Ashley Cimino-Mathews

PD-L1 (B7-H1) expression and the immune tumor microenvironment in primary and metastatic breast carcinomas.

Programmed death ligand 1 (PD-L1) expression by tumor-infiltrating lymphocytes (TILs) and tumor cells in breast cancer has been reported, but the relationships between PD-L1 expression by TIL, carcinoma cells, and other immunologic features of the breast tumor microenvironment remain unclear. We therefore evaluated the interrelationships between tumor cell surface and TIL PD-L1 expression, lymphocyte subpopulations, and patterns of immune cell infiltration in cohorts of treatment-naive, primary breast cancers (PBCs) (n = 45) and matched PBC and metastatic breast cancers (MBC) (n = 26). Seventy-eight percent of untreated PBCs contained PD-L1(+) TILs, but only 21% had PD-L1(+) carcinoma cells. Carcinoma PD-L1 expression localized to the tumor invasive front and was associated with high tumor grade (P = .04). Eighty-nine percent of PD-L1(+) carcinomas contained brisk TIL infiltrates, compared to only 24% of PD-L1(-) carcinomas; this included CD3(+) (P = .02), CD4(+) (P = .04), CD8(+) (P = .002), and FoxP3(+) T cells (P = .02). PD-L1(+) PBCs were more likely to contain PD-L1(+) TIL than PD-L1(-) PBCs (P = .04). Peripheral lymphoid aggregates were present in 100% of PD-L1(+) compared to 41% of PD-L1(-) PBC (P < .001). No patient with PD-L1(+) PBC developed distant recurrence, compared to 15% of patients with PD-L1(-) PBC. For the matched PBC and MBC cohort, 2 patients (8%) had PD-L1(+) tumors, with 1 case concordant and 1 case discordant for carcinoma PD-L1 expression in the PBC and MBC. Our data support PD-L1 expression by tumor cells as a biomarker of active breast tumor immunity and programmed death 1 blockade as a therapeutic strategy for breast cancer.

Neural crest transcription factor Sox10 is preferentially expressed in triple-negative and metaplastic breast carcinomas

The transcription factor Sox10 mediates the differentiation of neural crest-derived cells, and Sox10 labeling by immunohistochemistry (IHC) is used clinically primarily to support the diagnosis of melanoma. Sox10 expression by IHC has been previously documented in benign breast myoepithelial cells but not in breast carcinomas. Here, we report the first systematic study of Sox10 expression in invasive ductal carcinomas subclassified by IHC-defined molecular subtype (100 cases), as well as in 24 cases of ductal carcinoma in situ and 44 mammary fibroepithelial neoplasms. Tissue microarrays containing 168 primary breast tumors were subjected to IHC for Sox10. The extent of nuclear Sox10 labeling was scored by percentage labeling as follows: 0 (0%), 1+ (1%-25%), 2+ (25%-50%), 3+ (50%-75%), and 4+ (>75%). Overall, 40 (40%) of 100 invasive breast carcinomas demonstrated Sox10 immunoreactivity, which was seen primarily in the basal-like, unclassified triple-negative, and metaplastic carcinomas. Sox10 labeling was seen in 66% (38/58) of the basal-like, unclassified triple-negative, and metaplastic carcinomas as compared with 5% (2/42) of the luminal A, luminal B, and Her-2 carcinomas (P < .00001). Sox10 labeling was seen in 1 (4%) of 24 cases of ductal carcinoma in situ, which was negative for estrogen receptor/progesterone receptor. No labeling was seen in the stromal component of phyllodes tumors or fibroadenomas. These findings show that breast carcinoma must be considered in the differential diagnosis of melanoma for an S100-positive, Sox10-positive metastatic malignant neoplasm. Sox10 expression in the basal-like, unclassified triple-negative, and metaplastic carcinomas types supports the concept that these neoplasms show myoepithelial differentiation.

