Candidate tumor-specific CD8+ T cell subsets identified in the malignant pleural effusion of advanced lung cancer patients by single-cell analysis.

Isolation of tumor-specific T cells and their antigen receptors (TCRs) from malignant pleural effusions (MPE) may facilitate the development of TCR-transduced adoptive cellular immunotherapy products for advanced lung cancer patients. However, the characteristics and markers of tumor-specific T-cells in MPE are largely undefined. To this end, to establish the phenotypes and antigen specificities of CD8+ T cells, we performed single-cell RNA and TCR sequencing of samples from three advanced lung cancer patients. Dimensionality reduction on a total of 4,983 CD8+ T cells revealed 10 clusters including naïve, memory, and exhausted phenotypes. We focused particularly on exhausted T cell clusters and tested their TCR reactivity against neoantigens predicted from autologous cancer cell lines. Four different TCRs specific for the same neoantigen and one orphan TCR specific for the autologous cell line were identified from one of the patients. Differential gene expression analysis in tumor-specific T cells relative to the other T cells identified CXCL13, as a candidate gene expressed by tumor-specific T cells. In addition to expressing CXCL13, tumor-specific T cells were present in a higher proportion of T cells co-expressing PDCD1(PD-1)/TNFRSF9(4-1BB). Furthermore, flow cytometric analyses in advanced lung cancer patients with MPE documented that those with high PD-1/4-1BB expression have a better prognosis in the subset of 57 adenocarcinoma patients (p = .039). These data suggest that PD-1/4-1BB co-expression might identify tumor-specific CD8+ T cells in MPE, which are associated with patients' prognosis. (233 words).

Novel gene fusions in human oropharyngeal carcinoma.

Few reports have analyzed the fusion genes involved in carcinogenesis in the oropharynx, where the incidence of human papillomavirus-associated tumors is relatively low. The aim of this study was to identify novel driver fusion genes in patients with oropharyngeal cancer. The study enrolled fifty-seven patients who were diagnosed with oropharyngeal carcinoma. RNA sequencing data from fresh-frozen specimens were used to identify candidate fusion genes via the JAFFA, arriba, and STAR-Fusion pipelines. Candidate fusion genes were confirmed by direct sequencing. The expression level of a candidate fusion gene was compared to that of tumors without fusion genes. Finally, filtering was performed for driver genes using the annoFuse pipeline. In addition, the VIRTUS pipeline was used to analyze the presence of human papillomavirus in the tumors. We identified 5 (8.8 %) novel potential driver in-frame fusion genes, MKNK2::MOB3A, ICMT::RPS6KA3, ATP1B3::GRK7, CSNK2A1::KIF16B, and FGFR3::MAEA, and 1 (1.8 %) known in-frame fusion gene, FGFR3::TACC3, in 57 patients with pharyngeal carcinoma. Our results suggest that sporadic fusion genes may contribute to tumorigenesis in oropharyngeal carcinomas.

Clinicopathological importance of Bcl-2 and p53 in postmenopausal triple-negative breast carcinoma and association with age.

Appropriate biomarkers are required to predict the clinical outcome of triple-negative breast cancer (TNBC). In this study, we focused on the clinical importance of two representative tumor-associated proteins, Bcl-2 and p53. Bcl-2 expression is usually related to estrogen receptor expression and a favorable outcome in breast cancer. TNBC has been reported to show a high frequency of p53 positivity suggesting TP53 mutations. The expressions of Bcl-2 and p53 were immunohistochemically examined in TNBC involving two age groups of postmenopausal women (≥75 y/o, n = 75; 55-64 y/o, n = 47), who underwent surgery without neoadjuvant therapy. We examined their associations with each other, or with clinicopathological factors including the outcome. Bcl-2 expression was inversely correlated with androgen receptor, apocrine morphology, and p53 expressions, and was an independent predictor of a poor outcome in total or in younger women. p53 positivity was associated with a more favorable outcome than p53 negativity in the younger group. In combined analyzes, none of the twenty Bcl-2-negative/p53-positive cases in the younger group exhibited recurrence, resulting in the independent favorable predictive value of Bcl-2-negative/p53-positive. The anti-apoptotic nature of Bcl-2 may be apparent in TNBC. The excellent outcome of Bcl-2-negative/p53-positive cases in the younger group warrants further combined investigation of Bcl-2/p53 in TNBC.

Genomic landscape of glioblastoma without IDH somatic mutation in 42 cases: a comprehensive analysis using RNA sequencing data.

