STING inhibits the reactivation of dormant metastasis in lung adenocarcinoma.

Metastasis frequently develops from disseminated cancer cells that remain dormant after the apparently successful treatment of a primary tumour. These cells fluctuate between an immune-evasive quiescent state and a proliferative state liable to immune-mediated elimination1-6. Little is known about the clearing of reawakened metastatic cells and how this process could be therapeutically activated to eliminate residual disease in patients. Here we use models of indolent lung adenocarcinoma metastasis to identify cancer cell-intrinsic determinants of immune reactivity during exit from dormancy. Genetic screens of tumour-intrinsic immune regulators identified the stimulator of interferon genes (STING) pathway as a suppressor of metastatic outbreak. STING activity increases in metastatic progenitors that re-enter the cell cycle and is dampened by hypermethylation of the STING promoter and enhancer in breakthrough metastases or by chromatin repression in cells re-entering dormancy in response to TGFß. STING expression in cancer cells derived from spontaneous metastases suppresses their outgrowth. Systemic treatment of mice with STING agonists eliminates dormant metastasis and prevents spontaneous outbreaks in a T cell- and natural killer cell-dependent manner-these effects require cancer cell STING function. Thus, STING provides a checkpoint against the progression of dormant metastasis and a therapeutically actionable strategy for the prevention of disease relapse.

Pulmonary melanocytic nevus - A case report with a mutation analysis of common driver oncogenes.

Nevi are benign melanocytic tumors, and some nevi are considered to develop into malignant melanomas. Most nevi arise in the skin, but nevi occasionally occur in the conjunctiva, esophageal mucosa, or at other sites. Pulmonary melanocytic nevi are extremely rare, and only one case has been reported in the literature. Here, we present a case of pulmonary melanocytic nevus, involving a BRAF gene mutation (V600E), and we discuss the potential significance of this condition as a precursor to pulmonary malignant melanoma.

Update on the potential significance of psammoma bodies in lung adenocarcinoma from a modern perspective.

AIMS: Psammoma bodies are concentrically lamellated microscopic structures made of calcium. They are commonly observed in papillary carcinomas of the thyroid gland and serous papillary adenocarcinomas of the ovary, but are also occasionally detected in lung adenocarcinomas. Only one study, published in 1972, has systematically described the significance of psammoma bodies in lung adenocarcinomas. The aim of this study was to update the significance of psammoma bodies in lung adenocarcinomas from a modern perspective. METHODS AND RESULTS: Psammoma bodies were detected in 7.2% (59/822) of the adenocarcinomas examined, among which the papillary (20.3%, 12/59) and acinar (44.1%, 26/59) histological subtypes, with the feature of a terminal respiratory unit (91.5%, 54/59), were dominant. Malignant potential (cell growth activity measured by Ki67 labelling, lymph node metastasis, and postoperative survival) did not significantly differ between adenocarcinomas with and without psammoma bodies. On the basis of cytogenetic features, adenocarcinomas with psammoma bodies were preferentially affected by tyrosine kinase inhibitor (TKI)-targetable driver mutations [EGFR (69.8%, 37/53), ALK (13.2%, 7/53), and ROS1 (1.9%, 1/53)]. Multivariate analyses confirmed that psammoma bodies may constitute an independent predictor for these mutations, particularly EGFR and ALK mutations. CONCLUSIONS: Psammoma bodies may predict a favourable response of lung adenocarcinomas to TKIs.

A molecular pathological study of four cases of ciliated muconodular papillary tumors of the lung.

Ciliated muconodular papillary tumors (CMPTs) are a recently categorized benign or low-grade malignant neoplasm that develops in the peripheral lung. Only about 40 cases have been reported to date, and the clinicopathological characteristics have yet to be defined in detail. Here, we present four cases of CMPTs with a focus on their immunohistochemical profiles and driver gene mutations. These tumors were a papillary proliferation of a mixture of ciliated, mucous, and basal cells located in the peripheral lung. Ciliated, mucous and basal cells were positive for TTF-1 when using the clone SPT24, but negative for HNF-4α. Basal cells were positive for p40. Mucous cells in some tumors were positive for MUC5AC and MUC6. The Ki-67 index was less than 5%, and strong expression of p53 was not detected. Three of the four tumors had a BRAF (V600E) driver mutation, an EGFR (del E746-T751/S752V) driver mutation, or driver mutations in both EGFR (E709G) and KRAS (G12V). These mutation types are rare for any histological type of lung cancer. The present results confirmed that CMPT is a neoplasm with immunohistochemical features and driver gene mutations that are distinct from those of common lung tumors.

