3%) mixed, 2151 (2%) mucinous, 610 (0.6%) tubular, 382 (0.4%) micropapillary, 365 (0.3%) salivary, 208 (0.2%) papillary, 49 (0.04%) medullary, 26 (0.02%) metaplastic, 26 (0.02%) neuroendocrine, and 863 (0.8%) unknown. The distribution of low-, intermediate-, and high-risk RS was significantly different among HTs. Higher percentages of high-risk RS were identified in patients with ductal, medullary, and metaplastic types (P < .001). The odds of having high-risk RS were lower for some HTs, including micropapillary, after multivariable adjustment (P < .05). The low number of estrogen receptor-positive medullary and metaplastic carcinomas tested had higher odds of having high-risk RS. In T1 and T2 tumors, when ductal, lobular, mixed, and other types combined were compared, the mortality was different. CONCLUSIONS.­: This population-based study of RS in HTs showed high-risk RSs are identified in traditionally good prognostic subtypes. Micropapillary carcinoma has lower odds of high-risk RS even after multivariable adjustment.

21-gene recurrence score testing utilization among older women from different races: A population-based study.

OBJECTIVES: The influence of older age at diagnosis in combination with race/ethnicity on utilization and results of the 21-gene recurrence score (RS) assay for breast cancer (BC) patients is not fully understood. Our objectives were to evaluate the utilization of RS among older women with BC, the likelihood of a high-risk RS, and factors associated with breast cancer-specific mortality (BCSM) among older patients across different races. MATERIALS AND METHODS: We utilized the Surveillance, Epidemiology, and Results (SEER) database with linked RS results to evaluate women with estrogen receptor-positive BC diagnosed 2004-2015. Multivariable logistic regression was used to describe the differences in utilization of RS testing and the association of high-risk RS according to patient characteristics. The Cox proportional hazards model was used to analyze factors associated with BCSM. RESULTS: We found that 20.4% (109,244/536,555) of all women ≥18 and 14.3% (33,584/235,171) of women ≥65 underwent RS testing. Non-whites had lower odds of RS testing at younger ages whereas among women ≥65 there was no significant difference. After taking into account stage and grade, being ≥65 reduced the odds of high-risk RS in all races except American Indian/Alaskan Native. Age ≥ 65 was independently associated with increased hazard BCSM. Among women ≥65 with high-risk RS, chemotherapy was associated with lower hazard of BCSM in all races. CONCLUSIONS: Older women are less likely to be tested for RS, but also less likely to have high-risk RS. Older women with high-risk RS, when given chemotherapy have reduced BCSM across all races.

What to Expect From the New ASCO/CAP Guideline Recommendations for Hormone Receptor Testing in Breast Cancer: A National Reference Laboratory Experience.

The new ASCO/CAP guidelines on hormone receptor testing in breast cancer recommends standard operating procedures (SOPs) established to confirm or adjudicate estrogen receptor (ER) results with weak or ≤10% staining, and the status of internal controls (ICs) reported for cases with 0% to 10% staining. The aim of this study is to determine the frequency of ER testing with weak or ≤10% staining that may require additional steps following SOPs and to identify any correlation between hormone receptor status of the tumor and the likelihood of finding IC. Breast cancer cases between January 2014 and April 2019 were included to identify negative, low-positive and weak-positive cases. The presence/absence of IC was correlated to tumor type. Following ASCO/CAP guidelines, 29.8% of cases (374/1261) will need additional steps to confirm/adjudicate results due to negative, low, or weak positive ER status. The probability of finding IC is ~50% lower in cases of ER and progesterone receptor (PgR) negative tumors. Repeat testing may be warranted in 13.1% (92/700) of all cases due to lack of IC. In conclusion, the new ASCO/CAP guidelines recommend laboratories to establish and follow SOP to confirm or adjudicate ER results for about 30% of the cases before reporting hormone receptors status. Over 40% of cases with <10% tumor ER positivity lacked IC that may need a comment per the guidelines indicating a repeat testing may be warranted. However, the presence/absence of IC may be related to the subtype of breast cancer and should not necessarily bring into question the validity of the test.