PURPOSE: Glioblastoma is a malignant brain tumor with a poor prognosis. Genetic mutations associated with this disease are complex are not fully understood and require further elucidation for the development of new treatments. The purpose of this study was to comprehensively analyze genetic mutations in glioblastomas and evaluate the usefulness of RNA sequencing. PATIENTS AND METHODS: We analyzed 42 glioblastoma specimens that were resected in routine clinical practice and found wild-type variants of the IDH1 and IDH2 genes. RNA was extracted from frozen specimens and sequenced, and genetic analyses were performed using the CLC Genomics Workbench. RESULTS: The most common genetic alterations in the 42 glioblastoma specimens were TP53 mutation (28.6%), EGFR splicing variant (16.7%), EGFR mutation (9.5%), and FGFR3 fusion (9.5%). Novel genetic mutations were detected in 8 patients (19%). In 12 cases (28.6%), driver gene mutations were not detected, suggesting an association with PPP1R14A overexpression. Our findings suggest the transcription factors SOX10 and NKX6-2 are potential markers in glioblastoma. CONCLUSION: RNA sequencing is a promising approach for genotyping glioblastomas because it provides comprehensive information on gene expression and is relatively cost-effective.

Trastuzumab deruxtecan for human epidermal growth factor receptor 2-low advanced or metastatic breast cancer: recommendations from the Japanese Breast Cancer Society Clinical Practice Guidelines.

The Japanese Breast Cancer Society Clinical Practice Guidelines are published as timely guidance on clinical issues in breast cancer treatment in Japan. In the recent edition of these guidelines, we addressed a new clinical question 34 (CQ 34, systemic treatment part) "Is trastuzumab deruxtecan recommended for patients with unresectable or metastatic HER2-low breast cancer?" and a new future research question 7 (FRQ 7, pathological diagnosis part) "How is HER2-low breast cancer diagnosed for the indication of trastuzumab deruxtecan?". These questions address use of trastuzumab deruxtecan in patients with unresectable or metastatic HER2-low breast cancer who have previously received chemotherapy for metastatic disease. The strengths of evidence and recommendation were determined through a quantitative and qualitative systematic review using multiple outcomes, including efficacy and safety. We conclude that trastuzumab deruxtecan is recommended for this patient population (strength of recommendation: 1; strength of evidence: moderate; CQ34) and that HER2-low expression for the indication of trastuzumab deruxtecan should be diagnosed using companion diagnostics based on appropriate criteria (FRQ7).

CD30 expression in an emerging group of mesenchymal spindle cell neoplasms with ALK fusion detected by flow cytometry and immunohistochemistry.

An emerging group of spindle cell neoplasms harboring fusions involving NTRK or non-NTRK kinase genes often share characteristic S100 and/or CD34 expression; however, the diagnostic utility of immunohistochemical stains is not well established in this family owing to their lack of specificity. Recently, CD30 expression in spindle cell neoplasms with kinase gene fusions, such as NTRK, BRAF, RAF1, and RET, has been increasingly identified. We herein report a 10-year-old girl with high-grade spindle cell sarcoma of the neck. Prior to histopathological evaluation, flow cytometry (FCM) analysis and touch smear cytology of the tumor tissue revealed CD34+ and dimCD30+ spindle cell populations. Histopathologically, the case was characterized by monomorphic spindle-shaped cytomorphology with CD30, S100, and CD34 positivity and harbored close similarities with spindle cell neoplasms with NTRK or non-NTRK gene fusions. Subsequently, a comprehensive next-generation sequencing sarcoma panel identified a rare PLEKHH2::ALK fusion, and a diagnosis of ALK-rearranged spindle cell neoplasm was made. The patient showed significant tumor response to single-agent treatment with alectinib, an ALK-tyrosine kinase inhibitor. This case supports that CD30 is expressed in an ALK-rearranged mesenchymal neoplasm. The benefit of the early detection of CD30 expression by FCM for a prompt diagnosis and treatment is highlighted in the context of an aggressive clinical course. This case represents a learning experience regarding the need to the check the status of CD30 expression in these tumors and suggests the potential clinical benefits of CD30-targeted therapy.

Current diagnosis and treatment of salivary gland-type tumors of the lung.

