Insight into the significance of Foxp3 + tumor-infiltrating lymphocytes in squamous cell lung cancer.

PURPOSE: Although developing a better understanding of tumor-infiltrating Foxp3 + lymphocytes (Foxp3 + TILs) might provide essential knowledge to predict response to immunotherapy and prognosis, our current knowledge about Foxp3 + TILs is inadequate. This study investigated the prognostic significance of tumor-infiltrating Foxp3 + lymphocytes (Foxp3 + TILs) in squamous cell lung cancer (SQ-LC) objectively. METHODS: Among patients with SQ-LC surgically resected in our institution between 2011 and 2017, those with pathological stage IA3-IIIA were immunohistochemically studied to evaluate Foxp3 + TILs in their tumor stroma. The impact of Foxp3 + TILs on relapse-free survival (RFS) was analyzed with Kaplan-Meier survival analysis and multivariate analysis using a Cox proportional hazards model/Fine-Gray model. RESULTS: This study analyzed 100 patients. Multivariate analysis showed that a large number of Foxp3 + TILs in the stroma does not associate with a poor prognosis, rather that a large number of Foxp3 + TILs (≥ 64 cells) tend to be associated with a more favorable prognosis than a small number of Foxp3 + TILs (< 64 cells) (large vs small number: HR, 0.56; 95% CI, 0.17-1.83; P = 0.34). Exploratory analysis also showed that in the two populations divided by a difference in Foxp3 expression levels, similar trends to the main analysis were observed. CONCLUSION: Our results showed that a large number of Foxp3 + TILs in the stroma may not associate with a poor prognosis in SQ-LC. To use the seemingly complicated information of Foxp3 + TILs as biomarkers, better understanding the diversity and heterogeneity of Foxp3 + TILs and analyzing their subpopulations that increase in the TME may be needed.

Decreased Basophil Activation against House Dust Mite after Japanese Cedar Pollen Subcutaneous Immunotherapy: A Retrospective Study.

BACKGROUND: Allergen-specific immunotherapy (AIT), an established treatment for allergic diseases, prevents the development of other allergic manifestations. Although the mechanisms remain unclear, AIT has been shown to reduce basophil activation (BA) against nontarget allergens. OBJECTIVES: The aim of this study was to assess immunological changes in Dermatophagoides farinae (Der f) after Japanese cedar pollen (JCP)-based subcutaneous immunotherapy (SCIT) monotherapy. METHOD: The data of 16 patients (age: 6-37 years) with JCP-induced allergic rhinitis who were sensitive to Der f (serum Der f-specific immunoglobulin E [IgE] level >0.34 kUA/L) and received JCP-based SCIT for 5 years were reviewed retrospectively. BA by Der f and JCP extracts and serum-specific IgE and immunoglobulin G4 (IgG4) levels against these allergens were evaluated before and after completing 5 years of JCP-based SCIT monotherapy. RESULTS: The areas under the dose-response curves of BA by Der f and JCP extracts were significantly reduced (p = 0.02 and p = 0.002, respectively). JCP-specific IgE levels decreased and JCP-specific IgG4 levels increased significantly (p < 0.001 for both), whereas Der f-specific IgE and IgG4 levels did not change significantly. CONCLUSIONS: JCP-based SCIT monotherapy reduced Der f-specific BA. These findings suggest that JCP-based SCIT has the potential to modulate immune response toward nontarget allergens.

Micropapillary and solid components as high-grade patterns in IASLC grading system of lung adenocarcinoma: Clinical implications and management.

