Progressive changes in the protein expression profile of alveolar septa in early-stage lung adenocarcinoma.

BACKGROUND: Adenocarcinomas show a stepwise progression from atypical adenomatous hyperplasia (AAH) through adenocarcinoma in situ (AIS) to invasive adenocarcinoma (IA). Immunoglobulin superfamily containing leucine-rich repeat (ISLR) is a marker of tumor-restraining cancer-associated fibroblasts (CAFs), which are distinct from conventional, strongly α-smooth muscle actin (αSMA)-positive CAFs. Fibroblast activation protein (FAP) has been focused on as a potential therapeutic and diagnostic target of CAFs. METHODS: We investigated the changes in protein expression during adenocarcinoma progression in the pre-existing alveolar septa by assessing ISLR, αSMA, and FAP expression in normal lung, AAH, AIS, and IA. Fourteen AAH, seventeen AIS, and twenty IA lesions were identified and randomly sampled. Immunohistochemical analysis was performed to evaluate cancer-associated changes and FAP expression in the pre-existing alveolar structures. RESULTS: Normal alveolar septa expressed ISLR. The ISLR level in the alveolar septa decreased in AAH and AIS tissues when compared with that in normal lung tissue. The αSMA-positive area gradually increased from the adjacent lung tissue (13.3% ± 15%) to AIS (87.7% ± 14%), through AAH (70.2% ± 21%). Moreover, the FAP-positive area gradually increased from AAH (1.69% ± 1.4%) to IA (11.8% ± 7.1%), through AIS (6.11% ± 5.3%). Protein expression changes are a feature of CAFs in the pre-existing alveolar septa that begin in AAH. These changes gradually progressed from AAH to IA through AIS. CONCLUSIONS: FAP-positive fibroblasts may contribute to tumor stroma formation in early-stage lung adenocarcinoma, and this could influence the development of therapeutic strategies targeting FAP-positive CAFs for disrupting extracellular matrix formation.

KMT2A-rearranged sarcoma with unusual fusion gene CBX6::KMT2A::PYGO1.

Recently, rare sarcomas harboring KMT2A rearrangements have been reported. They occur in relatively young individuals, exhibit a sclerosing epithelioid fibrosarcoma-like morphology, and often have an aggressive prognosis. YAP1::KMT2A::YAP1 is the most common fusion gene, followed by VIM::KMT2A. We report the case of a 47-year-old man with a spindle cell tumor arising from the subcutaneous tissue of the right anterior chest. The tumor harbored an unusual novel fusion gene, CBX6::KMT2A::PYGO1. Histologically, the tumor consisted of proliferating spindle-shaped cells with uniform nuclei, which varied in cell density and the amount of intervening collagen fibers. After 2 years and 8 months without postoperative treatment, the patient showed no recurrence or metastasis. Although highly likely irreproducible, tumors with the CBX6::KMT2A::PYGO1 fusion gene were morphologically somewhat different from those containing the YAP1::KMT2A::YAP1. This suggests that KMT2A rearrangements with fusion gene partners different from YAP1 result in purely spindle-shaped cell tumors that produce collagen fibers.

Secondary leiomyosarcoma of the nasal cavity in a treated patient with possible hereditary retinoblastoma with germline reciprocal translocation of RB1 and DMXL1 and somatic TP53 mutation: A case report.

Retinoblastoma is a common primary intraocular malignant tumor that affects infants and young children. Radiation therapy for hereditary retinoblastoma increases the risk of secondary malignancy. The present report discusses the case of a retinoblastoma survivor who developed secondary leiomyosarcoma 42 years after receiving radiation therapy. The retinoblastoma of the patient was unilateral, and the patient had no family history of the disease. RNA and DNA panel sequencing of the leiomyosarcoma tissue was performed to elucidate the molecular mechanism of this secondary malignancy. The RNA panel sequencing detected a germline reciprocal translocation of RB1 and DMXL1, leading to a diagnosis of possible hereditary retinoblastoma. Furthermore, it detected a somatic fusion gene (RAD51-KNL1). The DNA panel sequencing identified various germline or somatic variants, including a somatic splice acceptor site mutation of TP53. We hypothesized that the molecular mechanism of the secondary malignancy of this patient was the combination of a germline reciprocal translocation of RB1 and DMXL1 and the accumulation of various somatic mutations containing the splice acceptor site mutation of TP53, which ultimately led to the development of a secondary leiomyosarcoma. Further prospective investigations are necessary to fully understand the role of reciprocal translocation of RB1 and DMXL1 or other mutations in the tumorigenesis of second malignancies in patients with hereditary retinoblastoma.

