Applied Immunohistochemistry & Molecular Morphology最新文献

Accurate diagnosis of cancer of unknown primary (CUP) poses a significant daily challenge for pathologists, necessitating reliable immunohistochemical (IHC) markers. SATB2 is a transcription factor primarily expressed in colorectal neoplasms. This study investigates the IHC expression of SATB2 in malignant melanomas (MM). Using tissue microarrays (TMAs) from Aalborg University Hospital, Denmark, comprising 56 primary and 12 metastatic MMs, we evaluated SATB2 expression through H-scores. We found that 48% of MM cases expressed SATB2, predominantly with weak to moderate staining intensity. Although no significant difference was observed between primary and metastatic MMs, a higher median H-score was noted in metastatic lesions. The results highlight the potential diagnostic pitfall of SATB2 expression in MM and underline the need for careful interpretation.

Distinction of metastasis to the breast from a breast primary, particularly high-grade triple-negative breast cancer (TNBC), can be challenging due to nonspecific morphology and immunohistochemical (IHC) profiles. Among metastases to the breast, high-grade serous carcinoma (HGSC) of müllerian origin is most likely to be misdiagnosed as TNBC. We assessed breast and müllerian markers on TNBC and HGSC, including keratin 7, keratin 20, GATA3, GCDFP15, mammaglobin, p53, PAX8 (MRQ50 and BC12 clones), TRPS1, SOX10, and WT1. Of 151 TNBC cases, TRPS1 had the highest sensitivity, showing expression in 149 (98.7%) cases, followed by SOX10 (110/151; 72.8%), GATA3 (102/151; 67.5%), GCDFP15 (29/151; 19.2%), and mammaglobin (27/151; 17.9%). PAX8 positivity was seen in 40.4% (61/151) of TNBC via the MRQ50 clone but was negative in all via the BC12 clone. Of 185 HGSC cases, PAX8 via the MRQ50 clone was the most sensitive (179/185; 96.8%), followed by WT1 (171/185; 92.4%) and PAX8 via the BC12 clone (164/185; 88.6%). In addition, TRPS1 positivity was seen in 75 HGSC cases (40.5%). Aberrant p53 patterns were seen in 64.9% (98/151) of TNBC and 94.1% (174/185) of HGSC. TRPS1 positivity in HGSC and PAX8 positivity via the MRQ50 clone in TNBC represent potential pitfalls in assessing high-grade carcinoma for which the differential diagnosis includes TNBC and HGSC. However, with this knowledge, utilization of a panel of breast and müllerian markers, including preferential use of the PAX8 BC12 clone, can facilitate accurate diagnosis.

Gastric gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms with variable behavior characterized by differentiation toward the interstitial cells of Cajal occurring anywhere in the gastrointestinal stromal tract. The management of GIST was revolutionized by the introduction of imatinib, a KIT inhibitor, which has become the standard first-line treatment for metastatic GIST. However, despite a clinical benefit rate of 80%, the majority of patients with GIST experience disease progression after 2 to 3 years of imatinib therapy. This shows the need for novel treatment approaches for imatinib refractory GISTs. The checkpoint proteins B7-H3 and B7-H4 inhibit the activation and function of T cells by potently suppressing the proliferation, cytokine production, and cytotoxicity of activated T cells, which is a mechanism for immune escape. This study aims to clarify B7-H3 and B7-H4 expression in gastric GISTs using immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). We confirmed B7-H3 expression (H-score ≥50 points) in 92% and B7-H4 expression in 0% of GIST samples. We examined B7-H3 mRNA expression in 3 representative GIST samples, each having their respective immunostained areas detected by RT-PCR. B7-H3 is expressed at a particularly high rate in GISTs. This suggests that B7-H3 might operate as part of an immune checkpoint in GISTs.

