Response to anti-HER2 neoadjuvant chemotherapy in HER2-positive invasive breast cancers with different HER2 FISH patterns.

AIMS: Human epidermal growth factor receptor 2 (HER2)-positive patients with breast cancer may have different HER2/CEP17 ratios and HER2 copy numbers, with inconsistent responses to anti-HER2 neoadjuvant chemotherapy (NACT). Our study aimed to explore the relationship between different HER2 fluorescence in situ hybridisation (FISH) patterns in HER2-positive patients with breast cancer and responses to anti-HER2 NACT. METHODS: 527 patients with HER2-positive invasive breast cancer who received anti-HER2 NACT from 2015 to 2022 were included and divided into three groups by FISH results, namely group A: HER2/CEP17<2.0 and HER2 copy numbers ≥6.0, HER2 immunohistochemistry 2/3+; group B: HER2/CEP17≥2.0 and HER2 copy numbers ≥4.0 and <6.0; group C: HER2/CEP17≥2.0 and HER2 copy numbers ≥6.0. We compared clinicopathological characteristics and pathological complete response (pCR) rates of different groups. RESULTS: According to HER2 FISH results, 12 patients (2.3%, 12/527) were in group A, 40 (7.6%, 40/527) were in group B and 475 (90.1%, 475/527) were in group C. The pCR rate was the lowest in group B (5.0%), while the pCR rates in group A and group C were 33.3% and 44.4%, respectively (p (group A vs. B) =0.021, p (group C vs. B) < 0.001). Both univariate and multivariate analyses revealed that HER2 FISH pattern was correlated with pCR rate (p (group C vs. B) < 0.001, p (group C vs. B) = 0.025). CONCLUSIONS: Patients with HER2/CEP17≥2.0 and HER2 copy numbers ≥4.0 and <6.0 do not benefit to the same extent from current anti-HER2 therapies as FISH-positive patients with other patterns.

A custom next-generation sequencing panel for 1p/19q codeletion and mutational analysis in gliomas.

The World Health Organization has updated their classification system for the diagnosis of gliomas, combining histological features with molecular data including isocitrate dehydrogenase 1 and codeletion of chromosomal arms 1p and 19q. 1p/19q codeletion analysis is commonly performed by fluorescence in situ hybridization (FISH). In this study, we developed a 57-gene targeted next-generation sequencing (NGS) panel including 1p/19q codeletion detection mainly to assess diagnosis and potential treatment response in melanoma, gastrointestinal stromal tumor, and glioma patients. Loss of heterozygosity analysis was performed using the NGS method on 37 formalin-fixed paraffin-embedded glioma tissues that showed 1p and/or 19q loss determined by FISH. Conventional methods were applied for the validation of some glioma-related gene mutations. In 81.1% (30 of 37) and 94.6% (35 of 37) of cases, 1p and 19q were found to be in agreement whereas concordance for 1p/19q codeletion and no 1p/19q codeletion was found in 94.7% (18 of 19) and 94.4% (17 of 18) of cases, respectively. Overall, comparing NGS results with those of conventional methods showed high concordance. In conclusion, the NGS panel allows reliable analysis of 1p/19q codeletion and mutation at the same time.

IRF4 rearrangement may predict favorable prognosis in children and young adults with primary head and neck large B-cell lymphoma.

PURPOSE: Large B-cell lymphoma with IRF4 rearrangement (LBCL, IRF4+) has been recently recognized as a specific entity that is frequently associated with young age and favorable prognosis. However, whether the good outcome of the disease is due to IRF4+ or other factors remains obscure. We thus analyzed 100 young patients with primary head and neck LBCL to see the clinicopathologic correlates of IRF4+. METHODS: The histopathology, immunophenotype, IRF4 status of the tumors, and clinical data were reviewed. RESULTS: Twenty-one tumors were diagnosed as LBCL, IRF4+, which were more frequently associated with a follicular growth pattern, medium-sized blastoid cytology, germinal center B-cell-like, and CD5+ phenotype, compared with IRF4- ones. While most of the patients received chemotherapy with or without radiation, eight IRF4+ patients received mere surgical resection of the tumor and exhibited excellent outcome. IRF4+ cases featured a significantly higher complete remission rate, and better survivals compared with IRF4- ones. Multivariate analysis confirmed IRF4+ correlates with a better survival. CONCLUSION: Our work confirmed the unique clinicopathologic features of LBCL, IRF4+, and disclosed for the first time the independent favorable prognostic impact of IRF4+. These findings may further unravel the heterogeneity of LBCL occurring in youth, and aid in risk stratification and tailoring the therapeutic strategy.