KDM2B-Rearranged Soft Tissue Sarcomas Expand the Concept of BCOR-Associated Sarcoma.

Sarcomas with BCOR genetic alterations (BCOR-associated sarcomas) represent a recently recognized family of soft tissue and bone tumors characterized by BCOR fusion, BCOR internal tandem duplication, or YWHAE::NUTM2B fusion. Histologically, the tumors demonstrate oval to spindle cell proliferation in a variably vascular stroma and overexpression of BCOR and SATB2. Herein, we describe 3 soft tissue sarcomas with KDM2B fusions that phenotypically and epigenetically match BCOR-associated sarcomas. The cases included 1 infant, 1 adolescent, and 1 older patient. All tumors showed histologic findings indistinguishable from those of BCOR-associated sarcomas and were originally diagnosed as such based on the phenotype. However, none of the tumors had BCOR or YWHAE genetic alterations. Instead, targeted RNA sequencing identified in-frame KDM2B::NUTM2B, KDM2B::CREBBP, and KDM2B::DUX4 fusions. KDM2B fusions were validated using reverse-transcription PCR, Sanger sequencing, and in situ hybridization assays. Genome-wide DNA methylation analysis matched all 3 tumors with BCOR-associated sarcomas using the Deutsches Krebsforschungszentrum (DKFZ) classifier and t-distributed stochastic neighbor embedding analysis. One localized tumor showed a flat genome-wide copy number profile, and the patient remained disease-free after treatment. The other tumors showed multiple copy number alterations, including MDM2/CDK4 amplification and/or CDKN2A/B loss, and both tumors metastasized, leading to the patient's death in one of the cases. When tested using KDM2B immunohistochemistry, all 3 KDM2B-rearranged sarcomas showed diffuse strong staining, and all 13 sarcomas with BCOR genetic alterations also demonstrated diffuse, strong, or weak staining. By contrast, among 72 mimicking tumors, only a subset of synovial sarcomas showed focal or diffuse weak KDM2B expression. In conclusion, our study suggests that KDM2B-rearranged soft tissue sarcomas belong to the BCOR-associated sarcoma family and expand its molecular spectrum. This may be related to the known molecular relationship between KDM2B and BCOR in the polycomb repressive complex 1.1. Immunohistochemical analysis of KDM2B is a potentially valuable diagnostic tool for BCOR-associated sarcomas, including those with KDM2B rearrangement.

Genetic dissection of intratumor heterogeneity of PD-L1 expression in EGFR-mutated lung adenocarcinoma.

In this study, we investigated the association between PD-L1 expression in tumor cells and underlying genetic mutations, which was analyzed in detail using laser microdissection and next-generation sequencing analysis. To investigate whether driver mutations are involved in the background of PD-L1 expression, the EGFR major activating mutation was selected as the most frequent driver mutation. Surgical resection specimens were used to extract sufficient amounts of nucleic acids for analysis, and the high tumor proportion score (TPS:100%) and low (TPS: 0%) PD-L1-expressing parts of the tumor were each laser microdissected to examine the association between PD-L1 expression heterogeneity and genetic mutations within the same tumor. The association between PD-L1 heterogeneity and gene mutations within the same tumor was investigated. Analysis showed no association between PD-L1 expression heterogeneity and genetic variants, which were found to be almost identical. However, PD-L1 expression was found to be associated with the number of tumor infiltrating lymphocytes (TILs) present in the tumor, which may be related to whether or not lymphocytes can infiltrate into the tumor depending on the tumor histological type (solid pattern, lepidic pattern, etc.) and other factors.

Analytical Performance of a Highly Sensitive System to Detect Gene Variants Using Next-Generation Sequencing for Lung Cancer Companion Diagnostics.

