Metaplastic breast cancers frequently express immune checkpoint markers FOXP3 and PD-L1.

BACKGROUND: Metaplastic breast carcinoma encompasses a heterogeneous group of tumours with differentiation into squamous and/or spindle, chondroid, osseous or rhabdoid mesenchymal-looking elements. Emerging immunotherapies targeting Programmed Death Ligand 1 (PD-L1) and immune-suppressing T cells (Tregs) may benefit metaplastic breast cancer patients, which are typically chemo-resistant and do not express hormone therapy targets. METHODS: We evaluated the immunohistochemical expression of PD-L1 and FOXP3, and the extent of tumour infiltrating lymphocytes (TILs) in a large cohort of metaplastic breast cancers, with survival data. RESULTS: Metaplastic breast cancers were significantly enriched for PD-L1 positive tumour cells, compared to triple-negative ductal breast cancers (P < 0.0001), while there was no significant difference in PD-L1 positive TILs. Metaplastic breast cancers were also significantly enriched for TILs expressing FOXP3, with FOXP3 positive intra-tumoural TILs (iTILs) associated with an adverse prognostic outcome (P = 0.0226). Multivariate analysis identified FOXP3 iTILs expression status as an important independent prognostic factor for patient survival. CONCLUSIONS: Our findings indicate the clinical significance and prognostic value of FOXP3, PD-1/PD-L1 checkpoint and TILs in metaplastic breast cancer and confirm that a subset of metaplastics may benefit from immune-based therapies.

Phenotypic and molecular dissection of metaplastic breast cancer and the prognostic implications.

Metaplastic breast carcinoma (MBC) is relatively rare but accounts for a significant proportion of global breast cancer mortality. This group is extremely heterogeneous and by definition exhibits metaplastic change to squamous and/or mesenchymal elements, including spindle, squamous, chondroid, osseous, and rhabdomyoid features. Clinically, patients are more likely to present with large primary tumours (higher stage), distant metastases, and overall, have shorter 5-year survival compared to invasive carcinomas of no special type. The current World Health Organisation (WHO) diagnostic classification for this cancer type is based purely on morphology - the biological basis and clinical relevance of its seven sub-categories are currently unclear. By establishing the Asia-Pacific MBC (AP-MBC) Consortium, we amassed a large series of MBCs (n = 347) and analysed the mutation profile of a subset, expression of 14 breast cancer biomarkers, and clinicopathological correlates, contextualising our findings within the WHO guidelines. The most significant indicators of poor prognosis were large tumour size (T3; p = 0.004), loss of cytokeratin expression (lack of staining with pan-cytokeratin AE1/3 antibody; p = 0.007), EGFR overexpression (p = 0.01), and for 'mixed' MBC, the presence of more than three distinct morphological entities (p = 0.007). Conversely, fewer morphological components and EGFR negativity were favourable indicators. Exome sequencing of 30 cases confirmed enrichment of TP53 and PTEN mutations, and intriguingly, concurrent mutations of TP53, PTEN, and PIK3CA. Mutations in neurofibromatosis-1 (NF1) were also overrepresented [16.7% MBCs compared to ∼5% of breast cancers overall; enrichment p = 0.028; mutation significance p = 0.006 (OncodriveFM)], consistent with published case reports implicating germline NF1 mutations in MBC risk. Taken together, we propose a practically minor but clinically significant modification to the guidelines: all WHO_1 mixed-type tumours should have the number of morphologies present recorded, as a mechanism for refining prognosis, and that EGFR and pan-cytokeratin expression are important prognostic markers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Claudin-4 immunohistochemistry is a useful pan-carcinoma marker for serous effusion specimens.