American Society of Clinical Oncology/College of American Pathologists 2018 Focused Update of Breast Cancer HER2 FISH Testing GuidelinesResults From a National Reference Laboratory.

OBJECTIVES: To review impact of the ASCO/CAP 2018 update on HER2 testing. METHODS: HER2 fluorescence in situ hybridization (FISH) test requests from primary and metastatic breast cancers between August 2018 and January 2019 were included. FISH results requiring a changed algorithm under the new guidelines (groups 2, 3, and 4) were identified and HER2:CEN17 ratios, average HER2, CEN17 signals/cell, and HER2 immunohistochemistry (IHC) results were recorded. RESULTS: Of the HER2 FISH cases 176/812(21.7%) fell within groups 2, 3, or 4; 0/12, 1/12, and 2/152 cases were positive (3+) by IHC, and 1/12, 2/12, and 6/152 cases were positive after targeted scoring from groups 2, 3, and 4, respectively. Following 2018 updates, 8.3%, 25%, and 5.3% of the groups 2, 3, and 4 were positive, respectively. CONCLUSIONS: Groups 2, 3, and 4 constituted over 20% of HER2 FISH tests in a reference laboratory. The 2018 ASCO/CAP update significantly decreased the HER2 positivity rate.

Differences in molecular features of triple-negative breast cancers based on the age at diagnosis.

BACKGROUND: Although the proportion of triple-negative breast cancers (TNBCs) diagnosed among older women is low, the number of TNBC cases is substantial because of the high incidence of breast cancer after the age of 65 years. The molecular features of TNBC in this age group have not been well described. METHODS: This study examined a population-based cohort of women with stage I to III TNBC diagnosed between the ages of 25 and 91 years with the PAM50 gene expression subtyping assay. The concordance between the TNBC classification by immunohistochemistry and the gene expression classification by PAM50, the expression of individual genes, and 5-year recurrence and breast cancer mortality in older women (≥65 years old) and younger women (<50 years old) was assessed. RESULTS: The molecular subtype distribution in TNBC was significantly different according to the age at diagnosis. TNBC was more likely to be classified as basal-like in women younger than 50 years (sensitivity, 0.91; 95% confidence interval, 0.77-0.97) than women 65 years old or older (sensitivity, 0.72; 95% confidence interval, 0.48-0.87); 35% of clinical TNBC cases in the latter group were the human epidermal growth factor receptor 2 (HER2)-enriched subtype by molecular classification. Older women with TNBC also had significantly higher expression of ERBB2 and lower expression of all 10 proliferation-associated genes tested (P < .01). The risk of breast cancer death within 5 years was significantly higher in women with TNBC in comparison with women with hormone receptor-positive cancers in all age groups. CONCLUSIONS: This study revealed differences in molecular subtypes among clinical TNBC cases based on patient age. A potentially targetable HER2-enriched group raises the possible need for intrinsic subtyping in older women with TNBC.

Impact of 2013 American Society of Clinical Oncology/College of American Pathologists Guidelines on HER2 Fluorescent In Situ Hybridization Testing in Breast Cancers : Experience From a National Reference Laboratory.

OBJECTIVES: We compared the impact of 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines on human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) testing results on breast cancers. METHODS: HER2 FISH testing performed between May 2015 and April 2016 following 2013 ASCO/CAP guidelines was included. HER2 to control probe ratios, mean HER2, and control probe copy numbers were used to reassign HER2 status using 2007 ASCO/CAP and US Food and Drug Administration (FDA) guidelines. RESULTS: HER2 FISH results were available in 2,017 cases. A total of 342 (17.0%) cases were amplified, 301 (14.9%) were equivocal, and 1,374 (68.1%) were nonamplified. After additional testing with the alternate probe, amplified cases increased to 21.6%. HER2 positivity rates following the 2013 ASCO/CAP guidelines were significantly higher compared with the 2007 ASCO/CAP and FDA guidelines. CONCLUSIONS: The 2013 ASCO/CAP guidelines lead to a higher number of HER2 FISH positive and equivocal cases. In a reference laboratory setting where an alternative control probe was used to resolve equivocal FISH cases, 31.2% of patients with initial equivocal results became HER2 positive.