The recent increase in the number of molecular targeted agents for lung cancer has led to the demand for the simultaneous testing of multiple genes. Although gene panels using next-generation sequencing (NGS) are ideal, conventional panels require a high tumor content, and biopsy samples often do not meet this requirement. We developed a new NGS panel, called compact panel, characterized by high sensitivity, with detection limits for mutations of 0.14%, 0.20%, 0.48%, 0.24%, and 0.20% for EGFR exon 19 deletion, L858R, T790M, BRAF V600E, and KRAS G12C, respectively. Mutation detection also had a high quantitative ability, with correlation coefficients ranging from 0.966 to 0.992. The threshold for fusion detection was 1%. The panel exhibited good concordance with the approved tests. The identity rates were as follows: EGFR positive, 100% (95% confidence interval, 95.5-100); EGFR negative, 90.9 (82.2-96.3); BRAF positive, 100 (59.0-100); BRAF negative, 100 (94.9-100); KRAS G12C positive, 100 (92.7-100); KRAS G12C negative, 100 (93.0-100); ALK positive, 96.7 (83.8-99.9); ALK negative, 98.4 (97.2-99.2); ROS1 positive, 100 (66.4-100); ROS1 negative, 99.0 (94.6-100); MET positive, 98.0 (89.0-99.9); MET negative 100 (92.8-100); RET positive, 93.8 (69.8-100); RET negative, 100 (94.9-100). The analytical performance showed that the panel could handle various types of biopsy samples obtained by routine clinical practice without requiring strict pathological monitoring, as in the case of conventional NGS panels.

Cutaneous syncytial myoepithelioma with folliculocentric growth and EWSR1 gene rearrangement: A case report.

Cutaneous syncytial myoepithelioma is a tumor type that was initially reported in 2013 as a syncytial variant of cutaneous myoepithelioma characterized by intradermal nodular proliferation of oval to spindle-shaped tumor cells in solid and syncytial patterns. Fusion of genes Ewing sarcoma breakpoint region 1 / EWS RNA binding protein 1 (EWSR1) and pre-B cell leukemia homeobox 3 (PBX3) is found in approximately 90% of the cases. We report a case of cutaneous syncytial myoepithelioma with diagnostic difficulty due to folliculocentric morphology and atypical immunohistochemical results, including diffuse positivity of α-smooth muscle actin and claudin 4 and negative immunoreactions for epithelial membrane antigen and S100 protein. In the present case, fluorescence in situ hybridization study demonstrated EWSR1 rearrangement. We further provide a discussion of differential diagnoses with a review of relevant literature.

Prevalence and characteristics of immune checkpoint inhibitor-related myocardial damage: A prospective observational study.

An increasing number of patients with cancer are being treated with immune checkpoint inhibitors. Consequently, the incidence of immune checkpoint inhibitor-related myocarditis has been increasing. Nonetheless, the diagnostic criteria for the immune checkpoint inhibitor-related myocarditis have not been sufficiently established. Therefore, the real-world incidence or prevalence of immune checkpoint inhibitor-related myocardial damage remains unknown. This was a single-center cohort study that included 100 patients admitted for immune checkpoint inhibitor therapy for any type of cancer. The patients underwent monthly measurement of cardiac troponin I and N-terminal pro-brain natriuretic peptide levels with electrocardiography. Additionally, echocardiography was performed every 3 months. Our protocol was continued until 6 months after the initiation of immune checkpoint inhibitors. We defined immune checkpoint inhibitor-related myocardial damage as an increase in cardiac troponin I levels by >0.026 ng/mL and/or a decrease in the left ventricular ejection fraction by >10% to <53% on echocardiography. The mean patient age was 64 years; 71% were men. The most commonly used immune checkpoint inhibitor was nivolumab (47%), followed by pembrolizumab (29%). Overall, 5% of patients received combination therapy. Among 100 patients, 10 (10%) were diagnosed with immune checkpoint inhibitor-related myocardial damage. Among them, five patients underwent endomyocardial biopsy. Of these patients, four were histopathologically observed to have lymphocyte infiltration in their myocardium. In conclusion, serial cardiac troponin I measurement during immune checkpoint inhibitor treatment could help detect early-phase myocardial damage. The prevalence of myocardial damage was much higher than previously expected.

TP53 Loss of Heterozygosity Induces De Novo SCLC Formation in EGFR-Mutated Lung Adenocarcinoma: A Case Report.

SCLC transformation in EGFR-mutated lung adenocarcinoma is one of the major phenotypic changes that is observed during the resistance to EGFR tyrosine kinase inhibitors. However, the mechanism of this transformation remains unclear. In this study, we found a small de novo SCLC component in surgically resected specimens of EGFR-mutated lung adenocarcinoma before EGFR tyrosine kinase inhibitor treatment. By using laser microdissection and whole-exome sequencing, TP53 loss of heterozygosity was found to be possibly involved in SCLC transformation.

