The frequency of microsatellite instability (MSI) is reportedly extremely low in breast cancer, despite widespread clinical expectations that many patients would be responsive to immune‐checkpoint ...inhibitors (ICI). Considering that some triple‐negative breast cancers (TNBC) responded well to ICI in a clinical trial and that a high density of tumor‐infiltrating lymphocytes (TILs) is frequently observed in other cancers with high levels of microsatellite instability (MSI‐H), we hypothesized that some TNBC with a high density of TILs would be MSI‐H. Medullary carcinoma (MedCa) of the breast, a rare histological type, is characterized by a high density of TILs. Considering that MedCa of the colon is often MSI‐H, we suspected that MedCa in breast cancer might also include MSI‐H tumors. Therefore, we conducted MSI tests on such breast cancers with a high density of TILs. The MSI status of 63 TIL‐high TNBC and 38 MedCa tumors, all from Asian women who had undergone curative surgery, were determined retrospectively. DNA mismatch repair (MMR) proteins and PD‐L1 expression were also investigated immunohistochemically. All samples were microsatellite stable, being negative for all microsatellite markers. TIL‐high TNBC with low MLH1 protein had higher levels of PD‐L1 in stromal immune cells (P = .041). MedCa tumors showed significantly higher PD‐L1 expression in immune cells than in TIL‐high TNBC (<.001). We found that MSI‐H tumors were absent in TIL‐high breast cancers. Examination of MMR proteins, not a purpose of Lynch syndrome screening, may merit further studies to yield predictive information for identifying patients who are likely to benefit from ICI.
We revealed that MSI‐H breast cancers do not correspond to TIL‐high tumors. Furthermore, MLH1 protein expression in triple‐negative breast cancer was inversely correlated with PD‐L1 expression in stromal immune cells. Therefore, we believe that the examination of MMR proteins may merit additional study, as these proteins might provide information for predicting which patients would are likely to benefit from immune‐checkpoint inhibitors.
Summary The impact of NGFI-A binding protein 2 ( NAB2) - signal transducer and activator of transcription 6 ( STAT6 ) fusion on the biological behavior and the mechanism of acquisition of malignant ...phenotype in solitary fibrous tumor (SFT) is not well understood. We examined variations of the NAB2-STAT6 fusion gene in 40 cases of SFT using formalin-fixed, paraffin-embedded tissues and secondary genetic alterations of tumor protein p53 (TP53), , platelet-derived growth factor receptor , β polypeptide ( PDGFRB) , and telomerase reverse transcriptase ( TERT) promoters. These gene variations were compared with the clinicopathological features. The 2-year and 5-year disease-free survival rates (DFSRs) were 91% and 83%, respectively. All 40 samples demonstrated nuclear staining for STAT6, including CD34-negative cases. Moreover, p53-positive staining was associated with a lower DFSR and was significantly associated with higher Ki-67 label index, higher mitotic rate (mitosis, >4/high-power field), and the presence of nuclear atypia/pleomorphism. NAB2-STAT6 fusions were detected in all of the cases; the NAB2 exon 4– STAT6 exon 2, the most common genotype, appeared in 18 cases, which was associated with thoracic tumor location and the less aggressive phenotype. In contrast, tumors with NAB2 exon 6– STAT6 exon 16/18 demonstrated an aggressive phenotype. Mutations in TP53 and PDGFRB were detected in 2 and 3 cases respectively, and these occurred in a mutually exclusive fashion. TERT promoter hot spot mutations were observed in 5 cases, which were associated with shorter DFSR. Two dedifferentiated SFT cases harbored both TP53 and TERT promoter mutations. TP53 mutations, which result in its overexpression, in combination with TERT promoter mutations seem to play an important role in the dedifferentiation process.
Tumor-infiltrating lymphocytes (TILs) are considered a prognostic marker for triple-negative breast cancer (TNBC). Immune checkpoint inhibitor (ICI)-based treatments are more effective for tumors ...with PD-L1-positive TILs, suggesting crucial roles of TILs in the local tumor immunity. However, factors attracting TILs are still largely unknown. Focusing on tumor antigenicity, we examined TNBC samples to identify the characteristics of TIL-high tumors.
Nine treatment-naïve TNBCs (TIL-high: five, TIL-low: four) were subjected to next-generation sequencing (NGS). Loss of heterozygosity (LOH) of PTEN was also analyzed.
