Clinical Proteomics for Solid Organ Tissues.

The evaluation of biopsied solid organ tissue has long relied on visual examination using a microscope. Immunohistochemistry is critical in this process, labeling and detecting cell lineage markers and therapeutic targets. However, while the practice of immunohistochemistry has reshaped diagnostic pathology and facilitated improvements in cancer treatment, it has also been subject to pervasive challenges with respect to standardization and reproducibility. Efforts are ongoing to improve immunohistochemistry, but for some applications, the benefit of such initiatives could be impeded by its reliance on monospecific antibody-protein reagents and limited multiplexing capacity. This perspective surveys the relevant challenges facing traditional immunohistochemistry and describes how mass spectrometry, particularly liquid chromatography-tandem mass spectrometry, could help alleviate problems. In particular, targeted mass spectrometry assays could facilitate measurements of individual proteins or analyte panels, using internal standards for more robust quantification and improved interlaboratory reproducibility. Meanwhile, untargeted mass spectrometry, showcased to date clinically in the form of amyloid typing, is inherently multiplexed, facilitating the detection and crude quantification of 100s to 1000s of proteins in a single analysis. Further, data-independent acquisition has yet to be applied in clinical practice, but offers particular strengths that could appeal to clinical users. Finally, we discuss the guidance that is needed to facilitate broader utilization in clinical environments and achieve standardization.

Multi-pilot implementation experiences of patient-centered pathology reports: lessons learned for the advancement of patient-centered tools for cancer decision-making.

PURPOSE: New federal legislation in the United States grants patients expanded access to their medical records, making it critical that medical records information is understandable to patients. Provision of informational summaries significantly increase patient perceptions of patient-centered care and reduce feelings of uncertainty, yet their use for cancer pathology is limited. METHODS: Our team developed and piloted patient-centered versions of pathology reports (PCPRs) for four cancer organ sites: prostate, bladder, breast, and colorectal polyp. The objective of this analysis was to identify common barriers and facilitators to support dissemination of PCPRs in care delivery settings. We analyzed quantitative and qualitative data from pilot PCPR implementations, guided by the RE-AIM framework to explore constructs of reach, effectiveness, adoption, implementation, and maintenance. RESULTS: We present two case studies of PCPR implementation - breast cancer and colorectal polyps-that showcase diverse workflows for pathology reporting. Cross-pilot learnings emphasize the potential for PCPRs to improve patient satisfaction, knowledge, quality of shared decision-making activities, yet several barriers to dissemination exist. CONCLUSION: While there is promise in expanding patient-centered cancer communication tools, more work is needed to expand the technological capacity for PCPRs and connect PCPRs to opportunities to reduce costs, improve quality, and reduce waste in care delivery systems.

Quantification of Human Epidermal Growth Factor Receptor 2 by Immunopeptide Enrichment and Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded and Frozen Breast Cancer Tissues.

BACKGROUND: Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by immunohistochemistry and/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. We evaluated the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin-fixed paraffin-embedded (FFPE) and frozen BC biopsies. METHODS: We used immunoaffinity-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM-MS and predicate immunohistochemistry and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor subcompartments (e.g., stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity. RESULTS: HER2 immuno-MRM-MS assay linearity was ≥103, assay coefficient of variation was 7.8% (FFPE) and 5.9% (frozen) for spiked-in analyte, and 7.7% (FFPE) and 7.9% (frozen) for endogenous measurements. Immuno-MRM-MS-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to glyceraldehyde-3-phosphate dehydrogenase. HER2 was quantified above the LLOQ in all tumors. CONCLUSIONS: Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.

Characterizing TP53 mutations in ovarian carcinomas with and without concurrent BRCA1 or BRCA2 mutations.