A novel EWSR1-HOXB13 rearrangement in a fibroblastic tumor from the abdomen of a young woman.

We describe a novel EWSR1-HOXB13-fusion in a fibroblastic tumor from the abdominal wall of a 29-year-old woman. This tumor caused intermittent intense pain and had grown to approximately 5 cm in size over two years. The tumor was located beneath subfascial section of the abdominal wall and was invading the abdominal cavity and pressing on the liver. The tumor was well-circumscribed and consisted of intersected fascicles of monomorphic spindle-shaped cells with uniform ovoid nuclei lacking nuclear pleomorphism or mitotic activity. This tumor was immunohistochemically negative for pan-cytokeratin AE1/AE3, desmin, SMA, S100, myogenin, MyoD1, CD34, melanosome, SOX10, STAT6, SS18-SSX, and ERG. H3K27me3 was retained. RNA sequencing revealed a unique EWSR1-HOXB13-fusion, and strong, diffuse nuclear immunostaining for HOXB13 was observed. No local recurrence or evident distant metastasis were observed over eight months without chemotherapy, implying that the behavior of this tumor is not yet known.

Primary Epithelioid Rhabdomyosarcoma of the Liver: Case Report of an Unusual Histological Variant.

Rhabdomyosarcoma (RMS) is a soft tissue tumor with striated muscle cell differentiation. It mostly occurs in children. While it can affect any part of the body, it commonly involves the urogenital organs, head and neck including the parameninges and orbit, and limbs. We describe an adult case of primary epithelioid RMS of the liver. A 71-year-old man presented with a 5.6 cm liver mass. Tumor histology revealed diffuse proliferation of small epithelioid cells and focal spindle cells. The tumor cells were immunohistochemically positive for myogenin (positive ratio 30%), desmin, Myo D1, and CD56. The tumor weakly expressed MDM2 and did not express CDK4. This suggested that dedifferentiated liposarcoma with a rhabdomyosarcomatous component was unlikely. There was no fusion gene of PAX3-FKHR or PAX7- FKHR to indicate alveolar RMS by RT-PCR. Subsequently, RNA Pan-Cancer Targeted sequencing was performed for 1385 genes revealed a single base substitution (c.742C>T) in TP53 that changes an amino acid (p.Arg248Trp). No fusion gene was found. No other primary RMS lesions were detected aside from the liver lesion. The tumor was diagnosed as a primary epithelioid RMS of the liver. His RMS already metastasized to the lymph nodes of the entire body. The patient declined further therapy and died one year later. This was the first case report of a primary epithelioid RMS of the liver.

Long-term survival in thymic carcinoma with postoperative pleural dissemination.

BACKGROUND: We report a patient with thymic squamous cell carcinoma who underwent multiple rounds of surgical resection and definitive radiotherapy for both primary tumor and postoperative recurrence. However, the patient remains well and healthy 18 years after initial diagnosis. Since long-term survival after postoperative recurrence of thymic carcinoma is extremely rare, we also present her immunohistochemical staining results, which suggested indolent disease. CASE PRESENTATION: A 42-year-old woman with thymic squamous cell carcinoma underwent en bloc resection of the tumor and thymus gland. Pleural dissemination was noted in the right thoracic cavity 3, 10, and 16 years postoperatively. Where possible, the nodules were resected surgically: during the postoperative 3rd and 16th years. Definitive radiotherapy was administered for all nodules that could not be excised during the postoperative 3rd and 10th years. Disease-free survival is 25 months. CONCLUSIONS: Local control of pleural dissemination may be beneficial in the treatment of postoperative recurrence of thymic carcinoma in limited cases of indolent disease.

α-Methylacyl-CoA racemase: a useful immunohistochemical marker of breast carcinoma with apocrine differentiation.