The immune microenvironment of breast ductal carcinoma in situ

The host immune response has a key role in breast cancer progression and response to therapy. However, relative to primary invasive breast cancers, the immune milieu of breast ductal carcinoma in situ (DCIS) is less understood. Here, we profile tumor infiltrating lymphocytes and expression of the immune checkpoint ligand programmed death ligand 1 (PD-L1) in 27 cases of DCIS with known estrogen receptor (ER), progesterone receptor, and human epidermal growth factor 2 (HER-2) expression using tissue microarrays. Twenty-four cases were pure DCIS and three had associated invasive ductal carcinoma. Tumors were stained by immunohistochemistry for PD-L1, as well as the lymphocyte markers CD3, CD4, CD8, FoxP3, and CD20. The expression of PD-L1 by DCIS carcinoma cells and tumor infiltrating lymphocytes was determined, and the average tumor infiltrating lymphocytes per high power field were manually scored. None of the DCIS cells expressed PD-L1, but 81% of DCIS lesions contained PD-L1+ tumor infiltrating lymphocytes. DCIS with moderate-diffuse tumor infiltrating lymphocytes was more likely to have PD-L1+ tumor infiltrating lymphocytes (P=0.004). Tumor infiltrating lymphocytes with high levels of PD-L1 expression (>50% cells) were seen only in triple-negative DCIS (P=0.0008), and PD-L1−tumor infiltrating lymphocytes were seen only in ER+/HER-2−DCIS (P=0.12). The presence of PD-L1+ tumor infiltrating lymphocytes was associated with a younger mean patient age (P=0.01). Further characterization of the DCIS immune microenvironment may identify useful targets for immune-based therapy and breast cancer prevention.

Metastatic triple-negative breast cancers at first relapse have fewer tumor-infiltrating lymphocytes than their matched primary breast tumors: a pilot study

Tumor-infiltrating lymphocytes (TILs) convey clinically relevant information for primary breast cancers (PBCs). However, limited data characterizing the immunobiology of metastatic breast cancers (MBCs) are available. Here, we examine TILs in surgically resected MBCs relative to their matched PBCs. Tissue microarrays of PBCs and MBCs were labeled for CD3 (total T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), FoxP3 (regulatory T cells), and CD20 (B cells) to characterize TILs. Expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor-2 (HER-2) classified the tumors as luminal (ER(+)/PR(+)/HER-2(-)), triple negative (ER(-)/PR(-)/HER-2(-)), or HER-2(+) (ER(-)/PR(-)/HER-2(+)). These analyses reveal 5 novel findings. First, MBCs overall contained fewer TILs (mean, 35.1 CD3(+) TILs/high-power field [hpf]) than their matched PBCs (mean, 23.6 CD3(+) TILS/hpf), with fewer CD20(+) cells than CD3(+) cells in PBC and MBC (P = .0247). Second, the number of CD3(+), CD8(+), CD4(+), and FoxP3 TILs was decreased in triple-negative MBCs relative to matched PBCs, whereas only CD8(+) TILs were decreased in luminal MBCs relative to matched PBCs. Third, triple-negative MBCs contain fewer TILS (mean, 16 CD3(+) TILs/hpf) than luminal MBCs (mean, 21.7 CD3(+) TILs/hpf). Fourth, brain metastases contained fewer TILs relative to MBC from other sites. Finally, in this series, a CD8(+)/FoxP3(+) T-cell ratio of 3 or greater in PBCs was associated with improved overall survival from diagnosis, whereas a CD8(+)/FoxP3(+) T-cell ratio less than 3 in MBCs at first relapse was associated with improved overall survival. These findings suggest that evaluating the immunologic microenvironment of both PBCs and MBCs may yield important clinical information to guide breast cancer prognosis and therapy.

Androgen receptor expression is usually maintained in initial surgically resected breast cancer metastases but is often lost in end-stage metastases found at autopsy.

Androgen receptor (AR) is expressed in approximately 70% of primary breast carcinomas (PBCs) and is a promising therapeutic target for metastatic breast carcinoma (MBC). Here, we examine AR expression in a population of initial surgically resected metastases and a separate cohort of end-stage metastases harvested at autopsy compared with their matched PBCs. Tissue microarrays of matched PBC and MBC were labeled by immunohistochemistry for AR, estrogen receptor (ER), progesterone receptor (PR), and Her2 and classified into the following previously described categories: luminal (ER/PR+/Her2-), triple negative (ER/PR/Her2-), Her2 (ER/PR-/Her2+), and luminal loss (ER/PR loss from primary to metastasis). In the cohort of surgically resected metastases (n = 16), AR was expressed in 12 of 16 PBC and maintained in 11 of 12 corresponding MBCs. Of these, 36% showed stronger AR labeling in the metastases and none showed a decrease. In the cohort of metastases harvested at autopsy (n = 16), AR was expressed in 11 of 16 primary carcinomas and maintained in only 5 of 11 corresponding metastases. Of these, none showed increased AR and 80% showed decreased AR labeling. AR expression is overwhelmingly concordant between matched PBC and MBC at initial presentation. These findings validate AR as a therapeutic target in MBC and suggest that AR may need to be reevaluated in metastases even if the primary is negative. However, similar to ER/PR, AR expression is often decreased with a trend toward complete loss in end-stage metastases, suggesting a shift of AR expression between initial and end-stage metastases. This suggests an opportunity for targeted antiandrogen therapy at an earlier stage of disease progression.