Histomorphological and molecular genetic characterization of different intratumoral regions and matched metastatic lymph nodes of colorectal cancer with heterogenous mismatch repair protein expression.

PURPOSE: To better understand the clinicopathological characteristics and molecular alterations in different intratumoral components of colorectal cancer (CRC) with heterogeneity of mismatch repair (MMR) protein expression and microsatellite instability (MSI) status. METHODS: The histopathological features, MSI status, and other molecular alterations were analyzed in separately microdissected intratumoral regions and matched metastatic lymph nodes in four cases with intratumoral heterogenous MMR expression screened from 500 CRC patients, using PCR-based MSI testing, MLH1 promoter methylation, and targeted next-generation sequencing (NGS). RESULTS: High microsatellite instability (MSI-H) was identified in MLH1/PMS2-deficient regions in Cases 1 to 3 and in MSH2/MSH6-deficient regions in Case 4, while microsatellite stability (MSS) was detected in all the intratumoral regions and metastatic lymph nodes with proficient MMR expression (pMMR). Intratumoral heterogeneity of MLH1 promoter methylation and/or other common driving gene mutations of CRC, such as KRAS and PIK3CA mutations, was identified in all four CRCs. Further, three cases (75%) showed heterogeneous histomorphological features in intratumoral components and metastatic lymph nodes (Cases 1, 2, and 4), and the corresponding metastatic lymph nodes showed moderate differentiation with MSS/pMMR (Cases 2 and 3). CONCLUSIONS: Intratumoral heterogeneous MSI status is highly correlated with intratumoral histomorphological heterogeneity, which is also an important clue for the intratumoral heterogeneity of drive gene mutations in CRC. Thus, it is essential to detect MMR protein expression and other gene mutations in metastases before treatment, especially for CRCs with intratumoral heterogenous MMR protein expression or heterogenous histomorphological features.

Concordance of the 21-gene assay between core needle biopsy and resection specimens in early breast cancer patients.

BACKGROUND: Adjuvant therapy decisions may be partly based on the results of a multigene quantitative reverse transcription-polymerase chain reaction (RT-PCR)-based assay: the 21-gene recurrence score (RS) test of resection specimens. When necessary, core needle biopsy (CNB) may be considered as a surrogate. Here, we evaluated the concordance in gene expression according to results from RT-PCR-based RS testing between paired CNBs and resection specimens. METHODS: CNBs and resection specimens from 50 breast cancer (BC) patients were tested to calculate RSs. First, we examined the concordance of the ER, PR and HER-2 status of tissue samples indicated by immunohistochemical (IHC) and RT-PCR analyses. Then, we compared the IHC findings of ER, PR, HER-2 and Ki-67 staining across paired samples. Ultimately, the RS and single-gene results for ER, PR, HER-2 and Ki-67 were explored between paired samples. RESULTS: The concordance between IHC and RT-PCR was 100%, 80.0% and 100% for ER, PR and HER-2, respectively, in both resection specimens and CNBs. The concordance for IHC ER, PR, HER-2 and Ki-67 status was 100%, 94.0%, 52.0% and 82.0%, respectively, between paired samples. RS results from paired samples showed a strong correlation. The overall concordance in RS group classification between samples was 74%, 72% and 78% based on traditional cutoffs, TAILORx cutoffs and ASCO guidelines, respectively. ER, PR, HER-2 and Ki-67 were modestly- to- strongly correlated between paired samples according to the RT-PCR results. CONCLUSION: A modest- to- strong correlation of ER, PR, HER-2 and Ki-67 gene expression and RS between CNBs and resection specimens was observed in the present study. The 21-gene RS test could be reliably performed on CNBs. ER, PR and HER-2 status showed remarkable concordance between the IHC and RT-PCR analyses. The concordance between paired samples was high for the IHC ER, PR and Ki-67 results and low for HER-2.

Mammary analog secretory carcinoma of the thyroid gland: A rare cancer harboring TRK fusion.