OBJECTIVES: Mutations in the TP53 tumor suppressor gene are common in ovarian carcinoma (OC) but their impact on outcomes is controversial. We sought to define the relationship of TP53 mutations to cancer outcomes and their interactions with co-occurrent BRCA1 or BRCA2 (BRCA) mutations, comparing three different TP53 mutation classification schemes. METHODS: We performed next generation sequencing on 393 cases of OC prospectively followed for survival. TP53 mutations were classified according to three schemes termed Structural, Functional, and Hotspot. Mutation distribution was compared between cases with and without BRCA mutations. In a subset of 281 cases of high grade serous carcinoma (HGSC), overall survival was compared using Kaplan-Meier curves, logrank testing, and multivariate Cox regression analysis, both stratified and adjusted for BRCA mutation status. Multivariate logistic regression was used to analyze the effects of TP53 mutation type on platinum resistance. RESULTS: TP53 mutations were identified in 76.8% of the total cohort (n = 302/393) and 87.9% of HGSC (n = 247/281). Cases with BRCA mutations demonstrated significantly higher TP53 mutation frequency overall (n = 84/91, 92.3% vs. n = 218/302, 72.2%, p < 0.001). TP53 mutations were not associated with overall survival, even when stratified by BRCA mutation. TP53 mutations were associated with platinum sensitivity, even after adjusting for BRCA mutation status (OR 0.41, p = 0.048). The choice of TP53 mutation classification scheme was not found to alter any significant outcome. CONCLUSIONS: BRCA mutations significantly co-occur with TP53 mutations. After adjusting for BRCA mutations, TP53 mutations are associated with platinum sensitivity, and this effect is not dependent on TP53 mutation type.

Pathologic findings and clinical outcomes in women undergoing risk-reducing surgery to prevent ovarian and fallopian tube carcinoma: A large prospective single institution experience.

OBJECTIVES: Risk-reducing salpingo-oophorectomy (RRSO) is recommended for women at increased risk of ovarian, fallopian tube (FT), and peritoneal carcinoma (collectively OC). We describe rates of occult neoplasia in the largest single-institution prospective cohort of women undergoing RRSO, including those with mutations in non-BRCA homologous repair (HRR) genes. METHODS: Participants undergoing RRSO enrolled in a prospective tissue bank between 1999 and 2017. Ovaries and FTs were serially sectioned in all cases. Participants had OC susceptibility gene mutations or a family history suggesting OC risk. Analyses were completed in Stata IC 15.1. RESULTS: Of 644 women, 194 (30.1%) had mutations in BRCA1, 177 (27.5%) BRCA2, 27 (4.2%) other HRR genes, and 15 (2.3%) Lynch Syndrome-associated genes. Seventeen (2.6%) had occult neoplasms at RRSO, 15/17 (88.2%) in the FT. Of BRCA1 carriers, 14/194 (7.2%) had occult neoplasia, 8/194 (4.1%) invasive. One PALB2 and two BRCA2 carriers had intraepithelial FT neoplasms. Occult neoplasm occurred more frequently in BRCA1/2 carriers ≥45 years of age (6.5% vs 2.2%, chi square, p = .04), and 211/371 (56.9%) BRCA1/2 carriers had surgery after guideline-recommended ages. Four in 8 (50%) invasive and 2/9 (22%) intraepithelial neoplasms had positive pelvic washings. None with intraepithelial neoplasms developed recurrence or peritoneal carcinoma. CONCLUSIONS: BRCA1 carriers have the highest risk of occult neoplasia at RRSO, and the frequency increased with age. Women with BRCA1/2 mutations often have RRSO beyond recommended ages. One PALB2 carrier had FT intraepithelial neoplasia, a novel finding. Serial sectioning is critical to identifying occult neoplasia and should be performed for all risk-reducing surgeries.

Inherited mutations in fallopian tube, ovarian and primary peritoneal carcinoma: Changes in diagnoses and mutational frequency over 20 years.

OBJECTIVES: Women with fallopian tube carcinoma (FTC) are reported to have a higher frequency of inherited BRCA mutations than those with ovarian carcinoma (OC) or primary peritoneal carcinoma (PPC). We hypothesized that routine serial sectioning of fallopian tubes would increase the proportion of cases designated as FTC and change the frequency of inherited mutations between carcinoma types. METHODS: Eight hundred and sixty-seven women diagnosed from 1998 to 2018 were enrolled at diagnosis into an institutional tissue bank. Germline DNA, available from 700 women with FTC (N = 124), OC (N = 511) and PPC (N = 65), was assessed using targeted capture and massively parallel sequencing for mutations in ovarian carcinoma susceptibility genes. Cases were divided between those prior to routine serial sectioning (1998-2008) and after (2009-2019), and the frequency of FTC and inherited mutations was assessed. RESULTS: The proportion of carcinomas attributed as FTC after 2009 was 28% (128/465), significantly higher than before 2009 [5% (21/402), p < .0001, OR 6.9, 95% CI 4.3-11.2], with subsequent decreases in OC and PPC. In the sequenced group, overall inherited mutation frequency in FTC (24/124, 19%), OC (106/511, 21%, p = .42), and PPC (16/65, 25%, p = .25) were similar. Germline mutation rates in FTC were lower after 2009,16/107 cases (15%), compared to 8/17 cases (47.1%) before 2009 (p = .005, OR 0.20, 95% CI 0.06-0.64). CONCLUSIONS: The prevalence of inherited mutations is similar in FTC compared to OC or PPC when using modern pathological assignment. Complete serial sectioning of fallopian tubes has significantly increased the diagnosis of FTC, and subsequently decreased the frequency of inherited mutations within this group.