The pathological features of idiopathic interstitial pneumonia-associated pulmonary adenocarcinomas.

AIMS: To investigate the pathological features of idiopathic interstitial pneumonia (IIP)-associated pulmonary adenocarcinoma. METHODS AND RESULTS: Surgically resected adenocarcinomas associated with IIP (the IIP group) and adenocarcinomas without IIP (the non-IIP group) were subjected to analysis. Adenocarcinomas in the IIP group were subdivided into two groups: one group included tumours connected to bronchiolar metaplasia in honeycomb lesions (the H-IIP group), and the other included tumours unrelated to honeycomb lesions (the NH-IIP group). Histomorphological appearance and immunohistochemical expression were compared among the H-IIP group, the NH-IIP group, and the non-IIP group. Most of the tumour cells in the H-IIP group had a tall, columnar shape that showed similar features to proximal bronchial epithelium, whereas tumour cells in the NH-IIP group and the non-IIP group had a club-like shape that showed similar features to respiratory bronchiolar/alveolar epithelium. Adenocarcinomas in the H-IIP group tended to be negative for thyroid transcription factor-1 (TTF-1) and positive for hepatocyte nuclear factor-4α (HNF-4α). The frequency of EGFR mutations was significantly lower in adenocarcinomas in the H-IIP group, although the frequencies of KRAS and ALK mutations did not differ among the three groups. CONCLUSIONS: Idiopathic interstitial pneumonia-associated pulmonary adenocarcinomas, especially those arising from honeycomb lesions, have distinct pathological features.

The potential significance of alpha-enolase (ENO1) in lung adenocarcinomas - A utility of the immunohistochemical expression in pathologic diagnosis.

We herein analyzed the relationships among the immunohistochemical expression of alpha-enolase (ENO1) and clinicopathological factors in order to define the significance of ENO1 in lung adenocarcinomas (ADCs). ENO1 expression was detected in most of the ADCs examined (95.8%), but not in bronchial and alveolar epithelia. ENO1 expression was typically observed in the cytoplasm among most ADCs (95.8%), but was also detected in the nucleus (56.3%). The levels were significantly higher in terminal respiratory unit (TRU) cytological subtype ADCs. Neither cytoplasmic nor nuclear expression was associated with any other clinicopathological factors including post-operative survival and growth activity. These results suggest that ENO1 is a crucial factor promoting neoplastic transformation exclusively in TRU subtype ADCs. We also investigated the potential utility of the immunohistochemical expression of ENO1 to differentiate TRU-type ADC cells from the reactive hyperplasia of pneumocytes and bronchiolar epithelial cells because difficulties are associated with discriminating these lesions in small biopsy specimens. The sensitivity and specificity of ENO1 (cytoplasmic/nuclear) were 87.5%/37.5% and 88.9%/100%, respectively, which are superior to those of p53 (18.8% and 100%). ENO1 has potential as a biomarker to assist in the histopathological detection of TRU subtype ADC cells.

Alterations in cathepsin L expression in lung cancers.

We herein investigated the potential role of cathepsin L in lung carcinogenesis. Lung cancer cell lines and surgically resected tumors were examined for the expression of the cathepsin L protein and copy number alterations in its gene locus. Cathepsin L was stably expressed in bronchiolar epithelial cells. Neoplastic cells expressed cathepsin L at various levels, whereas its expression was completely lost in most of the lung cancer cell lines (63.6%, 7/11) examined. Furthermore, expression levels were lower in a large fraction of lung tumors (69.5%, 139/200) than in bronchiolar epithelia. The expression of cathepsin L was lost in some tumors (16.0%, 32/200). In adenocarcinomas, expression levels were significantly lower in high-grade tumors than in low-grade tumors (one-way ANOVA, P < 0.0500). Copy number alterations were found in 18.0% (36 [32 gain + 4 loss] /200) of lung tumors. No relationship existed between cathepsin L protein expression levels and the copy number of its gene locus (Spearman's rank-order correlation, P = 0.3096). Collectively, these results suggest that the down-regulated expression of cathepsin L, which is caused by an undefined mechanism other than copy number alterations, is involved in the progression of lung adenocarcinomas.