Mammary analog secretory carcinoma (MASC), or secretory carcinoma of the thyroid is an extremely rare disease harboring ETV6-NTRK3 gene fusion with TRK activation. Here we report the twelfth case of MASC of the thyroid worldwide. A 36-year-old female was diagnosed with poor-differentiated thyroid carcinoma (PDTC). Pathology consultant and immunochemical workups showed the tumor cells were negative for TTF1, TG, PAX8, positive for S100, Vimentin, GATA-3, and focally positive for mammaglobin. Fluorescence in situ hybridization (FISH) assay using a dual-color break-apart probe showed ETV6 translocation t(12p13) (ETV6) was present and established the diagnosis of MASC. Next-generation sequencing (NGS) of a 47-gene panel identified exon 1-5 of ETV6 gene were fused with exons 15-19 of NTRK3 gene. The patient experienced three loco-regional recurrences within 12 months and eventually developed inoperable local disease as well as bilateral lung metastasis. She is currently receiving anti-TRK treatment with a follow-up time of 33 months. A literature review of MASC in the thyroid was also conducted.

Performance of Automated Dissection on Formalin-Fixed Paraffin-Embedded Tissue Sections for the 21-Gene Recurrence Score Assay.

This study aimed to compare the performance of MilliSect dissection and manual dissection. Twenty-five formalin-fixed paraffin-embedded (FFPE) breast cancer tissue blocks were selected for comparison. Specific areas of interest (AOIs) in invasive carcinoma on tissue sections were transferred to dissection slides by manual macrodissection or the MilliSect instrument. The comparison criteria were 1) the time required for dissection; 2) RNA concentration and purity; 3) RNA quantity of 5 housekeeping genes (by RT-qPCR); and 4) ER, PR, HER2, Ki-67 and recurrence score (RS) values (by the 21-gene assay). Then, tumor-adjacent tissues, including fibrocollagenous and epithelial tissues, from the same selected tissue blocks of 8 of 25 patients were scraped using the mesodissection method, and their RS values were assessed to evaluate the influence of tumor-adjacent tissues on the target AOIs. Ultimately, 4 AOIs of invasive ductal carcinoma (IDC) from 1 tissue block of another 4 patients with lymph node (LN) metastases each, LN tissue and a mixture of IDC and LN tissue from the other tissue block of the same 4 patients were mesodissected to evaluate the influence of infiltrating lymphocyte levels on the RS values of AOIs. In our experience, the MilliSect instrument, which provides process management documentation, required more time than manual macrodissection (on average, approximately 9.1 min per sample versus 5.8 min per sample, respectively). The RNA yield and quality of the dissected tissues were comparable for the 2 methods. However, the tumor-adjacent tissues of the AOIs may influence the RS to some extent. Tumor-infiltrating lymphocytes (TILs) can dramatically increase RSs, far exceeding the influence of tumor-adjacent fibrocollagenous and epithelial tissues. In conclusion, MilliSect mesodissection is comparable to manual dissection. This mesodissection tool may facilitate AOI alignment and the dissection process for the 21-gene RS assay. Samples whose adjacent tissues are intermixed with TILs warrant special attention.

Malignant Gastrointestinal Neuroectodermal Tumor: Clinicopathologic, Immunohistochemical, and Molecular Analysis of 19 Cases.

A malignant gastrointestinal neuroectodermal tumor (GNET) is rare, and it is therefore yet to be completely understood. This study aimed to present the clinicopathologic features of GNET, including treatment information. We included 19 patients with GNET with a mean tumor size of 4.2 cm. The most common site of tumor origin was the small intestine (57.9%), followed by the stomach (15.8%), colon (10.5%), ileocecal junction (5.3%), lower esophagus (5.3%), and anal canal (5.3%). Microscopically, the tumors were composed of epithelioid cells with eosinophilic or clear cytoplasm arranged in nest, sheet-like, papillary, or pseudoalveolar patterns and/or spindle tumor cells with eosinophilic cytoplasm arranged in a fascicular pattern. Immunohistochemically, the tumor cells stained positively for S100 (19/19,100%), SOX10 (14/15, 93.3%), vimentin (17/17, 100%), synaptophysin (Syn) (7/17, 41.2%), CD56 (4/13, 30.8%), CD99 (1/5, 20%), and CD117 (1/15, 6.7%), and negatively for HMB45, Melan A, DOG1, CD34, AE1/AE3, CAM5.2, chromogranin A, smooth muscle actin, and desmin. In total, 14/15 (93.3%) cases showed split Ewing sarcoma breakpoint region 1 gene (EWSR1) signals consistent with a chromosomal translocation involving EWSR1. Within a mean follow-up of 29.7 months (range: 3 to 63 mo), 2/15 (13.3%) patients died of disease, 5 (33.3%) were alive with disease, and 8 (53.3%) had no evidence of disease. Two and 1 patients showed partial response to apatinib and anlotinib, respectively. In conclusion, GNET has distinctive morphologic, immunohistochemical, and molecular genetic features and should be distinguished from other gastrointestinal tract malignancies. Apatinib and anlotinib might be effective for the treatment of advanced GNET and could prolong patient survival.