Multiplex immunofluorescence (mIF) staining plays an important role in profiling biomarkers and allows investigation of co-relationships between multiple biomarkers in the same tissue section. The Cell DIVE mIF platform (Leica Microsystems) employs an alkaline solution of hydrogen peroxide as a fluorophore inactivation reagent in the sequential staining, imaging, and bleaching protocol for use on FFPE sections. Suboptimal bleaching efficiency, degradation of tissue structure, and loss of antigen immunogenicity occasionally are encountered with the standard bleaching process. To overcome these impediments, we adopted a modified photochemical bleaching method, which utilizes an intense LED light exposure concurrent with the application of hydrogen peroxide. Repeated stain/bleach rounds with different antibodies were performed on breast tissue and other tissue sections. Residual signal after conventional bleaching and the modified technique were compared and tissue integrity and antigen immunogenicity were assessed. The modified technique effectively eliminates fluorescence signal from previous staining rounds and produces consistent results for multiple rounds of staining and imaging. With the modified method, photochemical treatments did not destroy tissue sub-cellular contents, and the tissue antigenicity was well preserved during the entire mIF process. Overall processing time was reduced from 36 to 30 hours in an mIF procedure with 8 rounds. With the conventional method, tissue quality was highly degraded after 8 rounds. The new technique allows reduced turn-around time, provides reliable fluorophore removal in mIF with excellent maintenance of tissue integrity, facilitating studies of the co-localization of multiple biomarkers in tissues of interest.

This study aimed to evaluate the predictive values of phosphoglucomutase-1 (PGM1) expression for prognosis in patients with hepatocellular carcinoma (HCC). PGM1 expression was assessed by immunohistochemistry in tissue microarrays. The relationship of PGM1 expression level with pathologic parameters and prognosis values was respectively analyzed by χ 2 test and Cox regression. The accuracy of independent risk factors in predicting prognosis was calculated by receiver operating characteristic curve. HCC patient-derived xenograft models were performed to evaluate the nuclear PGM1 antitumor effect. The results showed that PGM1 expression was low in HCC tissues. Nuclear PGM1 was an independent prognostic factor for overall survival and time to recurrence. Cox regression showed that nuclear PGM1, serum α-fetoprotein, liver cirrhosis, and TNM staging stage were independent risk predictors for HCC. Receiver operating characteristic curve demonstrated that combination of independent predictors had better prognostic value than TNM staging alone. Moreover, patient-derived xenograft models showed antitumor effect of nuclear PGM1. We found that low expression of nuclear PGM1 was detected in HCC tissues and associated with poor prognostic. Nuclear PGM1 was an independent prognostic factor in patients with HCC. Furthermore, nuclear PGM1 combining other independent risk factors showed a better prognostic value. Nuclear PGM1 was a useful prognostic biomarker for patients with HCC.

Accurate assessment of HER2 expression levels is paramount for determining eligibility for targeted therapies. HER2 immunohistochemistry provides a semiquantitative measurement of HER2 protein overexpression. Historically, little focus has been on the lower end of the HER2 expression range. The advent of novel therapeutic molecules that require fewer membrane epitopes to be effective has prompted a reevaluation of the current immunohistochemistry testing protocols, with special emphasis on the detection limit. Here, we have used Boston Cell Standards technology to determine the sensitivity of 2 commercially available HER2 immunohistochemistry assays, including a lower limit of detection.

BRCA1/2 are tumor suppressor genes which regulate the DNA repair mechanism. Mutations in BRCA1/2 may increase the risk of breast cancer in patients. In the present study frequency of BRCA1/2 mutations in triple negative breast cancer (TNBC) patients was assessed and correlated with molecular subtypes of TNBC. Blood samples from 65 confirmed cases of TNBC were collected. DNA was isolated from whole blood and libraries were prepared using a BRCA1/2 custom panel. Sequencing was done on Ion torrent S5 sequencer and ion reporter was used for data analysis. Further molecular subtyping of mutation positive TNBC cases was done using immunohistochemistry markers CK5/6; CK4/14; Vimentin and E-Cadherin and androgen receptor (AR) using tissue microarray. Twenty five of 65 patients had heterozygous pathogenic mutations, alterations with conflicting interpretation of pathogenicity, variants of uncertain significance and variants of unknown significance. Nine patients had pathogenic mutation in BRCA 1 gene only and 2 patients had pathogenic mutations in BRCA2 gene. Two patients were transheterozygous for BRCA mutations, that is, had pathogenic mutations in both BRCA1/2 genes simultaneously and 5 were compound heterozygous (involving BRCA2 gene in all the cases). Prevalent subtypes among BRCA positive cases were unclassified subtype (n=4, 33%), Basal like (n=5, 41%), and mesenchymal subtype (n=3, 25%). None of the LAR subtype showed BRCA1/2 mutations. The present study observed that the BRCA1 mutation is more frequent than BRCA2 mutation in TNBC. BRCA1/2 mutations do not correspond to BRCAness or basal phenotype. Considering high incidence of breast cancer and lack of correlation of basal morphology with BRCA1/2 mutation, the molecular methods should be used for screening for BRCA1/2 mutations. This will not only help in familial screening but also in deciding targeted therapy with PARP (poly-ADP ribose polymerase) inhibitors.