Leptomeningeal melanomatosis associated with neurocutaneous melanosis: an autopsy case report.

An autopsy case of leptomeningeal melanomatosis associated with neurocutaneous melanosis (NCM) involving a 44-year-old male is reported. The autopsy showed that the leptomeningeal surface of the brain and the spinal cord were covered with a diffuse black lesion. A histological examination detected diffusely distributed, proliferating, melanin-containing cells and demonstrated that the lesion consisted of three different components; i.e. regions of melanomatosis, melanocytosis, and melanocyte hyperplasia. In the leptomeningeal melanomatosis component, tumor cells with pleomorphic nuclei and prominent nucleoli had infiltrated into the cerebral parenchyma via Virchow-Robin spaces. The Ki-67 labeling index and the nuclear accumulation of p53 and p16 protein were immunohistochemically examined in each component. The Ki-67 labeling indices of the melanomatosis, melanocytosis, and melanocyte hyperplasia components were 8.7%, 0.8%, and 0%, respectively. Immunostaining of nuclear p16 produced a negative result in the melanomatosis component, but positive results in the melanocytosis and melanocyte hyperplasia components, whereas nuclear p53 expression was not detected in any of the components. This case suggests that p16(INK4) /CDKN2 may play a significant role in progression of leptomeningeal melanocytic neoplasms. We also reviewed previously reported cases of leptomeningeal neoplasms associated with NCM and discussed the relationship between the biological behavior and proliferative activity of such lesions.

The potential role of microRNA-31 expression in early colorectal cancer.

The expression of microRNA-31 (miR-31) has been implicated in the progression of some human malignancies including colorectal cancer. However, the clinical significance of the expression of miR-31 in submucosally invasive (T1) colorectal cancer remains unclear. The aim of the present study was to delineate the relationship between clinicopathological features and the oncogenic modulator miR-31 in submucosally invasive colorectal cancer. We investigated the expression of miR-31 in 50 submucosally invasive colorectal cancer specimens, along with the corresponding non-tumoral mucosa specimens, using a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The relationships between miR-31 expression levels and clinicopathological characteristics were assessed. The miR-31 host gene locus was investigated using fluorescence in situ hybridization. qRT-PCR revealed that the expression of miR-31 was higher in colorectal cancer tissue than in non-tumoral tissue (P = 0.0002). The up-regulated expression of miR-31 may play an oncogenic role in the early stage of carcinogenesis in colorectal cancers.

A case of pulmonary hamartoma with distinctive histopathological features: a discussion of its differential diagnosis and histogenesis.

We herein describe a case of a benign pulmonary tumor with distinctive histopathological features. A 55-year-old Japanese male presented with a well-demarcated tumor in the left upper lobe of his lung, which gradually increased in size from 18 to 21 mm over 24 months. The resected tumor consisted of an epithelial component of compact irregular glands and mesenchymal component of fascicles between the glands. The differentiation of pneumocytes and smooth muscle cells was immunohistochemically detected in the epithelial component and the mesenchymal component, respectively. No mitosis, necrosis, bleeding, or invasion was observed. A histopathologic diagnosis of fibroleiomyomatous hamartoma was made. We also review previously reported tumors with similar histopathological features and discuss their differential diagnosis and histogenesis.

Expression of multidrug resistance 1 gene in B-cell lymphomas: association with follicular dendritic cells.

AIMS: Multidrug resistance (MDR) in B-cell lymphomas still constitutes a major obstacle to the effectiveness of chemotherapy even in the anti-CD20 antibody therapy era. The aim of this study was to investigate the expression of MDR-associated molecules in reactive lymphadenopathy (RL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). METHODS AND RESULTS: The expression of mRNA for ABC-transporter family genes was determined by real-time RT-PCR in lymph nodes from RL, FL, and DLBCL cases. MDR1 exhibited significantly stronger expression in RL, FL, and DLBCL than Raji B-cell lymphoma cells. RL and FL showed significantly higher expression than DLBCL. Immunohistochemically, MDR1 positive cells were localized in the germinal centers of RL and center of the nodular lesions of FL showing associations with CD21 positive follicular dendritic cells (FDCs). Raji cells were co-cultured with FDC sarcoma-derived cells and the expression of MDR1 and drug resistance were analyzed. The co-culture of Raji cells with FDCs induced strong expression of MDR1 and introduced resistance to doxorubicin-induced apoptosis. CONCLUSIONS: These results suggest that FDCs induce MDR1 expression in reactive as well as neoplastic B-cells. Inhibition of the interaction of FDCs with B-cells may provide a novel strategy for treating the chemotherapy resistant fraction.