Salivary gland-type tumors of the lung are thought to originate from the submucosal exocrine glands of the large airways. Due to their rare occurrence, reports of their study are limited to small-scale or case reports. Therefore, daily clinical practices often require a search for previous reports. In the last 20 years, several genetic rearrangements have been identified, such as MYB::NF1B rearrangements in adenoid cystic carcinoma, CRTC1::MAML2 rearrangements in mucoepidermoid carcinoma, EWSR1::ATF1 rearrangements in hyalinizing clear cell carcinoma and rearrangements of the EWSR1 locus or FUS (TLS) locus in myoepithelioma and myoepithelial carcinoma. These molecular alterations have been useful in diagnosing these tumors, although they have not yet been linked to molecularly targeted therapies. The morphologic, immunophenotypic, and molecular characteristics of these tumors are similar to those of their counterparts of extrapulmonary origin, so clinical and radiologic differential diagnosis is required to distinguish between primary and metastatic disease of other primary sites. However, these molecular alterations can be useful in differentiating them from other primary lung cancer histologic types. The management of these tumors requires broad knowledge of the latest diagnostics, surgery, radiotherapy, bronchoscopic interventions, chemotherapy, immunotherapy as well as therapeutic agents in development, including molecularly targeted agents. This review provides a comprehensive overview of the current diagnosis and treatment of pulmonary salivary gland tumors, with a focus on adenoid cystic carcinoma and mucoepidermoid carcinoma, which are the two most common subtypes.

Expression of CD5 in salivary gland tumors: an ancillary marker for carcinoma showing thymus-like differentiation (CASTLE) of the major salivary gland.

Recently, cases of carcinoma showing thymus-like differentiation (CASTLE) occurring in major salivary glands have been identified. To assess the diagnostic value of CD5 immunohistochemistry in distinguishing salivary CASTLE from other types of salivary gland tumors, we evaluated CD5 expression in 109 salivary gland tumors, encompassing 23 different histological types, including salivary CASTLE. In addition, we reviewed 10 previously reported cases of salivary CASTLE. Most salivary CASTLE cases (10/11, 91%) showed strong CD5 expression. In contrast, 104 of 108 (96%) non-salivary CASTLE tumors were negative for CD5, while the remaining four tumors (3.7%), all of which were histologically Warthin tumors, showed focal positivity for CD5 with weak to moderate intensity. In conclusion, the findings in this study support the potential use of CD5 immunohistochemistry for distinguishing salivary CASTLE from other histological types of salivary gland tumors. Aberrant CD5 expression in this tumor may be linked to the tumor microenvironment.

Indications for sentinel lymph node biopsy in node-negative oral cancers.

BACKGROUND: We aimed to define the indications for sentinel lymph node biopsy (SLNB), the third option for cervical treatment in oral cancer with negative cervical lymph nodes. METHODS: The greatest depth of invasion (DOI) and long diameter (LD) of the primary site were used as exposures. SLN metastasis was considered the outcome. RESULTS: In three trials conducted between 2009 and 2016, 158 patients were eligible and reassigned to this study group. The scatterplot based on the respective values of DOI and LD would eventually be divided into three sections. In cases of sections T1, T2, and T3, the proportions of SLN metastasis positivity were 21.3%, 35.3%, and 51.2%, respectively. In certain cases of T1 with 2 mm < DOI ≤ 5 mm and 8 mm < LD ≤ 20 mm, the proportion of SLN metastasis positivity was 40.9%. CONCLUSIONS: SLNB-navigated or assisted neck dissection can be added as an effective procedure for N0 neck control.

Multiple bronchiolar adenomas/ciliated muconodular papillary tumors of the bilateral lung with tumor budding and potential malignant transformation into squamous cell carcinoma: a case report and literature review.

Background: Bronchiolar adenoma (BA)/ciliated muconodular papillary tumor (CMPT) is a rare lung tumor characterized by ciliated, mucous and basal cells. Recently, some cases of driver mutations or malignant transformations have been reported. However, the nature of BA/CMPT remains controversial. Here, we report a case of bilateral pulmonary multiple BAs with tumor budding and squamous metaplasia. Case Description: A 55-year-old man presented with multiple small nodules in the lower lobes of the bilateral lungs on physical examination 7 years prior. During the past 3 years of regular follow-up, some nodules had slightly enlarged. Because the nodules were mostly solid, the patient underwent video-assisted thoracoscopic segmentectomy of the left lower lung. A postoperative pathological diagnosis of BA was made. In all lesions, the fusion and mutation of major driver genes were not detected by next-generation sequencing (NGS). No recurrence or metastasis was observed after 37 months of follow-up. Notably, all five resected lesions were BA/CMPT, and one lesion was accompanied by squamous metaplasia and tumor budding. Conclusions: Our report found that BA/CMPT with squamous metaplasia and tumor budding has the potential to transform into lung squamous cell carcinoma, expanding its connection with malignant transformation. Smoking may be one of the risk factors. We also found that BA/CMPT can be multiple lesions rather than a solitary lesion.