Carcinoma with apocrine differentiation is an androgen receptor (AR)-positive subset of triple-negative breast carcinomas. In addition to carcinoma with apocrine differentiation, other AR-positive triple-negative breast carcinomas occur, albeit less frequently. We found that α-methylacyl-CoA racemase (AMACR), also known as P504S, is overexpressed in carcinoma with apocrine differentiation and non-neoplastic apocrine metaplasia. We aimed to evaluate AMACR as a possible marker of carcinoma with apocrine differentiation. We immunohistochemically examined the expression of AMACR in carcinoma with apocrine differentiation and nonapocrine carcinomas and compared it with that of gross cystic disease fluid protein-15 (GCDFP-15). In total, 212 breast carcinomas were evaluated: 39 carcinomas with apocrine differentiation, 28 ductal carcinomas in situ with apocrine morphology (ADCIS), and 145 nonapocrine breast carcinomas. AMACR was expressed in 38 of 39 (97.4%) carcinomas with apocrine differentiation and in 27 of 28 (96.4%) ADCIS, consistent with the expression of GCDFP-15. However, in nonapocrine carcinomas, AMACR expression was observed in 32 of 145 (22.0%) lesions, whereas GCDFP-15 expression was observed in 91 of 145 (62.7%) lesions. For carcinoma with apocrine differentiation, AMACR was as sensitive as GCDFP-15 (both 97.1%) but more specific (77.9% versus 37.2%). In selected cases, AMACR messenger RNA (mRNA) levels were quantitatively determined relative to that of TATA-binding protein mRNA, and they comprised 5.23, 1.33, and 0.60 for carcinomas with apocrine differentiation, nonapocrine carcinomas, and normal breast tissue, respectively. CONCLUSION: Our findings demonstrate that AMACR expression may be used for differentiating carcinoma with apocrine differentiation from nonapocrine carcinomas and indicate that AMACR is a more sensitive carcinoma with apocrine differentiation marker than GCDFP-15.

Atezolizumab with bevacizumab, paclitaxel and carboplatin was effective for patients with SMARCA4-deficient thoracic sarcoma.

SMARCA4-deficient thoracic sarcoma (DTS) is a recently noted progressive thoracic malignancy. We recently experienced three cases of SMARCA4-DTS who were treated with atezolizumab in combination with bevacizumab, paclitaxel and carboplatin (ABCP) as the first-line therapy. Immunohistopathological analysis revealed absent expression of SMARCA4 in all cases. The tumor mutational burden was over 11/Mb and mutations in SMARCA4 and TP53 were detected in all three cases. Partial response to ABCP treatment was observed in all three cases, with a progression-free survival of approximately 6 months or longer and a continuous response of 1 year or longer in one case. The first-line ABCP treatment demonstrated durable efficacy in SMARCA4-DTS regardless of the degree of PD-L1 expression.

Lay abstract Lung cancer is the leading cause of cancer-related death worldwide. Among them, SMARCA4-deficient thoracic sarcoma (DTS), which lacks SMARCA4 expression and exhibits an undifferentiated carcinoma histology, is a recently identified subtype of lung cancer. It tends to occur in younger people with heavy smoking status and has been reported to recur quickly and have a poor prognosis even after chemotherapy, radiation therapy or surgery. There is no effective molecularly targeted agent for SMARCA4-DTS and the identification of an effective therapy is required. Here, we report the clinical features and genomic information of three SMARCA4-DTS cases in which atezolizumab with bevacizumab, paclitaxel and carboplatin treatment was effective. This report suggests the efficacy of atezolizumab with bevacizumab, paclitaxel and carboplatin treatment compared with conventional chemotherapy.

Primary breast angiosarcoma with disseminated intravascular coagulation is successfully treated with self-subcutaneous unfractionated heparin calcium injection: A case report.

Angiosarcoma is a rare sarcoma with a poor prognosis and is prone to disseminated intravascular coagulation (DIC), where DIC often interferes with chemotherapy. Primary angiosarcoma of the breast (PASB) is a type of angiosarcoma that is located in mammary parenchyma and is not associated with radiation exposure. The current study reported a 47-year-old female with DIC associated with PASB. The DIC of the patient relapsed during mono-chemotherapy with paclitaxel (PTX) after first-line anticoagulant therapy using thrombomodulin-α. The second-line danaparoid sodium therapy, followed by self-subcutaneous injection of unfractionated heparin calcium (UFH), resulted in long-term stabilization of DIC. Under this second-line anticoagulant therapy, the patient continued chemotherapy and chemoradiotherapy for >13 months in the outpatient setting without impairment of quality of life. The present case suggested that self-subcutaneous injections of UFH may be a useful therapeutic option for long-term control of DIC associated with PASB. However, further prospective clinical trails are needed to verify the efficacy of self-subcutaneous injection of UFH in similar settings.