Hepatocyte nuclear factor-1 α inactivated hepatocellular adenomas in patient with congenital absence of the portal vein: a case report.

Hepatocellular adenomas (HCAs) have been recognized recently as a heterogeneous group, and are subclassified according to genotype as well as morphological characteristics. We report a case of a 35-year-old Japanese woman who exhibited hepatocyte nuclear factor (HNF)-1α-inactivated HCA in the background of the congenital absence of the portal vein (CAPV). On a dynamic contrast computed tomography (CT) scan, the hypovascular tumor enlarged from 1 cm to 3 cm and another tumor emerged in the course of 7 years. Because the possibility of hepatocellular carcinoma (HCC) with multiple metastases was not excluded, partial hepatectomy was performed. On a cut section, two well-demarcated tumors were observed and one tumor had a central fibrous scar. The histological features of these tumors were similar to those of focal nodular hyperplasia (FNH) with a central scar and HCA; however, these tumors were diagnosed as HNF-1α-inactivated HCA by immunohistochemistry according to the criteria of the current World Health Organization (WHO) classification. In non-tumorous liver tissue, an abnormal architecture of the vessels and a vague nodular appearance of lobuli were observed, which were likely to be those of nodular regenerated hyperplasia (NRH). We discuss its pathogenesis and relationship with CAPV.

Abnormal RNA structures (RNA foci) containing a penta-nucleotide repeat (UGGAA)n in the Purkinje cell nucleus is associated with spinocerebellar ataxia type 31 pathogenesis.

Spinocerebellar ataxia type 31 (SCA31) is an autosomal-dominant cerebellar ataxia showing a Purkinje cell (PC)-predominant neurodegeneration in humans. The mutation is a complex penta-nucleotide repeat containing (TGGAA)n , (TAGAA)n , (TAAAA)n and (TAGAATAAAA)n inserted in an intron shared by two different genes BEAN1 and TK2 located in the long arm of the human chromosome 16. Previous studies have shown that (TGGAA)n is the critical component of SCA31 pathogenesis while the three other repeats, also present in normal Japanese, are not essential. Importantly, it has been shown that BEAN1 and TK2 are transcribed in mutually opposite directions in the human brain. Furthermore, abnormal RNA structures called "RNA foci" are observed by a probe against (UAGAAUAAAA)n in SCA31 patients' PC nuclei, indicating that the BEAN1-direction mutant transcript appears instrumental for the pathogenesis. However, it is not known whether the critical repeat (TGGAA)n contributes to the formation of RNA foci, neither do we understand how the RNA foci formation is relevant to the pathogenesis. To address these issues, we investigated two SCA31 cerebella by fluorescence in situ hybridization using a probe against (UGGAA)n . We also asked whether the mutant BEAN1-transcript containing (UGGAA)n exerts toxicity compared to the other three repeats in cultured cells. Histopathologically, we confirm that the PC is the main target of SCA31 pathogenesis. We find that the RNA foci containing (UGGAA)n are indeed observed in PC nuclei of both SCA31 patients, whereas similar foci were not observed in control individuals. In both transiently and stably expressed cultured cell models, we also find that the mutation transcribed in the BEAN1-direction yields more toxicity than control transcripts and forms RNA foci detected with probes against (UGGAA)n and (UAGAAUAAAA)n . Taking these findings together, we conclude that the RNA foci containing BEAN1-direction transcript (UGGAA)n are associated with PC degeneration in SCA31.

Application of high-throughput single-nucleus DNA sequencing in pancreatic cancer.