OBJECTIVE: To evaluate the utility of claudin-4 as a pan-carcinoma marker in cell-blocks of effusion specimens and compare results with Ber-Ep4 staining. METHODS: Effusion cell-blocks (n = 284) were stained for claudin-4 and results compared with Ber-Ep4. Cases included 172 metastatic malignancies (137 adenocarcinomas, 20 small cell lung tumours, eight metastatic melanoma, four squamous cell carcinoma, three urothelial cell carcinoma), 49 benign reactive cases and 63 mesotheliomas. RESULTS: All 49 benign effusions were negative. Only 1/63 (1.6%) mesotheliomas was positive for claudin-4. Claudin-4 staining was positive in 131/137 (95.6%) adenocarcinoma cases. Cases negative for claudin-4 included single cases of metastases from breast, colon, stomach, prostate, kidney and ovary. Claudin-4 outperformed Ber-Ep4. Sensitivity (95.6% vs 85.4%), specificity (99.1% vs 86.6%), negative predictive value (94.9% vs 82.9%) and positive predictive value (99.2% vs 88.6%) were all higher for claudin-4 compared with Ber-Ep4, respectively. Only two cases were claudin-4-/Ber-Ep4+. Significantly (P < .0064) more cases of metastatic adenocarcinoma stained positive for claudin-4 (131/137; 95.6%) than Ber-Ep4 (117/137; 86.2%). Claudin-4 staining was present in 15/20 (75%) of neuroendocrine carcinomas, 3/4 (75%) squamous cell carcinoma and 3/3 (100%) urothelial cell carcinoma. All eight cases of melanoma were negative for both claudin-4 and Ber-Ep4. CONCLUSIONS: Claudin-4 staining is a useful addition to IHC panels for effusions specimens with superior performance to Ber-Ep4.

Investigation of lymphoid lesions of the head and neck using combined fine needle aspiration cytology and flow cytometry: Accuracy and pitfalls.

OBJECTIVE: We reviewed the diagnostic utility of combined fine needle aspiration cytology (FNAC) and flow cytometry (FC) in the diagnosis of lymphoid lesions of the head and neck. METHOD: In total, 1402 patients with combined FNAC-FC reports were correlated with follow-up information. Rapid on-site evaluation (ROSE) of cytological specimens was performed in 52% of cases. RESULTS: In total, 211 lymphoid malignancies were identified, including 198 non-Hodgkin lymphoma (NHL) and 13 Hodgkin lymphoma (HL). Accuracy measures for NHL were: sensitivity 95.5%; specificity 99.9%; PPV 99.5%; NPV 99.2%; accuracy 99.3%. Only seven of 13 cases of HL were detected by FNAC-FC. False negative cases included HL (six cases), diffuse large B-cell lymphoma (four), T-cell lymphoma (two), follicular lymphoma (one), marginal zone cell lymphoma (one) and B-cell NHL, not otherwise specified (one). Two false positive results were identified: one immunoblastic hyperplasia reported as suspicious for HL and one case reported as suggestive of NHL that was found to be reactive hyperplasia. Cases collected with ROSE had a significantly lower rate (P < 0.0001) of insufficient cells for FC analysis (7.0%) than cases where ROSE was not performed (16.4%). Sensitivity (P < 0.0001) and NPV (P = 0.0023) were significantly higher for ROSE-collected specimens. None of the false-negative NHL cases had ROSE performed. CONCLUSIONS: FNAC-FC is a highly sensitive and specific test for NHL. Diagnostic errors mostly involved HL, large cell lymphomas and T-cell lymphomas. ROSE results in a significantly higher adequacy rate for FC and higher sensitivity for NHL.

An epithelial to mesenchymal transition programme does not usually drive the phenotype of invasive lobular carcinomas.