Association Between 18F-Fluorodeoxyglucose Uptake and Mutation Status of Epidermal Growth Factor Receptor in Sinonasal Tract Cancer.

BACKGROUND/AIM: The Warburg effect of cancer has been applied to detect various carcinomas though the 18F-fluorodeoxyglucose (18F-FDG) uptake on positron emission tomography with computed tomography (PET/CT). 18F-FDG-PET/CT in lung cancer predicted the mutation status of epidermoid growth factor receptor (EGFR). This study aimed to investigate whether 18F-FDG uptake parameters were significantly related to EGFR mutation status in patients with sinonasal tract squamous cell carcinoma (STSCC). PATIENTS AND METHODS: Twenty-nine tumor specimens of primary STSCC from patients with definitive treatment were collected. RESULTS: The 18F-FDG uptake from primary tumors was not different between mutant- and wild-status of EGFR on either Mann-Whitney U-test or the receiver operating curve. A metabolic tumor volume of ≥25 with the minimum p-value from the log-rank test for STSCC-specific survival was associated with a significantly shorter STSCC-specific, disease-free, local recurrence-free survival on the univariate and multivariate analyses adjusted for the clinical stage, treatment, and EGFR status. CONCLUSION: 18F-FDG-PET/CT did not predict mutation of the EGFR status in STSCC.

Diagnostic utility of DNA integrity number as an indicator of sufficient DNA quality in next-generation sequencing-based genomic profiling.

OBJECTIVES: DNA integrity number (DIN) is a metric for assessing DNA degradation, calculated based on electrophoresis using the Agilent TapeStation System. The utility of DIN as a diagnostic indicator of sufficient DNA quality in clinical next-generation sequencing (NGS) has not been well described. METHODS: We evaluated the DINs of 166 tumor formalin-fixed, paraffin-embedded (FFPE) tissue samples submitted for 124-gene panel sequencing. We also investigated a new metric on the electropherogram that could improve the predictive accuracy of the DIN. RESULTS: A DIN cutoff of 2.5 discriminated samples with successful analysis (n = 143) from samples with failed analysis (n = 23), with a sensitivity of 0.84 and a specificity of 0.78 (area under the curve [AUC] = 0.88). The DIN was positively correlated with the mean coverage (r = 0.72, P < .0001) but could not discriminate success from failure when the DIN was below 2.5 (negative predictive value, 0.44). We introduced a new metric, the peak/base ratio, that distinguished success from failure with higher accuracy than the DIN (cutoff = 1.6; sensitivity = 0.98, specificity = 0.83, and AUC =0.96). CONCLUSIONS: To predict successful NGS, the DNA quality of FFPE tissue can be easily and reliably assessed using the DIN and peak/base ratio.

Mucous Gland Adenoma of the Lung: A Neoplastic Counterpart of Mucinous Bronchial Glands.

Mucous gland adenoma (MGA) is a rare benign tumor that usually arises in the proximal airway and consists of mucus-secreting cells resembling bronchial glands. Here, we report 2 cases of MGAs and describe their morphologic, immunohistochemical, and molecular profiles in comparison with 19 pulmonary tumors of 5 other histologic types with mucinous cells (invasive mucinous adenocarcinoma, mucoepidermoid carcinoma, mixed squamous cell and glandular papilloma, bronchiolar adenoma/ciliated muconodular papillary tumor, and sialadenoma papilliferum). Two MGAs were found in 1 male patient and 1 female patient, located in the bronchus and trachea, respectively. One MGA was examined by RNA sequencing, and no putative driver mutations (including BRAF, KRAS, and AKT1 mutations) or gene fusions were identified. In another case of MGA, V600E mutations of BRAF and E17K mutations of AKT1 were not detected by allele-specific real-time PCR or digital PCR, respectively. However, a gene expression analysis revealed that the MGA presented a specific RNA expression profile with multiple genes enriched in the salivary gland. The gene expression of NKX3.1 was significantly higher in the MGA case in comparison to normal control lungs (P < .001). We then examined NKX3.1 immunohistochemistry for 2 MGAs and 19 tumors of 5 other histologic types. NKX3.1 was positive in MGA (2/2, 100%), whereas all constituent cells, including mucinous cells, were negative for NKX3.1 in other histologic types (0%, 0/19). In normal lung tissue, NKX3.1 was positive for mucinous acinar cells of the bronchial glands. In conclusion, the gene expression profile, taken together with the histologic similarity between MGA and bronchial glands, and the preferred location of the tumors (proximal airways with submucosal glands) suggest that MGA is a neoplastic counterpart of mucinous bronchial glands. NKX3.1 immunohistochemistry can be a sensitive and specific ancillary marker that distinguishes MGA from other histologic mimics.