OBJECTIVES: The grading system proposed by the International Association for the Study of Lung Cancer is based on a combination of predominant histologic subtypes and the proportion of high-grade components with a cutoff of 20%. We aimed to examine the clinical implications of the grading system beyond the discrimination of patient prognosis, while assessing the biological differences among high-grade subtypes. METHODS: We retrospectively reviewed 648 consecutive patients with resected lung adenocarcinomas and examined their clinicopathologic, genotypic, and immunophenotypic features and treatment outcomes. Besides the differences among grades, the clinical impact of different high-grade components: micropapillary (MIP) and solid (SOL) patterns, was individually evaluated. RESULTS: Survival outcomes were well-stratified according to the grading system. Grade 3 tumors exhibited aggressive clinicopathologic features, while being an independent prognostic factor in multivariable analysis. A small proportion (<20 %) of high-grade components in grade 2 had a negative prognostic impact. The prognostic difference bordering on the 20 % cutoff of the MIP proportion was validated; however, the proportion of SOL component did not affect prognosis. A survival benefit from adjuvant chemotherapy was observed in grade 3 tumors regardless of histologic subtype, but not in grade 1-2 tumors. The molecular and immunophenotypic features were different among grades, but still heterogeneous in grade 3, with MIP harboring frequent EGFR mutation and SOL exhibiting high PD-L1 expression. The treatment outcome after recurrence was worse in grade 3, but tumors with MIP pattern had an equivalent prognosis to that of grade 1-2 tumors, reflecting the high frequency of molecular targeted therapy. CONCLUSIONS: In addition to stratifying patient prognosis, the current grading system could discriminate clinical course, therapeutic effects of adjuvant chemotherapy, and molecular and immunophenotypic features. Further stratification based on biological heterogeneity in grade 3 remains necessary to enhance the role of the grading system in guiding patient management.

Insight into the significance of CD8+ tumor-infiltrating lymphocytes in squamous cell lung cancer.

BACKGROUND: Although there are great expectations regarding the use of tumor-infiltrating lymphocytes (TILs) to predict effects of immunotherapies and prognosis, knowledge about TILs remains insufficient for clinical application. METHODS: We objectively investigated the prognostic significance of tumor-infiltrating CD8 + lymphocytes (CD8 + TILs) in squamous cell lung cancer (SQ-LC). Among patients who underwent surgical resection of SQ-LC in 2011-2017, 100 patients with pathological stage IA3-III were immunohistochemically studied to evaluate CD8 + TILs in the tumor stroma and parenchyma. The impact of CD8 + TILs on relapse-free survival was analyzed using a Kaplan-Meier survival analysis and multivariate analyses using Fine-Gray and Cox proportional hazards models. RESULTS: The multivariate analysis showed that large and small numbers, but not intermediate numbers, of CD8 + TILs in the tumor stroma may be related to a more favorable prognosis (small vs. intermediate: HR, 0.64; 95% CI: 0.29-1.41, p = 0.27; large vs. intermediate: HR, 0.48; 95% CI: 0.21-1.09, p = 0.08). In contrast, a large number of CD8 + TILs in the tumor parenchyma was associated with a poor prognosis (HR, 2.60; 95% CI: 0.91-7.42, p = 0.075). An exploratory analysis showed a potentially strong association between an extremely large number of CD8 + TILs in the tumor parenchyma and a poor prognosis, even with a large number of CD8 + TILs in the tumor stroma. CONCLUSION: Our study provided partial but important information on the significance of CD8 + TILs in SQ-LC. To use CD8 + TILs as biomarkers, a better understanding of CD8 + TILs as well as other important components in the tumor microenvironment and the inflammatory phenotypes they form may be needed.

Clinical risk factors at 3 months of age for the development of bronchial asthma at 36 months of age.

BACKGROUND: We examined the associations between factors evident at the routine 3-month well-child visit (WCV) and the risk of developing 36-month parent-reported physician-diagnosed bronchial asthma (BA). METHODS: This longitudinal study was conducted in Nagoya City, Japan, and included 40,242 children who qualified for the 3-month WCVs in the city between April 1, 2016 and March 31, 2018. In total, 22,052 (54.8%) questionnaires linked to their 36-month WCVs were analyzed. RESULTS: The prevalence of BA was 4.5%. The multivariable Poisson regression model identified male sex (adjusted risk ratio [aRR], 1.59; 95% confidence interval [CI]: 1.40-1.81), born in autumn (aRR, 1.30; 95% CI: 1.09-1.55), having at least one sibling (aRR, 1.31; 95% CI: 1.15-1.49), wheeze history before 3-month WCVs, with clinic/hospital visit: aRR, 1.99; 95% CI: 1.53-2.56; hospitalization: aRR, 2.99; 95% CI: 2.09-4.12, eczema with itch (aRR, 1.51; 95% CI: 1.27-1.80), paternal history of BA (aRR, 1.98; 95% CI: 1.66-2.34), maternal history of BA (aRR, 2.11; 95% CI: 1.77-2.49), and rearing pets with fur (aRR, 1.35; 95% CI: 1.15-1.58) were independent risk factors for BA at 36 months of age. The combination of severe wheeze history (with clinic/hospital visit or hospitalization) and maternal and paternal BA could identify high-risk infants whose prevalence of BA was 20%. CONCLUSIONS: The combined assessment of important clinical factors enabled us to identify high-risk infants set to derive optimal benefit from health guidance provided to the parent or caregiver of the child or infant at WCVs.