An Alternate Diagnostic Algorithm for the Diagnosis of Intraepithelial Fallopian Tube Lesions.

Intraepithelial fallopian tube neoplasia is thought to be a precursor lesion to high-grade serous carcinoma of the Müllerian adnexae, particularly in women with BRCA1 or BRCA2 mutations. This association has led to recommendations to assess fallopian tubes for intraepithelial atypia. However, the diagnostic reproducibility of a diagnosis of intraepithelial neoplasia is unclear. In this study, 2 gynecologic pathologists independently evaluated sections of fallopian tubes from a sample of women (N=198, 623 slides) undergoing salpingectomy. A total of 101 (54%) women were undergoing risk-reducing salpingo-oophorectomy. Pathologists were blinded to patient histories and prior diagnoses. Pathologists rendered one of three diagnoses for each slide: "negative for fallopian tube intraepithelial neoplasia (FTIN)," "indeterminate for FTIN," or "definite for FTIN." Cases that were considered by histology definite for FTIN or suspicious for FTIN were stained with p53 and Ki67. Pathologists agreed on the diagnosis of "definite for FTIN" 61.5% of the time. There was no agreement on any cases for the diagnosis of "indeterminate for FTIN." Fifteen "indeterminate for FTIN" and 12 "definite for FTIN" cases were stained with p53 and Ki67. Two of the "indeterminate" cases (13%) had p53-positive foci. Five of the "definite" cases had p53-positive foci. In 3 of the other 8 "definite" cases, there was obvious carcinoma present, but the carcinoma did not stain with p53, suggesting a possible null phenotype. We propose that immunostains should only be used to aid in the diagnosis of FTIN in cases with indeterminate histology. The use of p53 immunohistochemistry in cases that were considered "definite for FTIN" by histology was minimally helpful, and in fact often served to further confuse the diagnosis.

Tumor infiltrating lymphocytes and homologous recombination deficiency are independently associated with improved survival in ovarian carcinoma.

OBJECTIVE: The presence of tumor infiltrating lymphocytes (TIL) and defects in homologous recombination (HR) are each important prognostic factors in ovarian carcinoma (OC). We characterized the association between HR deficiency (HRD) and the presence of TILs in a cohort of OC patients and the relative contribution to overall survival. METHODS: Patients with carcinoma of the ovary, fallopian tube, or peritoneum were prospectively enrolled. Malignant neoplasm and serum samples were collected. Immunohistochemistry for CD3+ T cells and CD68+ tumor associated macrophages (TAMs) was performed on specimens collected at primary surgery. Damaging germline and somatic mutations in genes in the HR-mediated repair (HRR) pathway were identified using BROCA sequencing. HRD was defined as a damaging mutation in one of 12 genes in the HRR pathway or promoter hypermethylation in BRCA1 or RAD51C. RESULTS: Ninety-eight of 250 patients included in the analysis had HRD OC (39.2%). HRD OC were enriched for CD3+ TILs and CD68+ TAMs. High CD3+ TIL was present in 65.3% of HRD OC compared to 43.4% of non-HRD OC (P = 0.001). High CD68+ TAM was present in 66.3% of HRD OC compared to 50.7% of non-HRD OC (P = 0.015). Patients with HRD OC and high CD3+ TILs had the longest median overall survival compared to non-HRD OC with low CD3+ TILs (70.9 vs. 35.8 months, adjusted HR 0.38, 95% CI (0.25-0.59)). CONCLUSIONS: Patients that have both CD3+ TILs and HRD OC are afforded the greatest improvement in overall survival. This finding may have therapeutic implications for OC patients treated with emerging immunotherapies.