Despite insights gained by bulk DNA sequencing of cancer it remains challenging to resolve the admixture of normal and tumor cells, and/or of distinct tumor subclones; high-throughput single-cell DNA sequencing circumvents these and brings cancer genomic studies to higher resolution. However, its application has been limited to liquid tumors or a small batch of solid tumors, mainly because of the lack of a scalable workflow to process solid tumor samples. Here we optimize a highly automated nuclei extraction workflow that achieves fast and reliable targeted single-nucleus DNA library preparation of 38 samples from 16 pancreatic ductal adenocarcinoma patients, with an average library yield per sample of 2867 single nuclei. We demonstrate that this workflow not only performs well using low cellularity or low tumor purity samples but reveals genomic evolution patterns of pancreatic ductal adenocarcinoma as well.

Clinico-genomic Characterization of ATM and HRD in Pancreas Cancer: Application for Practice.

PURPOSE: Characterizing germline and somatic ATM variants (gATMm, sATMm) zygosity and their contribution to homologous recombination deficiency (HRD) is important for therapeutic strategy in pancreatic ductal adenocarcinoma (PDAC). EXPERIMENTAL DESIGN: Clinico-genomic data for patients with PDAC and other cancers with ATM variants were abstracted. Genomic instability scores (GIS) were derived from ATM-mutant cancers and overall survival (OS) was evaluated. RESULTS: Forty-six patients had PDAC and pathogenic ATM variants including 24 (52%) stage III/IV: gATMm (N = 24), and sATMm (N = 22). Twenty-seven (59%) had biallelic, 15 (33%) monoallelic, and 4 indeterminate (8%) variants. Median OS for advanced-stage cohort at diagnosis (N = 24) was 19.7 months [95% confidence interval (CI): 12.3-not reached (NR)], 27.1 months (95% CI: 22.7-NR) for gATMm (n = 11), and 12.3 months for sATMm (n = 13; 95% CI: 11.9-NR; P = 0.025). GIS was computed for 33 patients with PDAC and compared with other ATM-mutant cancers enriched for HRD. The median was lower (median, 11; range, 2-29) relative to breast (18, 3-55) or ovarian (25, 3-56) ATM-mutant cancers (P < 0.001 and P = 0.003, respectively). Interestingly, biallelic pathogenic ATM variants were mutually exclusive with TP53. Other canonical driver gene (KRAS, CDKN2A, SMAD4) variants were less frequent in ATM-mutant PDAC. CONCLUSIONS: ATM variants in PDAC represent a distinct biologic group and appear to have favorable OS. Nonetheless, pathogenic ATM variants do not confer an HRD signature in PDAC and ATM should be considered as a non-core HR gene in this disease.

Frequent DYRK2 gene amplification in micropapillary element of lung adenocarcinoma - an implication in progression in EGFR-mutated lung adenocarcinoma.

The present study aimed to discern the molecular alterations involved in the progression of EGFR-mutated lung adenocarcinoma (LADC). We previously demonstrated that the micropapillary (mPAP) element is the most important histological factor for assessing malignant grades in LADCs. Therefore, mPAP and other elements were separately collected from three cases of EGFR-mutated LADC using laser capture microdissection and subjected to a comprehensive mRNA expression analysis. We focused on DYRK2 in this study because its level showed a substantial increase in EGFR-mutated LADCs with mPAP. We also immunohistochemically examined 130 tumors for the expression of DYRK2. The results confirmed a strong expression of DYRK2 in EGFR-mutated LADC with mPAP. Fluorescent in situ hybridization (FISH) analyses targeting the DYRK2 locus revealed frequent gene amplification in EGFR-mutated LADC, specifically occurring in the high-grade components, like mPAP. In summary, the results of this study suggest that DYRK2 overexpression through gene amplification is one of the molecular mechanisms responsible for promoting the progression of EGFR-mutated LADC.

Expression of Toll-like receptor 9 in bone marrow cells of myelodysplastic syndromes is down-regulated during transformation to overt leukemia.