A variety of copy number variations were observed, and no genes differed significantly between TIL-high and -low groups. However, PTEN loss was more frequently observed in the TIL-high group: 60% compared to 25% in TIL-low tumors. NGS correlated well with LOH analysis in identifying PTEN loss. All three tumors with PTEN loss in the TIL-high group showed high PD-L1. All nine samples were microsatellite-stable.
Frequent PTEN loss and high expression of PD-L1 in TIL-high TNBC suggest that PTEN mutation may be a biomarker for ICIs.
Anaplastic lymphoma kinase (ALK)-positive histiocytosis is a rare emerging entity characterized by systemic or localized proliferation of histiocytes harboring ALK rearrangements. Breasts are ...reportedly affected by ALK-positive histiocytosis. Here, we evaluated 2 localized cases of breast ALK-positive histiocytosis through a comprehensive clinicopathologic, molecular, and genomic analysis to further delineate this entity and better understand its pathogenesis. The cases involved 2 undiagnosed ALK-positive spindle-cell breast lesions. Both cases were Asian women aged 30s to 40s who underwent excisions for asymptomatic breast masses. Macroscopically, both lesions were well-circumscribed, solid masses. Microscopically, both lesions were predominantly composed of fascicles with uniform, bland spindle cells, admixed with epithelioid histiocyte-like cells and lymphoid aggregates. Immunohistochemically, the spindle and epithelioid cells coexpressed ALK and histiocytic markers (eg, CD68, CD163). Genetically, both lesions harbored KIF5B-ALK, confirmed by fluorescence in situ hybridization and polymerase chain reaction-direct sequencing analyses. Combining these results, both cases were successfully diagnosed as ALK-positive histiocytosis. Furthermore, no common or previously annotated somatic alterations were identified by whole-exome sequencing. One case harbored clonal immunoglobulin gene rearrangements according to the polymerase chain reaction-based BIOMED-2 protocol. Therefore, ALK-positive histiocytosis can be accurately diagnosed through a combination of morphologic, immunohistochemical, and molecular analyses. In this entity, breast cases may have distinct clinicopathologic features: Asian women aged 30s to 40s, asymptomatic masses, and predominant spindled morphology. For pathogenesis, ALK rearrangements could be the driver alteration, and a subset of ALK-positive histiocytosis may harbor a lymphoid lineage. These findings can be utilized to improve the diagnosis of ALK-positive histiocytosis and better understand its pathogenesis.
Perivascular epithelioid cell tumors (PEComas) are mesenchymal neoplasms with immunoreactivity for both melanocytic and smooth muscle markers. PEComas occur at multiple sites, and malignant PEComas ...can undergo metastasis, recurrence and aggressive clinical courses. Although the lung is a common metastatic site of PEComas, they usually appear as multiple nodules but rarely become cystic or cavitary. Here, we describe a female patient whose lungs manifested multiple cystic, cavity-like and nodular metastases 3 years after the resection of uterine tumors tentatively diagnosed as epithelioid smooth muscle tumors with uncertain malignant potential. This patient's subsequent pneumothorax necessitated video-assisted thoracoscopic surgery, and examination of her resected lung specimens eventually led to correcting the diagnosis, i.e., to a PEComa harboring tuberous sclerosis complex 1 (TSC1) loss-of-heterozygosity that originated in the uterus and then metastasized to the lungs. The administration of a gonadotropin-releasing hormone analogue later stabilized her clinical course. To the best of our knowledge, the present case is the first in the literature that associates PEComas with a TSC1 abnormality. Additionally, the pulmonary manifestations, including imaging appearance and pneumothorax, somewhat resembled those of lymphangioleiomyomatosis, a representative disease belonging to the PEComa family. Although PEComas are rare, clinicians, radiologists and pathologists should become aware of this disease entity, especially in the combined clinical setting of multiple cystic, cavity-like, nodular lesions on computed tomography of the chest and a past history of the tumor in the female reproductive system.