Epithelial to mesenchymal transition (EMT) is a cellular phenotype switching phenomenon which occurs during normal development and is proposed to promote tumour cell invasive capabilities during tumour progression. Invasive lobular carcinoma (ILC) is a histological special type of breast cancer with a peculiar aetiology - the tumour cells display an invasive growth pattern, with detached, single cells or single files of cells, and a canonical feature is the loss of E-cadherin expression. These characteristics are indicative of an EMT or at the very least that they represent some plasticity between phenotypes. While some gene expression profiling data support this view, the tumour cells remain epithelial and limited immunohistochemistry data suggest that EMT markers may not feature prominently in ILC. We assessed the expression of a panel of EMT markers (fibronectin, vimentin, N-cadherin, smooth muscle actin, osteonectin, Snail, Twist) in 148 ILCs and performed a meta-analysis of publically available molecular data from 154 ILCs. Three out of 148 (2%) ILCs demonstrated an early and coordinated alteration of multiple EMT markers (down-regulation of E-cadherin, nuclear TWIST, and up-regulation of vimentin, osteonectin, and smooth muscle actin). However, the data overall do not support a role for EMT in defining the phenotypic peculiarities of the majority of ILCs. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Expression profiling identifies genes involved in neoplastic transformation of serous ovarian cancer.

BACKGROUND: The malignant potential of serous ovarian tumors, the most common ovarian tumor subtype, varies from benign to low malignant potential (LMP) tumors to frankly invasive cancers. Given the uncertainty about the relationship between these different forms, we compared their patterns of gene expression. METHODS: Expression profiling was carried out on samples of 7 benign, 7 LMP and 28 invasive (moderate and poorly differentiated) serous tumors and four whole normal ovaries using oligonucleotide microarrays representing over 21,000 genes. RESULTS: We identified 311 transcripts that distinguished invasive from benign tumors, and 20 transcripts that were significantly differentially expressed between invasive and LMP tumors at p < 0.01 (with multiple testing correction). Five genes that were differentially expressed between invasive and either benign or normal tissues were validated by real time PCR in an independent panel of 46 serous tumors (4 benign, 7 LMP, 35 invasive). Overexpression of SLPI and WNT7A and down-regulation of C6orf31, PDGFRA and GLTSCR2 were measured in invasive and LMP compared with benign and normal tissues. Over-expression of WNT7A in an ovarian cancer cell line led to increased migration and invasive capacity. CONCLUSION: These results highlight several genes that may play an important role across the spectrum of serous ovarian tumorigenesis.

Gata3 Immunohistochemical Staining is A Useful Marker for Metastatic Breast Carcinoma in Fine Needle Aspiration Specimens.

AIMS: The utility of GATA3 immunohistochemistry (IHC) as an aid to the cytological diagnosis of metastatic breast carcinoma in fine needle aspiration (FNA) specimens was investigated. MATERIALS AND METHODS: Cell block sections from 111 FNA cases of metastatic malignancy were stained for GATA3, including metastases from 43 breast and 44 nonmammary adenocarcinomas, 19 melanomas, 4 urothelial carcinomas, and 1 thyroid medullary carcinoma. Sites sampled included lymph nodes (87), bone (8), liver (5), lung (6), superficial masses (4), and pelvic mass (1). RESULTS: Ninety-one percent (39/43) of metastatic breast carcinoma cases were positive for GATA3. All estrogen receptor (ER)-positive were also GATA3 positive cases. The majority (9/14; 64%) of ER-negative and 37% (3/8) of triple-negative cases were positive for GATA3. All nonmammary adenocarcinoma cases were negative with the exception of one case of metastatic pancreatic adenocarcinoma. Metastatic melanoma cases were all negative but 75% (3/4) urothelial carcinomas expressed GATA3. CONCLUSIONS: GATA3 IHC staining is a useful addition to IHC panels for FNA samples in specific settings such as distinguishing metastatic breast from lung carcinoma or melanoma.

Which are more correctly diagnosed: conventional Papanicolaou smears or Thinprep samples? A comparative study of 9 years of external quality-assurance testing.