MYC gene amplification is often acquired in lethal distant breast cancer metastases of unamplified primary tumors.

In breast cancer, amplification of MYC is consistently observed in aggressive forms of disease and correlates with poor prognosis and distant metastases. However, to date, a systematic analysis of MYC amplification in metastatic breast cancers has not been reported. Specifically, whether the MYC amplification status may change in metastases in comparison to the corresponding primary breast tumor, and potential variability among different metastases within the same patient have also not been assessed. We generated single patient tissue microarrays consisting of both primary breast carcinomas and multiple matched systemic metastases from 15 patients through our previously described rapid autopsy program. In total, the 15 tissue microarrays contained 145 primary tumor spots and 778 spots derived from 180 different metastases. In addition, two separate tissue microarrays were constructed composed of 10 matched primary breast cancers and corresponding solitary metastases sampled not at autopsy but rather in routine surgical resections. These two tissue microarrays totaled 50 primary tumor spots and 86 metastatic tumor spots. For each case, hormone receptor status, HER2/neu, EGFR and CK5/6 expression were assessed, and the cases were characterized as luminal, basal-like or HER2 based on published criteria. Both fluorescence in situ hybridization and immunohistochemistry for MYC was performed on all cases. Of the 25 cases, 24 were evaluable. While 4 of 24 primary tumors (16%) demonstrated MYC amplification, an additional 6 (25% of total evaluable cases) acquired MYC amplification in their systemic metastases. Of note, there was remarkably little heterogeneity in MYC copy number among different metastases from the same patient. MYC immunoreactivity was increased in metastases relative to matched primaries in the surgical cohort, although there was no perfect correlation with MYC amplification. In conclusion, amplification of MYC is a frequent event in breast cancer, but occurs more frequently as a diffuse, acquired event in metastatic disease than in the corresponding primary. These observations underscore the importance of MYC in breast cancer progression/metastasis, as well as its relevance as a potential therapeutic target in otherwise incurable metastatic disease.

The role of GATA3 in breast carcinomas: a review

GATA3 is a zinc-binding transcription factor that regulates the differentiation of many human tissue types, including the mammary gland. In surgical pathology, immunohistochemistry for GATA3 is largely used to support urothelial or breast origin in a carcinoma of unknown origin. GATA3 is sensitive but not entirely specific in this setting. Although GATA3 labeling is highest in estrogen receptor-positive carcinomas, it also labels estrogen receptor-negative carcinomas and thus has particular diagnostic utility in the setting of triple-negative breast carcinomas, which are typically negative for other mammary-specific markers.

Diagnostic use of PAX8, CAIX, TTF-1, and TGB in metastatic renal cell carcinoma of the thyroid.

Clear-cell renal cell carcinoma (ccRCC) may present with metastatic lesions in patients with a concurrent undiagnosed primary or a remote history of ccRCC. The thyroid is not uncommonly involved by metastatic ccRCC, in which a metastasis could be misinterpreted as a clear-cell change in adenomatoid nodules, follicular adenomas, or parathyroid glands. PAX8 is a transcription factor expressed by thyroid and renal-lineage cells. No previous study has evaluated the diagnostic use of PAX8 and ccRCC marker carbonic anhydrase IX (CAIX) in this setting. Cases of metastatic ccRCC in the thyroid (n=12), parathyroid glands and adenomas with clear-cell change (n=6), papillary thyroid carcinoma (n=6), thyroid follicular adenomas (n=5), and adenomatoid nodules with clear-cell change (n=5) were studied. Cases were assessed by standard immunohistochemistry for thyroid transcription factor-1 (TTF-1), thyroglobulin (TGB), PAX8, and CAIX. The extent and intensity of nuclear or cytoplasmic immunoexpression were assessed, with any labeling considered as a positive result. All metastatic ccRCCs were positive for PAX8 (moderate-to-strong, patchy-to-diffuse) and CAIX (strong, diffuse), and were negative for TTF-1 and TGB. All primary thyroid lesions labeled strongly and diffusely for TTF-1, TGB, and PAX8, and were negative for CAIX. Parathyroid tissues were negative for TTF-1, TGB, PAX8, and CAIX. An immunoprofile of “TTF1(−)/TGB(−)/CAIX(+)” was 100% sensitive and specific for metastatic ccRCC of the thyroid. The reverse profile “TTF1(+)/TGB(+)/CAIX(−)” supported a primary thyroid lesion. PAX8 was not useful in distinguishing metastatic ccRCC from thyroid lesions.