Soft tissue sarcomas are a heterogeneous group of rare malignant tumors. Advanced soft tissue sarcomas have a poor prognosis, and effective systemic therapies have not been established. Tyrosine ...kinases are increasingly being used as therapeutic targets for a variety of cancers and soft tissue sarcomas. Although complex karyotype sarcomas typically tend to carry more potentially actionable genetic alterations than do translocation-associated sarcomas (fusion gene sarcomas), based on our database review, we found that leiomyosarcoma and malignant peripheral nerve sheath tumors have lower frequencies of potential targets than other nontranslocation soft tissue sarcomas. We theorized that both leiomyosarcoma and malignant peripheral nerve sheath tumors might be included in any unique translocations. Furthermore, if tyrosine kinase imbalances, especially fusion genes, occur in patients with leiomyosarcomas and malignant peripheral nerve sheath tumors, tyrosine kinase inhibitors might be a drug development target for this sarcoma. In this study, we used a tyrosine kinase screening system that could detect an imbalance in mRNA between 5'- and 3'-sides in tyrosine kinase genes to identify potential novel therapeutic tyrosine kinase targets for soft tissue sarcomas.
(1) Are there novel therapeutic tyrosine kinase targets in tumors from patients with soft tissue sarcomas that are detectable using mRNA screening focusing on imbalance expressions between the 5' and 3' end of the kinase domain? (2) Can potential targets be verified by RNA sequencing and reverse transcription PCR (RT-PCR)? (3) Will potential fusion gene(s) transform cells in in vitro assays? (4) Will tumors in mice that have an identified fusion gene respond to treatment with a therapeutic drug directed at that target?
We used mRNA screening to look for novel tyrosine kinase targets that might be of therapeutic potential. Using functional assays, we verified whether the identified fusion genes would be good therapeutic candidates for soft tissue sarcomas. Additionally, using in vivo assays, we assessed whether suppressing the fusion's kinase activity has therapeutic potential. Study eligibility was based on a patient having high-grade spindle cell and nontranslocation sarcomas, including leiomyosarcoma, malignant peripheral nerve sheath tumor, and high-grade myxofibrosarcoma. Between 2015 and 2019, of the 172 patients with soft tissue sarcomas treated with surgical resection at Juntendo University Hospital, 72 patients had high-grade nontranslocation sarcomas. The analysis was primarily for leiomyosarcoma and malignant peripheral nerve sheath tumors, and there was a limitation of analysis size (reagent limitations) totaling 24 samples at the start of the study. We collected additional samples from a sample bank at the Tokyo Medical and Dental University to increase the number of sarcomas to study. Therefore, in this study, a total of 15 leiomyosarcoma samples, five malignant peripheral nerve sheath tumors samples, and four high-grade myxofibrosarcoma samples were collected to achieve the sample size of 24 patients. To identify tyrosine kinase fusion genes, we designed a NanoString-based assay (NanoString Technologies Inc, Seattle, WA, USA) to query the expression balances regarding transcripts of 90 tyrosine kinases at two points: the 5' end of the kinase domain and within the kinase domain or 3' end of the kinase domain. The tumor's RNA was hybridized to the NanoString probes and analyzed for the expression ratios of outliers from the 3' to 5' end of the kinase domain. Presumed novel fusion events in these positive tumors that were defined by NanoString-based assays were confirmed tyrosine kinase fusion genes by RNA sequencing and confirmatory RT-PCR. Functional analyses consisting of in vitro and in vivo assays were also performed to elucidate whether the identified tyrosine kinase gene fusions were associated with oncogenic abilities and drug responses.
We identified aberrant expression ratios regarding the 3' to 5' end of the kinase domain ratios in ROS1 transcripts in a leiomyosarcoma in a 90-year-old woman. A novel MAN1A1-ROS1 fusion gene was identified from her thigh tumor through RNA sequencing, which was confirmed with real-time PCR. In functional assays, MAN1A1-ROS1 rearrangement revealed strong transforming potential in 3T3 cells. Moreover, in an in vivo assay, crizotinib, a ROS1 inhibitor, markedly inhibited the growth of MAN1A1-ROS1 rearrangement-induced transformed cells in a dose-dependent manner.
We conducted tyrosine kinase screening to identify new therapeutic targets in soft tissue sarcomas. We found a novel MAN1A1-ROS1 fusion gene that may be a therapeutic target in patients with leiomyosarcoma. This study demonstrates that the mRNA screening system may aid in the development of useful therapeutic options for soft tissue sarcomas.