The Absence of Cancer in the Location of a Breast Tissue Marker After Neoadjuvant Chemotherapy may Predict Pathological Complete Response with High Accuracy: Results from a Phase II Trial.

BACKGROUND: It is difficult to determine pathological complete response (pCR) before surgery in clinical complete response (cCR) cases by imaging alone. We designed a prospective study to evaluate whether a breast tissue marker placed in a tumor before neoadjuvant chemotherapy (NAC) can predict a pCR, possibly removing the need for surgery. METHODS: We recruited patients with primary invasive breast cancer assigned to undergo curative surgery and possible NAC. A breast marker (UltraClip®) was placed in the primary tumor before standard NAC. We evaluated the probability of no cancer in the marker but cancer in removed specimens from a cCR group. RESULTS: A total of 102 patients were enrolled. Patients were categorized by cancer stage and subtypes. Seventy-two patients (70.6%) received standard NAC; 23 (34.3%) attained cCR, of whom pCR was obtained in 12 (52.2%). The probability of no cancer in the marker's location but cancer in the removed specimens was 4.3% (95% confidence interval, 0.1-21.9). The false-negative rate was 9.1% (1/11), and the negative predictive value was 92.3% (12/13). In only one case, no cancer was found in the marker's location, but cancer cells were present in the removed specimen. CONCLUSIONS: The absence of cancer in the location of a breast tissue marker after NAC predicted pCR with high accuracy. Therefore, the rebiopsy of a marker's location might mean surgery is unnecessary.

Biological Difference between L858R and Exon 19 Deletion Contributes to Recurrence-Free Survival of Resected Non-Small Cell Lung Cancer.

INTRODUCTION: The differences in biological characteristics among different genotypes of classical EGFR mutations have not been clarified. This study aimed to clarify the clinical and biological differences between L858R and 19 deletion in NSCLC. METHODS: We analyzed a cohort of 191 consecutive cases of surgically resected NSCLC harboring EGFR driver mutations (L858R or 19 deletion) in which curative resection was performed in Aichi Cancer Center Hospital, Nagoya, Japan, from January 2006 to September 2021 and in which recurrence subsequently developed. We also subjected 61 surgically resected NSCLC specimens harboring EGFR driver mutations (L858R or 19 deletion) to an RNA sequencing analysis. RESULTS: In patients with stage I disease, the median time to recurrence did not differ to a statistically significant extent between the types of EGFR mutations; however, among those with stage II and III disease, the median time to recurrence in patients with the L858R genotype tended to be shorter in comparison to those with 19 deletion (log-rank test, p = 0.47 and 0.46, respectively). In comparison to 19 deletion tumors, L858R tumors had higher cytological malignancy (e.g., mitotic ability) and showed stronger immunogenicity. CONCLUSION: L858R and 19 deletion tumors are likely to have a slight difference in the time to recurrence. They suggest that even in EGFR driver tumors, which are treated as the same disease category, the biological characteristics of the tumors are different, which may leave room for innovations in postoperative treatment and treatment at recurrence.

Synchronous driver gene alterations (EGFR L858R, T790M, and ROS1) rearrangements in a patient with early-stage lung adenocarcinoma.

Concurrent EGFR mutation and ROS1 rearrangement is a rare event in early-stage non-small-cell lung cancer. In addition, a co-occurring de novo EGFR T790M mutation in such a case is extremely rare. We encountered a 72-year-old woman who developed 3 early-stage lung lesions synchronously, one each harboring EGFR L858R, ROS1 rearrangement, and EGFR L858R and de novo T790M. These three nodules were pathologically time-matched lepidic predominant adenocarcinoma with small invasive lesions, which may reflect the concept of field cancerization with driver mutations.