E2F1 induces LSINCT5 transcriptional activity and promotes gastric cancer progression by affecting the epithelial-mesenchymal transition.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been shown to play important regulatory roles in human cancer. We previously verified that the lncRNA long stress-induced noncoding transcript 5 (LSINCT5) is overexpressed in gastric cancer (GC) cells and closely correlated with cell proliferation and patient prognosis. However, whether aberrant LSINCT5 expression has an important effect on GC progression is unclear, and the potential mechanisms remain unknown. In GC, E2F1 expression is also aberrant, but the biological functions of E2F1 are controversial, and the correlation between E2F1 and lncRNAs remains unknown. MATERIALS AND METHODS: Expression of LSINCT5 was analyzed in metastatic GC tissues compared with nonmetastatic tissues using quantitative real-time PCR (qRT-PCR) assays. Gain and loss of function approaches were used to investigate the biological role of LSINCT5 in GC cell migration and invasion. A computational screen of LSINCT5 promoter was conducted to search for transcription factor-binding sites. LSINCT5 promoter activities were examined by ChIP and luciferase reporter assays. qRT-PCR and western blotting assays were performed to detect the expression of multiple EMT markers in cells in which LSINCT5 was overexpressed or knocked down. RESULTS: An integrated quantitative analysis revealed that LSINCT5 was significantly over-expressed in metastatic GC tissues. Forced LSINCT5 expression promoted cell migration and invasion, whereas loss of LSINCT5 function decreased cell migration and invasion. Mechanistic investigations showed that LSINCT5 is a direct transcriptional target of E2F1. Moreover, LSINCT5 overexpression was found to play an important role in the epithelial-to-mesenchymal transition by regulating the expression of E-cadherin, N-cadherin, vimentin, and matrix metalloproteinase-2. CONCLUSION: These data suggest that E2F1-mediated activation of LSINCT5, a regulator of cell migration and invasion, constitute the mechanistic link between the E2F1-mediated pathway and lncRNA that regulates cell migration and invasion. Thus, LSINCT5 may be a target for new GC therapies.

Microcystic stromal tumour of the ovary: frequent mutations of ß-catenin (CTNNB1) in six cases.

AIMS: To analyse the clinicopathological, immunohistochemical and ß-catenin (CTNNB1) mutation characteristics of six cases of microcystic stromal tumour of the ovary (MCST). METHODS AND RESULTS: Six Chinese patients with MCST who ranged in age from 29 to 69 years (mean 50 years) were included in the study. Five patients were detected with a pelvic mass during routine health examinations and one patient presented initially with opsomenorrhoea. All tumours involved the left ovary, with solid-cystic cut surface in five cases and cystic cut surface in one case. Microscopically, microcysts, solid nests and hyaline degenerated fibrous stroma were variably mixed. Immunohistochemically, the tumour cells in all cases were diffusely positive for CD10, vimentin and WT-1 and negative for α-inhibin and calretinin. ß-catenin expression was observed in both the nucleus and the cytoplasm in five cases and only in the cytoplasm in one case. The results of CTNNB1 mutation analysis revealed four missense point mutations in four cases, which were c.97T>C, c.101G>A, c.110C>G and c.122C>T. CONCLUSIONS: MCST shows a unique morphology with characteristic immunophenotype. ß-catenin expression in the nucleus and ß-catenin mutations were identified in the majority of cases, which suggests that the Wnt/ß-catenin pathway may play a crucial role in the tumorigenesis of MCST.