Evaluation of Intraoperative Assessments in the Management of Endometrial Carcinoma: A Retrospective Quality Assurance Review.

Intraoperative assessment (IA) of uteri is often used to help determine whether to perform lymphadenectomy in patients with endometrial carcinoma. We sought to perform a quality assurance review of the practice of IA at our institution. In a 1-yr period, 107 hysterectomies had an IA performed. Grade of neoplasm in preoperative endometrial biopsy, neoplasm size, depth of myometrial invasion at IA, operative management, and final histologic features were recorded. Operative reports were reviewed to assess the surgeon's interpretation of the IA and the effect on surgical management. The sensitivity and specificity for IA of deep myometrial invasion (>50% myometrial thickness) compared with final histology was 76.9% and 91.1%. The positive predictive value was 71.4%, negative predictive value 93.2% and accuracy 88%. Neoplasm size was provided in 47% of cases. In 10% of patients lymphadenectomy was performed despite low-risk features. IA results were included in the operative report in 87% of cases. There were differences in 8.4% of cases between the IA diagnosis and the surgeon's operative report. IA of deep myometrial invasion is reliable at our institution. Several metrics for quality improvement have been identified as a result of this retrospective review. These include but are not limited to more reliable reporting of neoplasm size, documentation, and communication with gynecologic oncologists.

Error Reduction and Diagnostic Concordance in Breast Pathology.

Errors in anatomic pathology can result in patients receiving inappropriate treatment and poor patient outcomes. Policies and procedures are necessary to decrease error and improve diagnostic concordance. Breast pathology may be more prone to diagnostic errors than other surgical pathology subspecialties due to inherit borderline diagnostic categories such as atypical ductal hyperplasia and low-grade ductal carcinoma in situ. Mandatory secondary review of internal and outside referral cases before treatment is effective in reducing diagnostic errors and improving concordance. Assessment of error through amendment/addendum tracking, implementing an incident reporting system, and multidisciplinary tumor boards can establish procedures to prevent future error.

Radiologic and Pathologic Correlation for Lactating Adenomas.

Lactating adenomas are benign breast lesions that occur in pregnant, lactating, and postpartum women. These lesions have no associated malignant potential; their origin is disputed with no consensus on whether they represent hyperplastic or neoplastic processes. On ultrasound, lactating adenomas are classically described as solid, circumscribed, parallel masses with iso/hypoechoic internal echotexture and posterior enhancement. Histologically, lactating adenomas appear as circumscribed nodules of tightly packed lobular acini with extensive lactational change during pregnancy or the postpartum period. Masses in pregnant and lactating women with probably benign imaging characteristics-oval, circumscribed, parallel, iso/hypoechoic-can be managed with short interval follow-up (BI-RADS 3) rather than biopsy. However, lactating adenomas can also demonstrate characteristics that overlap with pregnancy-associated breast cancer, such as margins that are not circumscribed, prompting biopsy to exclude pregnancy-associated carcinoma. Breast imaging radiologists must be aware of the variable appearances of lactating adenomas to appropriately manage pregnant and lactating women presenting with palpable lumps.

Idiopathic Granulomatous Mastitis of the Breast: Radiologic-Pathologic Correlation.

Granulomatous inflammation is an uncommon inflammatory condition of the breast that includes both infectious (bacterial, fungal, parasitic) and noninfectious (autoimmune, sarcoidosis, idiopathic granulomatous mastitis [IGM], reaction to foreign materials) etiologies. IGM is the most common subset of granulomatous inflammation where no underlying etiology is established. Infectious causes of granulomatous inflammation should be excluded, as these have established treatments that can significantly improve patient outcomes. IGM should be considered in the differential when mastitis is refractory to antibiotics. Patients usually present with an erythematous, tender, palpable unilateral breast mass. The most common mammographic presentation is a focal or global asymmetry. The imaging appearance mimics breast cancer, therefore diagnosis usually requires tissue sampling with histopathologic analysis and cultures to exclude infection. When patients are diagnosed with IGM, this poses a clinical dilemma as there are a variety of treatment options available, including oral and intralesional steroids. The time course of the disease is often prolonged by multiple recurrences, and specific treatment remains an area of ongoing research. The purpose of this article is to review the range of clinical features, imaging manifestations, associated histopathology, and management of IGM.