Primary Cystic Pleuropulmonary Synovial Sarcoma Presenting as Recurrent Pneumothorax.

Primary pleuropulmonary synovial sarcomas are quite rare, representing 0.1-0.5% of all pulmonary malignancies. We report an entirely cystic monophasic synovial sarcoma in a 25-year-old male who presented with recurrent pneumothorax and no evidence of a mass lesion on imaging. The purpose of this case report is to increase awareness of neoplasms clinically presenting as a pneumothorax with no imagining evidence of a mass-forming lesion and emphasize the significance of fluorescent in situ hybridization testing in nontypical synovial sarcoma cases.

RAI1 Alternate Probe Identifies Additional Breast Cancer Cases as Amplified Following Equivocal HER2 Fluorescence In Situ Hybridization Testing: Experience From a National Reference Laboratory.

CONTEXT: -In 2013 the American Society of Clinical Oncology and College of American Pathologists updated the HER2 guidelines and changed the equivocal category for HER2 in situ hybridization testing to an average HER2 copy number of 4.0 to 5.9 with a HER2:CEP17 ratio of less than 2.0 and proposed retesting, with an option of using another control probe to avoid false-negative results. RAI1, located at band position 17p11.2, is a popular alternate probe locus for retesting equivocal changes. OBJECTIVE: -To review experience with the RAI1 alternate probe in HER2 fluorescence in situ hybridization equivocal breast cancers. DESIGN: -Primary and metastatic breast cancers with equivocal HER2 fluorescence in situ hybridization, retested with an alternate (RAI1) probe, were identified. HER2, RAI1, and CEP17 copy numbers, HER2 to control probe ratios, and genetic heterogeneity were recorded. Hematoxylin-eosin-stained slides were reviewed for type and grade of cancer. RESULTS: -Of 876 cases tested with CEP17 as the reference probe, 97 (11.1%) had equivocal HER2 fluorescence in situ hybridization results. Additional testing with the RAI1 probe classified 39.2% cases (38 of 97) as amplified with a HER2:RAI1 ratio ranging from 2.0 to 3.2 (mean, 2.37); 3.1% (3 of 97) were still unclassifiable because of a deletion of RAI1. CONCLUSIONS: -RAI1 identified close to 40% of original HER2 fluorescence in situ hybridization equivocal cases as amplified, making these patients eligible for targeted therapies. It is not known whether guidelines for US Food and Drug Administration-approved probes can be extrapolated to alternate probes when an alternate control probe shows losses or gains. Because of the lack of guidelines for reporting HER2 status with alternate probes, laboratories face challenges in interpreting results.

Quantification of Estrogen Receptor Expression in Normal Breast Tissue in Postmenopausal Women With Breast Cancer and Association With Tumor Subtypes.

Estrogen exposure is important in the pathogenesis of breast cancer and is a contributing risk factor. In this study we quantified estrogen receptor (ER) alpha expression in normal breast epithelium (NBR) in women with breast cancer and correlated it with breast cancer subtypes. Tissue microarrays were constructed from 204 breast cancer patients for whom normal breast tissue away from tumor was available. Slides stained with ER were scanned and expression in normal terminal duct lobular epithelium was quantitated using computer-assisted image analysis. ER expression in normal terminal duct lobular epithelium of postmenopausal women with breast cancer was significantly associated with estrogen and triple (estrogen, progesterone receptors, and HER2) negative phenotypes. Also increased age at diagnosis was significantly associated with ER expression in NBR. ER positivity in normal epithelium did not vary by tumor size, lymph node status, tumor grade, or stage. On the basis of quantitative image analysis, we confirm that ER expression in NBR increases with age in women with breast cancer, and report for the first time, a significant association between ER expression in NBR with ER-negative and triple-negative cancers in postmenopausal women.