Examination of the effectiveness of local therapy for oligo-recurrence of EGFR-mutated NSCLC.

BACKGROUND: The effectiveness of local therapy has been reported in patients with oligo-recurrence of non-small cell lung cancer (NSCLC), a metachronous recurrence with a limited number of recurrences, which can be treated with local therapy. Conversely, remarkable progress has been made in systemic therapy for NSCLC with the advent of molecular targeted therapy. In particular, epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are very effective in the treatment of EGFR-mutated NSCLC. There is currently no consensus on treatment for oligo-recurrence of EGFR-mutated NSCLC. METHODS: From 2004 to 2014, 811 patients underwent complete resection for NSCLC at Kitasato University Hospital and, of these, 244 patients developed recurrence. Oligo-recurrence was defined as the presence of two or less recurrent lesions, and 34 patients presented with EGFR-mutated oligo-recurrence. RESULTS: We retrospectively examined and compared the effects of EGFR-TKIs with those of radical local therapy in patients with oligo-recurrent EGFR-mutated NSCLC. The five-year post-recurrence survival (PRS) rates of patients with EGFR-mutated oligo-recurrence who received radical local therapy (n = 23) and those who did not (n = 11) were 59.4 and 45.5%, respectively (p = 0.777). Multivariate analysis revealed no favorable prognostic factors associated with prolonged PRS, and radical local therapies did not improve PRS in patients with oligo-recurrence (p = 0.551). CONCLUSION: Radical local therapy did not affect PRS in patients with oligo-recurrent EGFR-mutated NSCLC. Even in cases of oligo-recurrence, the administration of local therapy in patients with EGFR-mutated NSCLC might be carefully considered.

Evaluation of peripheral basophil activation during exercise provocation test for desensitized patients.

Some food allergic patients who have undergone oral immunotherapy develop exercise-induced allergic reactions on desensitization (EIARDs). This study investigated basophil activation status during the exercise provocation test (EPT) performed to diagnose EIARD. EPT was performed on 20 participants, and in vivo basophil activation status was analyzed using activation markers CD203c and CD63. The results showed that there was no significant difference between EPT-positive and negative subjects for basophil activation status throughout EPT. Consequently, in vivo basophil activation after ingestion of the causative food may not be associated with EIARDs. New tests are desired for predicting EIARDs.

Comprehensive Sequence Analysis of Parvalbumins in Fish and Their Comparison with Parvalbumins in Tetrapod Species.

Parvalbumins are small molecules with important functions in Ca2+ signaling, but their sequence comparisons to date, especially in fish, have been relatively poor. We here, characterize sequence motifs that distinguish parvalbumin subfamilies across vertebrate species, as well as those that distinguish individual parvalbumins (orthologues) in fish, and map them to known parvalbumin structures. As already observed by others, all classes of jawed vertebrates possess parvalbumins of both the α-parvalbumin and oncomodulin subfamilies. However, we could not find convincing phylogenetic support for the common habit of classifying all non-α-parvalbumins together as "ß-parvalbumins." In teleost (modern bony) fish, we here distinguish parvalbumins 1-to-10, of which the gene copy number can differ between species. The genes for α-parvalbumins (pvalb6 and pvalb7) and oncomodulins (pvalb8 and pvalb9) are well conserved between teleost species, but considerable variation is observed in their copy numbers of the non-α/non-oncomodulin genes pvalb1-to-5 and pvalb10. Teleost parvalbumins 1-to-4 are hardly distinguishable from each other and are highly expressed in muscle, and described allergens belong to this subfamily. However, in some fish species α-parvalbumin expression is also high in muscle. Pvalb5 and pvalb10 molecules form distinct lineages, the latter even predating the origin of teleosts, but have been lost in some teleost species. The present study aspires to be a frame of reference for future studies trying to compare different parvalbumins.

Generalized allergic reaction in response to exercise due to strawberry gibberellin-regulated protein: a case report.