Toll-like receptors (TLRs) play a crucial role in the host defense against invading microorganisms by recognizing pathogen-associated molecular patterns. Recently, a number of endogenous molecules have been reported to be ligands of TLRs. Some of these molecules are known to be expressed in cancer tissue and activate intracellular signal pathways via TLRs during cancer progression. Thus, in the present study, we analyzed the expression dynamics of TLRs in the bone marrow of myelodysplastic syndromes (MDS) during the course of transformation to overt leukemia (OL) using real-time RT-PCR. MDS bone marrow cells at the time of initial diagnosis tended to express higher levels of TLR2, TLR4 and TLR9 than control bone marrow cells. Among these TLRs, TLR9 exhibited a significant decrease of expression at the time of transformation to OL. The expression of TLR9 and TNF-alpha showed significant correlation in bone marrow cells from patients with MDS and OL. Immunohistochemically, TLR2 was mostly localized to neutrophils of the control and MDS bone marrow. TLR4 was observed in a subset of neutrophils and a few mononuclear cells in control and MDS bone marrow. In addition, TLR4 was weakly expressed in nearly half of immature myeloid cells of MDS cases. TLR9 was mainly localized to neutrophils in the control and RA bone marrow and strongly expressed in the immature myeloid cells of RAEB cases, although the blastic cells of OL cases did not express TLR9. Bone marrow cells in MDS exhibit frequent apoptosis, while OL cells are prone to be immortal. Thus, TLR9 might be associated with regulation of apoptotic/proliferative signals via TNF-alpha in the MDS bone marrow.

Differential expression of Toll-like receptors in follicular lymphoma, diffuse large B-cell lymphoma and peripheral T-cell lymphoma.

Although Toll-like receptors (TLRs) in mammals are well-known to play important roles in innate immunity, newer roles for the TLRs have suggested that cells with aberrant TLR expression may have a survival advantage over normal cells. Lymphocytes are one of a small number of cell types that express many of the TLRs, suggesting that abnormal TLR levels/signaling may potentially influence the progression of malignant lymphomas. Thus, frozen samples of 51 lymph nodes from patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphoma (PTCL) were analyzed for the expression of TLR1 to 9 using quantitative real-time PCR, and compared to those in reactive lymphadenopathy (RL) samples. TLR2 was over-expressed in both DLBCL and PTCL but not in FL when compared to RL. TLR1 and TLR4 expression was up-regulated in PTCL, while TLR8 was highly expressed in DLBCL. Although TLR5 showed lower expression in FL, expression of TLR3, TLR6, TLR7 and TLR9 did not vary significantly between different lymphoma subtypes. Double immunostaining revealed an increase in the number of TLR2 and/or TLR8 expressing lymphoma cells in DLBCL. In PTCL, TLR2 and TLR4 expression was localized to neoplastic T cells. TLR expression is highly variable among lymphoma subtypes. However, despite this some significant differences exist that may prove useful in the development of novel therapeutic strategies.

Specific expression of MUC21 in micropapillary elements of lung adenocarcinomas - Implications for the progression of EGFR-mutated lung adenocarcinomas.

We investigated the significance of MUC21 in EGFR-mutated lung adenocarcinoma (LADC). Two-hundred forty-one surgically resected LADCs (116 EGFR-mutated and 125 wild-type tumors) were examined for immunohistochemical expression of MUC21 protein. A polyclonal antibody and two monoclonal antibodies (heM21C and heM21D) that bind differentially glycosylated MUC21 epitopes were used, and MUC21 proteins detected by these antibodies were named MUC21P, MUC21C, and MUC21D, respectively. MUC21 mRNA levels were semi-quantified and classified into "high" and "low". Among the immunohistochemical expression detected by three different antibodies, high expressors tended to be related to EGFR mutations. The three varieties of the immunohistochemical expressions were related to different histological elements in the EGFR-mutated LADCs. Either MUC21P or MUC21C high expressors had a higher proportion of lepidic elements with low papillary structure and micropapillary elements. MUC21D high expressors had a significantly higher proportion of micropapillary elements (Mann-Whitney test P ≤0.0001). Furthermore, MUC21D high expressors showed high incidence of lymphatic canal invasion and lymph node metastasis (Pearson x2 test, P = 0.0021, P = 0.0125), and a significantly higher recurrence rate (5-year recurrence-free survival 50.7% vs. 73.8%, log-rank test P = 0.0495). MUC21 proteins with a specific glycosylation status may be involved in the progression of EGFR-mutated LADCs, particularly at the stage where tumors are transforming from pure lepidic to micropapillary through low papillary lepidic lesions.