A subset of malignant phyllodes tumors express p63 and p40: A diagnostic pitfall in breast core needle biopsies

Breast phyllodes tumors are rare fibroepithelial neoplasms of variable grade, and one key differential of malignant phyllodes on core biopsy is sarcomatoid carcinoma. p63 is reported to be sensitive and specific for sarcomatoid carcinoma, with rare expression in phyllodes in limited series. The p63 deltaNp63 isoform, p40, is postulated to be more specific for squamous differentiation but has not previously been evaluated in breast phyllodes or sarcomatoid carcinoma. Tissue microarrays containing 34 unambiguous phyllodes tumors (10 benign, 10 borderline, 14 malignant), 13 sarcomatoid carcinomas, and 10 fibroadenomas were labeled by immunohistochemistry for p63, p40, CD34, and cytokeratins AE1/AE3, 34betaE12, and CK8/18. No borderline phyllodes tumor, benign phyllodes tumor, or fibroadenoma labeled with p63, p40, or cytokeratin. However, p63 labeled 57% malignant phyllodes tumors and 62% sarcomatoid carcinomas, and p40 labeled 29% malignant phyllodes (focal) and 46% sarcomatoid carcinomas. Among established markers, cytokeratins labeled 21% malignant phyllodes tumors (focal) and 100% sarcomatoid carcinomas. CD34 labeled 57% malignant phyllodes tumors and no sarcomatoid carcinomas. Focal p63, p40, and cytokeratin labeling can be seen in malignant phyllodes tumors but not in lower-grade fibroepithelial lesions, and immunoreactivity with these markers alone is not diagnostic of sarcomatoid carcinoma on core needle biopsy. In the differential diagnosis of malignant phyllodes, p40 is a more specific but less sensitive marker of sarcomatoid carcinoma than p63. These results are consistent with the sarcoma literature in which p63 labeling has been increasingly reported and suggest caution in classifying malignant spindle cell tumors of the breast on core biopsy.

GATA-3 immunohistochemistry in the differential diagnosis of adenocarcinoma of the urinary bladder.

GATA-3 is a newly described marker that labels urothelial and breast carcinoma. However, no prior study has evaluated the expression of GATA-3 in primary bladder adenocarcinoma. Tissue microarrays (TMAs) containing 46 primary bladder adenocarcinomas were constructed. They contained 19 signet ring cell (SRC) and 27 conventional adenocarcinomas. Three additional cases of SRC using routine sections were included resulting in a total of 22 SRCs. In addition, TMAs containing 32 primary gastric signet ring adenocarcinomas and 36 primary lobular breast carcinomas were evaluated. The TMAs were subjected to immunohistochemical analysis for GATA-3, with nuclear labeling scored by intensity and percentage labeling. Breast and urothelial TMAs were also labeled for estrogen receptor, progesterone receptor, and gross cystic duct fluid protein. Diffuse nuclear GATA-3 labeling was seen in 9/22 (41.0%) SRCs and in 2/27 (7.0%) conventional adenocarcinomas (P=0.01). Extracellular mucin production was seen in 12 SRCs. One of 12 (8.0%) SRCs with extracellular mucin was GATA-3 positive, and 8/10 SRCs without extracellular mucin was GATA-3 positive (P=0.005). No nuclear GATA-3 labeling was seen in any gastric signet ring carcinoma. Diffuse, moderate to strong nuclear GATA-3 labeling was seen in 36/36 (100%) primary lobular breast carcinomas. Nuclear GATA-3 labeling is a useful marker for primary adenocarcinomas of the urinary bladder with signet ring features and can be helpful in distinguishing primary signet ring carcinomas of the urinary bladder from gastric signet ring carcinomas. GATA-3 is rarely positive in bladder adenocarcinomas that lack signet ring features and in SRCs displaying extracellular mucin production.