Accuracy of Preoperative Breast MRI Versus Conventional Imaging in Measuring Pathologic Extent of Invasive Lobular Carcinoma.

OBJECTIVE: To determine whether invasive lobular carcinoma (ILC) extent is more accurately depicted with preoperative MRI (pMRI) than conventional imaging (mammography and/or ultrasound). METHODS: After IRB approval, we retrospectively identified women with pMRIs (February 2005 to January 2014) to evaluate pure ILC excluding those with ipsilateral pMRI BI-RADS 4 or 5 findings or who had neoadjuvant chemotherapy. Agreement between imaging and pathology sizes was summarized using Bland-Altman plots, absolute and percent differences, and the intraclass correlation coefficient (ICC). Rates of underestimation and overestimation were evaluated and their associations with clinical features were explored. RESULTS: Among the 56 women included, pMRI demonstrated better agreement with pathology than conventional imaging by mean absolute difference (1.6 mm versus -7.8 mm, P < 0.001), percent difference (10.3% versus -16.4%, P < 0.001), and ICC (0.88 versus 0.61, P = 0.019). Conventional imaging more frequently underestimated ILC span than pMRI using a 5 mm difference threshold (24/56 (43%) versus 10/56 (18%), P < 0.001), a 25% threshold (19/53 (36%) versus 10/53 (19%), P = 0.035), and T category change (17/56 (30%) versus 7/56 (13%), P = 0.006). Imaging-pathology size concordance was greater for MRI-described solitary masses than other lesions for both MRI and conventional imaging (P < 0.05). Variability of conventional imaging was lower for patients ≥ the median age of 62 years than for younger patients (SD: 12 mm versus 22 mm, P = 0.012). CONCLUSION: MRI depicts pure ILC more accurately than conventional imaging and may have particular value for younger women.

The Long and Winding Road for Breast Cancer Biomarkers to Reach Clinical Utility.

Tumor data from the ABCSG 5 trial of chemotherapy versus endocrine therapy for premenopausal ER+ breast cancer supports molecular subtyping by Ki-67 IHC as a prognostic marker. But while this tissue was handled uniformly, Ki-67 testing overall is unstandardized, complicating clinical utility. Increasing potential biomarkers herald more challenges in biomarker validation.See related article by Bago-Horvath et al., p. 5682.

Risk for Upgrade to Malignancy After Breast Core Needle Biopsy Diagnosis of Lobular Neoplasia: A Systematic Review and Meta-Analysis.

PURPOSE: Lobular neoplasia (LN) detected on breast core needle biopsy is frequently managed with surgical excision because of concern for undersampled malignancy. The authors performed a systematic review and meta-analysis to estimate the risk for upgrade to malignancy in the setting of imaging-concordant classic LN diagnosed on core biopsy. METHODS: PubMed and Embase were searched for original articles published from 1998 to 2020 that reported rates of upgrade to malignancy for classic LN, including atypical lobular hyperplasia (ALH) and classic lobular carcinoma in situ (LCIS). Two reviewers extracted study data and assessed the following quality criteria: exclusion of variant LCIS, exclusion of imaging-discordant lesions, and outcome reporting for ≥70% of lesions. For studies meeting all criteria, pooled risks for upgrade to any malignancy (invasive carcinoma or ductal carcinoma in situ) and invasive malignancy for all LN, ALH, and LCIS were estimated using random-effects models. RESULTS: For 65 full-text articles included in the review, the risk for upgrade to any malignancy ranged from 0% to 45%. Among the 16 studies that met all quality criteria for the meta-analysis, pooled risks for upgrade to any malignancy were 3.1% (95% confidence interval [CI], 1.8%-5.2%) for all LN, 2.5% (95% CI, 1.6%-3.9%) for ALH, and 5.8% (95% CI, 2.9%-11.3%) for LCIS. Risks for upgrade to invasive malignancy were 1.3% (95% CI, 0.7%-2.4%) for all LN, 0.4% (95% CI, 0.0%-4.2%) for ALH, and 3.5% (95% CI, 2.0%-5.9%) for LCIS. CONCLUSIONS: The risk for upgrade to malignancy for LN found on breast biopsy is low. Imaging surveillance can likely be offered as an alternative to surgical management for LN, particularly for ALH.