If novel tyrosine fusions such as MAN1A1-ROS1 fusion can be found in sarcomas from other patients, they could offer avenues for new molecular target therapies for sarcomas that currently do not have effective chemotherapeutic options. Therefore, the establishment of a screening system that includes both genomic and transcript analyses in the clinical setting is needed to verify our discoveries and take the developmental process of treatment to the next step.
Recurrent hot-spot mutations in the telomerase reverse transcriptase (TERT) promoter have been reported in various types of tumor. In several tumor types, TERT promoter mutations are associated with ...poor clinical outcomes. TERT promoter mutations are reported to be rare in soft tissue tumors, with the exception of myxoid liposarcoma (MLS). Our previous study reported that TERT promoter mutations occurred in a subset of solitary fibrous tumors (SFTs) and were associated with adverse clinical outcomes in Japanese individuals. The site-specific frequency (e.g. central nervous or soft tissue origin) of TERT promoter mutations in our SFT cases appeared to be different from previously reported values in a European population. These findings prompted the present study to elucidate the potential role of ethnic background in the different frequencies of TERT promoter mutations in bone and soft tissue sarcomas. In the present study, TERT promoter mutations were examined in 180 cases of bone and soft tissue sarcomas. TERT promoter region mutations were identified in 10 cases 5 SFTs, 3 MLSs, 1 undifferentiated pleomorphic sarcoma (UPS) and 1 malignant granular cell tumor. All mutations were C228T. The frequencies of TERT promoter mutation in MLS and UPS were 23.1 (3/13) and 5% (1/20), respectively. Only 1/5 patients with TERT-mutated tumors experienced local recurrence or distant metastasis. The present study revealed the first case of a malignant granular cell tumor with a TERT promoter mutation and revealed that the frequency of TERT promoter mutations in MLSs of Japanese patients is lower compared with that reported in German patients, providing evidence of a possible ethnic difference in the frequency of TERT promoter mutations.
Solitary fibrous tumors (SFTs), initially observed in the pleura, were later found to develop in almost any extrapleural site. Dedifferentiation within SFTs was characterized only recently. We report ...a case of dedifferentiated SFT arising within the pelvis of a 70-year-old Japanese woman. Macroscopically, the resected tumor measured 17 × 17 × 13 cm. Histologically, the tumor displayed distinct heterologous osteosarcomatous and chondrosarcomatous components on a background of conventional SFT. Immunohistochemistry uncovered a loss of CD34 expression in the dedifferentiated area, whereas the nuclear expression of signal transducer and activator of transcription-6 (
STAT6
) and NGFI-A-binding protein 2 (
NAB2
) was maintained in both components. The
p53
mutation 158 CGC > CAC (A158H) was found only in the dedifferentiated component. Furthermore, a fusion gene of
NAB2(exon6)-STAT6(exon18)
was detected in both the conventional and dedifferentiated components. The patient died of the disease 4 months after surgery. This case identifies a possible role of
p53
dysfunction in the dedifferentiation process of SFT as reported in other sarcomas.
Adipophilin, a lipid droplet-associated protein that regulates lipid droplet structure and formation, is expressed in a wide variety of tumors. The aims of this study were to evaluate the frequency ...and distribution pattern of adipophilin expression in gastric epithelial neoplasia and to correlate these variables with clinicopathological features and the mucin phenotype. We retrospectively examined 159 cases of gastric epithelial neoplasia, which were classified according to the Vienna classification system as 52 noninvasive low-grade adenoma (category 3), 65 noninvasive high-grade neoplasia (category 4), and 42 invasive neoplasia (category 5). Immunohistochemistry for adipophilin was performed, and phenotypic marker expression was determined by immunohistostaining with MUC2, MUC5AC, CD10, MUC6, and villin. Adipophilin was expressed in 41 of the 52 (79 %) category 3 cases, in 42 of the 65 (65 %) category 4 cases, and in 23 of the 42 (55 %) category 5 cases. Expression of adipophilin was only present in lesions with complete or incomplete intestinal phenotypes. Adipophilin was expressed more frequently in the surface epithelium in category 3, whereas there was a stepwise increase in cryptal staining of adipophilin from category 3 to category 5. In conclusion, adipophilin expression is closely related to the intestinal differentiation of the tumor. The pattern of immunostaining for adipophilin might be a useful new marker for discriminating adenomas from adenocarcinomas.
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