BACKGROUND: The Royal College of Pathologists of Australasia Cytopathology Quality Assurance Program offers external testing in gynecologic cytology to Australasian and international laboratories. Laboratory interpretation of conventional Papanicolaou (Pap) smears is compared with interpretation of liquid-based cervical cytologic (ThinPrep) samples. METHODS: Conventional Pap smears and ThinPrep samples were distributed to participating laboratories annually over 9 years (from 2004 to 2012), and a range of entities was tested. Target responses and major error rates and diagnostic trends over time were explored. RESULTS: In total, 23,373 conventional Pap smears and 14,104 ThinPrep samples were reported. Both Australasian (P = .003) and international (P < .001) laboratories achieved a higher percentage of the target diagnosis of squamous dysplasia with ThinPrep samples. Australasian laboratories more accurately diagnosed endocervical adenocarcinoma in situ with conventional smears (P = .036), whereas international laboratories performed better with ThinPrep samples (P = .006). Sampling of the lower uterine segment was more accurately diagnosed by both Australasian (P < .001) and international (P = .001) laboratories using conventional Pap smears. Significant improvements in achieving the target diagnosis over time for squamous dysplasias using both modalities were observed for Australasian and international laboratories (P < .001 for both). There was improvement in diagnosing high sampling using ThinPrep for both groups (P = .001 and P = .015, respectively). Australasian performance declined over time in reaching the target of normal (no infections) for both conventional (P = .001) and ThinPrep (P < .001) techniques and for international laboratories with the ThinPrep technique (P < .001). CONCLUSIONS: Participation in external proficiency testing in cervical cytology allows an analysis of performance, the identification of areas of diagnostic difficulty, a review of trends over time, and the highlighting of topics for ongoing education.

GATA3: a promising marker for metastatic breast carcinoma in serous effusion specimens.

BACKGROUND: The usefulness of GATA3 (GATA-binding protein 3 to DNA sequence [A/T]GATA[A/G]) as a marker for metastatic breast carcinoma in serous effusion specimens was investigated. METHODS: Cell block sections from 74 serous effusion specimens (32 ascitic, 2 pericardial, and 40 pleural fluids) were stained with an anti-GATA3 murine monoclonal antibody. The specimens included 62 confirmed metastatic carcinomas from the breast (30 specimens), female genital tract (13 specimens), gastrointestinal tract (7 specimens), lung adenocarcinoma (9 specimens), pancreas (1 specimen), kidney (1 specimen), and bladder (1 specimen). The breast carcinoma cases included 15 ductal carcinomas and 8 lobular carcinomas; the histology subtype was not available for 7 specimens. Twelve cases containing florid reactive mesothelial cells were also stained. The breast carcinoma cases were also stained for mammaglobin and gross cystic disease fluid protein of 15 kilodaltons (GCDFP-15) to compare their sensitivity with GATA3. RESULTS: Positive nuclear staining for GATA3 was found to be present in 90% of metastatic breast carcinoma specimens (27 of 30 specimens). All nonbreast metastatic carcinomas tested were negative with the exception of the single case of metastatic urothelial carcinoma. No staining was observed in any of the benign reactive cases or in benign mesothelial cells present in the malignant cell block preparations. Two cases demonstrated weak positivity of benign lymphoid cells. Staining results were unambiguous because all positive cases demonstrated intense nuclear staining in > 50% of tumor cells. Mammaglobin (57% staining; 17 of 30 cases) and GCDFP-15 (33% staining; 10 of 30 cases) were found to be less sensitive markers of breast carcinoma. If used in a panel, mammaglobin and GCFP-15 staining would have identified only 1 additional case compared with those stained with GATA3. CONCLUSIONS: GATA3 may be a useful addition to immunostaining panels for serous effusion specimens when metastatic breast carcinoma is a consideration.

Fibroadenoma and intraduct papilloma--a common pathogenesis?

This report describes two cases, each showing fibroepithelial proliferation, admixed with intraduct papillomas. Case 1 was from a 13-year-old female who presented with a breast mass, while case 2 was from a 60-year-old female who also had a breast lump. Case 1 showed proliferative breast disease with multiple small papillomas, some with leaf-like contours. Broad-based parenchymal proliferations protruding into ducts were also seen, and there were overlap features between the two ends of this spectrum. Case 2, similarly, demonstrated a mixed intraductal proliferation, with papillomas on narrow stalks together with low-grade phyllode areas extending into ducts from a wider base. That fibroadenoma and intraduct papilloma may have a common pathogenesis is discussed.