Improving the impact of clinical documentation through patient-driven co-design: experiences with cancer pathology reports.

OBJECTIVE: With the unprecedented rise of patient access to clinical documentation through electronic health records, there is a need for health systems to understand best practices for redesigning clinical documentation to support patient needs. This study used an experience-based co-design approach to inform the redesign of cancer pathology reports to improve their patient-centeredness and impact on patient engagement. MATERIALS AND METHODS: Multiple methods for data collection and stakeholder engagement were used, including Delphi prioritisation with breast and colorectal cancer experts (n=78) and focus groups with patients with cancer (n=23) in the Seattle area. Iterative rounds of consensus generation and reflection were used to elicit themes and design recommendations for the development of patient-centred pathology reports on cancer care. RESULTS: Although each cancer type had nuanced elements to consider, common design requirements emerged around two key themes: (1) clinical documentation language should be framed in a way that informs and engages patients, and (2) clinical documentation format should be leveraged to enhance readability and information flow. Study activities illuminated detailed recommendations to improve the patient-centeredness of pathology reports based on patients' and clinicians' lived experience. DISCUSSION: The design requirements that emerged from this study provide a framework that can guide the rapid development of patient-centred pathology reports for all cancer types. Even further, health systems can replicate these methods to guide experience-based co-design of clinical documentation for contexts beyond cancer care. CONCLUSION: This work offers practice-based learnings that can more effectively guide health systems in their clinical documentation redesign efforts.

Ductal Carcinoma in Situ: Current Concepts in Biology, Imaging, and Treatment.

Ductal carcinoma in situ (DCIS) of the breast is a group of heterogeneous epithelial proliferations confined to the milk ducts that nearly always present in asymptomatic women on breast cancer screening. A stage 0, preinvasive breast cancer, increased detection of DCIS was initially hailed as a means to prevent invasive breast cancer through surgical treatment with adjuvant radiation and/or endocrine therapies. However, controversy in the medical community has emerged in the past two decades that a fraction of DCIS represents overdiagnosis, leading to unnecessary treatments and resulting morbidity. The imaging hallmarks of DCIS include linearly or segmentally distributed calcifications on mammography or nonmass enhancement on breast MRI. Imaging features have been shown to reflect the biological heterogeneity of DCIS lesions, with recent studies indicating MRI may identify a greater fraction of higher-grade lesions than mammography does. There is strong interest in the surgical, imaging, and oncology communities to better align DCIS management with biology, which has resulted in trials of active surveillance and therapy that is less aggressive. However, risk stratification of DCIS remains imperfect, which has limited the development of precision therapy approaches matched to DCIS aggressiveness. Accordingly, there are opportunities for breast imaging radiologists to assist the oncology community by leveraging advanced imaging techniques to identify appropriate patients for the less aggressive DCIS treatments.

Unexpected PAX8 Immunoreactivity in Metastatic High-grade Breast Cancer.

Immunohistochemistry (IHC) is often critical for distinction between metastatic carcinomas of Mullerian organ and breast origin. Paired box family protein 8 (PAX8) has been described as a transcription factor highly specific to neoplasms derived from Mullerian organs, thyroid, and kidney. PAX8 IHC with polyclonal and monoclonal antibody reagents was performed on 27 primary and 22 metastatic breast carcinomas. Eight of 27 primary breast carcinomas (30%) were positive for PAX8 with the monoclonal antibody reagent only; 0 of 22 were polyclonal anti-PAX8 immunoreactive. Substantial numbers of metastases had positive immunoreactivity for polyclonal anti-PAX8 (23%). Each of these metastases and additional cases (45% total) also had positive immunoreactivity for monoclonal anti-PAX8, including 5 of 7 brain metastases. IHC with monoclonal anti-PAX8 was positive on 6 of 7 primary breast carcinomas corresponding to PAX8-positive metastases. Together, these results indicate a significant fraction of breast carcinoma metastases and corresponding primary neoplasms have immunoreactivity for PAX8, and positivity rates depend on the antibody used. Diagnoses of metastatic breast carcinoma were achieved with the aid of clinical history and additional IHC in cases of PAX8 immunoreactivity. Contextual interpretation is imperative for PAX8 IHC, particularly when the differential diagnosis includes metastatic breast carcinoma with limited diagnostic material available.