Inconsistent Results With Different Secondary Reflex Assays for Resolving HER2 Status.

OBJECTIVES: Guidelines suggest that secondary reflex testing may be useful for resolving HER2 status in breast cancers with equivocal results by both immunohistochemistry (IHC) and in situ hybridization (ISH). We compared two reflex ISH assays and a polymerase chain reaction (PCR) assay for this application. METHODS: Twenty-nine breast cancers with equivocal IHC and ISH results were retested two ways: (1) ISH using differentially labeled probes targeting ERBB2 ( HER2 , 17q12) and either RAI1 (17p11.2) or ORC4 (2q22.3-2q23.1) in two separate assays and (2) real-time quantitative PCR amplification of ERBB2 and a control locus ( EIF5B , 2q11.2). RESULTS: Results of the HER2 / RAI1 and HER2 / ORC4 ISH assays were concordant for 21 (72%) cases, and results of all three secondary reflex assays were concordant for only 18 (62%) cases. Result discrepancies between the two ISH readers were observed for cases close to the cutoff threshold. CONCLUSIONS: Use of different control loci for ISH is associated with discordant results, and PCR is more likely to classify cases as nonamplified, possibly due to contamination with nontumor cells. While resolution of HER2 -equivocal results is desirable from a clinical perspective, different secondary reflex assays yield different results, and the correlation of these results with clinical outcomes is unknown.

Genomic Copy Number Analysis of HER2-Equivocal Breast Cancers.

OBJECTIVES: Guidelines for HER2 testing define an equivocal range for HER2 using two approved testing methods, immunohistochemistry (IHC) and in situ hybridization (ISH). We investigated genome-wide copy number alterations in this subgroup. METHODS: Ten breast cancers with equivocal HER2 status by both IHC and ISH were analyzed by single-nucleotide polymorphism cytogenomic microarray (SNP array). DNA ploidy analysis by flow cytometry was performed on nine cases with sufficient material remaining. RESULTS: SNP array analysis showed uniform gain of chromosome 17 (polysomy) in one case and segmental copy number gains encompassing HER2 and the centromere in five other cases. Flow cytometry revealed hyperdiploidy in six cases, all but one of which also had HER2 gains on SNP array. Although there was no evidence of HER2 amplification by SNP array, six cases showed amplification of other genomic regions, including known oncogenes in four cases. CONCLUSIONS: A combination of hyperdiploidy and segmental copy number gains contributes to HER2 ISH-equivocal results in most breast cancers. Cases in which HER2 copy number gain is not corroborated by genomic analysis suggest the presence of other contributing variables influencing ISH results. Genomic copy number analysis also implicates non-HER2 oncogenic drivers in many cases that are HER2 equivocal.

Use of in situ hybridization for HPV in head and neck tumors: experience from a national reference laboratory.

The human papillomavirus (HPV) status of head and neck squamous cell carcinomas (SCCs) is a frequent request for Anatomic Pathology labs. However, prognostic value of HPV status is limited to identification of high risk HPV in oropharyngeal SCCs. The purpose of this study is to investigate the ordering practices of in situ hybridization (ISH) for HPV in head and neck tissues at our national reference laboratory. All testing orders for low risk, high risk, and combined low and high risk HPV-ISH tests requested at ARUP Laboratories between January 2010 and November 2013 had their results reviewed and were grouped by anatomic location of the tested tissue. The H&E and HPV-ISH slides from a sample of the most recent 123 tests were reviewed by two pathologists. A total of 1,128 HPV-ISH tests were ordered during the study period. Testing for combined low and high risk HPV was the most commonly ordered test. The positivity rate for high risk HPV was highest in oropharyngeal tissues. 49 of 123 reviewed cases had testing requested on non-malignant tissue, 11 of which were non-neoplastic. Unnecessary HPV-ISH ordering is prevalent in head and neck tissues. Dual testing for low and high risk HPV, frequent testing outside of the oropharynx, and testing non-neoplastic tissues appear to be common practices.