For patients with metastatic triple-negative breast cancer (TNBC), treatment with pembrolizumab is dependent on the accurate determination of programmed death ligand 1 (PD-L1) expression using immunohistochemistry (IHC). This study evaluated the interobserver concordance in assessing PD-L1 expression on TNBC samples using the commercial 22C3 IHC assay and an in-house assay based on the E1L3N antibody. Concordance between the 22C3 and the E1L3N IHC assays was evaluated on TNBC samples read by a commercial laboratory and a Brigham and Women's Hospital breast pathologist (BWH reader). Each slide was given a PD-L1 combined positive score (CPS) and was considered PD-L1 positive or negative based on the CPS cutoff of 10. Interobserver concordance for the assays was also evaluated on a subset of samples between 2 and 3 independent readers. On 71 samples, 2 independent readers (1 BWH reader and commercial laboratory) using E1L3N and 22C3, respectively, reached agreement on PD-L1 status (positive/negative) on 64 samples (90.1%). Using 22C3, 2 independent readers reached agreement on PD-L1 status on 30 of 36 samples (83.3%), and 3 independent readers reached agreement on 16 of 27 samples (59.3%). Using E1L3N, 2 BWH readers reached agreement on PD-L1 status on 18 of 27 samples (66.7%). Three BWH readers reached an agreement on 2 of 12 of the most challenging samples (16.7%). In conclusion, concordance between E1L3N and 22C3 testing using CPS for PD-L1 in metastatic TNBC was >90%. However, certain cases were challenging to agree upon using current threshold criteria, highlighting the need for more standardized evidence-based methods to assess PD-L1 expression.

Cervical cancer remains one of the leading causes of death from malignant diseases in women worldwide. Primary and secondary prevention have led to better outcomes in developed countries, whereas in developing countries, cervical cancer continues to be responsible for an unjustifiably high number of fatalities. The discovery of new tumor biomarkers can lead to earlier diagnosis, better therapeutic decisions, and improved treatment methods. IMP3 is a protein responsible for invasiveness and other aggressive characteristics of tumor processes. Its highly specific expression has been proven in various malignant processes. The level of IMP3 expression in cervical cancer cells could be used as a prognostic factor for a worse disease course. In this study, IMP3 expression was examined in 80 patients who underwent surgery for squamous cell cervical cancer in the first FIGO stage of the disease, and its association with disease-free period and overall survival was investigated. Data analysis did not show a statistically significant association between IMP3 expression and the mentioned primary outcomes, despite its association with clinical-pathological indicators of advanced disease. In conclusion, the analysis of IMP3 protein expression in patients with early-stage cervical cancer is of limited utility.

Merkel cell carcinoma (MCC) is a rare, highly aggressive skin cancer of neuroendocrine origin that is typically associated with either the presence of Merkel cell polyomavirus or chronic exposure to ultraviolet (UV) light. We report a case of relapsed MCC that presented with new symptoms of fatigue, back pain, and myeloid left shift identified during scheduled follow-up. The patient was found to have circulating neoplastic cells in the peripheral blood and bone marrow metastasis. Immunohistochemistry for synaptophysin, CD56, INSM-1, CK20, CD117 were positive, whereas CD34, TdT, Chromogranin, CD10, myeloperoxidase, CD3 and CD19 were negative. Flow cytometry of the peripheral blood confirmed the presence of an abnormal nonhematopoietic cell population expressing CD56 positivity. A next-generation sequencing (NGS) panel revealed the presence of variants in RB1, TP53, and other genes, some of which have not been previously described in MCC. This rare presentation highlights the challenges in the diagnosis and management of MCC.