BACKGROUND: The Rosaceae family includes fruits, such as peach, apple, Japanese apricot, cherry (Prunoideae subfamily), and strawberry (Rosoideae subfamily). The allergens responsible for Rosaceae fruit allergies have been reported to include Bet v 1 and profilin, which mainly cause oral symptoms, and lipid transfer protein (LTP). Recently, gibberellin-regulated protein (GRP) has been identified as an allergen that induces generalized symptoms in peach-, orange-, and plum-related allergies. Most patients with food allergies induced by GRP show allergic symptoms accompanied by cofactors, such as exercise or drugs. To date, there are very few reports of generalized symptoms induced by strawberry. CASE PRESENTATION: We evaluated the reactivity of strawberry GRP in a 15-year-old boy who was confirmed to have generalized symptoms induced by strawberry with exercise using an oral food challenge test (OFCT). The patient's serum exhibited a strong positive reaction to strawberry GRP but not to peach GRP or peach LTP. The patient's basophils reacted to strawberry and peach GRP but not to peach LTP. CONCLUSIONS: Strawberry GRP may be a causative component for strawberry with exercise-induced generalized symptoms in this patient. This is the first study to investigate the role of GRP in strawberry with cofactor-induced allergic symptoms. Further epidemiological and clinical researches are necessary to improve diagnostic and therapeutic approaches for patients with strawberry allergy.

Determination of Severe Peach Allergens, Gibberellin-Regulated Protein, and Lipid Transfer Protein, Using Monoclonal Antibodies.

In this study, monoclonal antibodies against two major fruit allergens-gibberellin-regulated protein (GRP) and lipid transfer protein (LTP)-were established. Sandwich enzyme-linked immunosorbent assays (ELISAs) for the quantification of peach GRP and LTP were constructed using these antibodies. Both ELISAs reacted with the respective antigens when heated at 100ºC for 20 min, but not when reduced with sodium sulfite, indicating that GRP and LTP are heat-stable, while disulfide bonds play an important role in their native steric structures. GRP and LTP in peaches and peach-containing foods were quantified by these ELISAs. In both cases, there were few differences among peach cultivars normally available on the market; however, concentrations were higher when the peach was ripe. GRP was localized in the pulp of the peach, while LTP was present in the peel. They could be quantified in peach-containing beverages, as well as in dried and canned peaches. GRP in Japanese apricots could also be determined using this ELISA, as its amino acid sequence is the same as that of peach GRP. Then, high concentrations of GRP were detected in umeboshi, a traditional Japanese pickled apricot. Peach leaves were found to have a high LTP content, accordingly, LTP was also observed in lotions containing peach leaf extract. The ability to quantitatively detect GRP and LTP in this study will, therefore, contribute to the improvement of component-resolved diagnoses and quality of life in patients allergic to peaches.

Prevalence of infantile wheezing and eczema in a metropolitan city in Japan: A complete census survey.

Infantile wheezing and eczema are associated with the subsequent onset of asthma and other atopic diseases. However, there are no large population-based surveys on infantile allergic symptoms in Japan. The objective of the study was to determine the prevalence of wheezing and asthma in infants in Nagoya, Japan. This population-based cross-sectional study was performed in the metropolitan city of Nagoya, Japan. We surveyed parents to ascertain the prevalence of wheezing and eczema in infants who attended group health checkups at 3, 18, and 36 months of age. Their parents completed modified questionnaires from the International Study of Asthma and Allergies in Childhood. More than 90% of the approximately 40,000 children in each study group living in the target area were included in the survey. The prevalence of wheezing was 8%, 17%, and 13% at 3, 18, and 36 months, respectively, and was characterized by birth season. The prevalence of eczema was 24%, 30%, and 31%, at 3, 18, and 36 months, respectively. Participants born in autumn and winter had a higher incidence of eczema in each age group. Three-quarters of the children had a parental history of allergic conditions. Parental allergic diseases and male gender are risk factors for wheezing and eczema in children. This survey had a high response rate and covered almost the entire population of the target age groups in a large city. We believe that the results of this study, therefore, provide a much higher level of confidence regarding the prevalence of allergies in infants in Japan than that in previous studies with limited cohorts.

Serological examination for clinical cross-reactivity between salmon roe and pollock roe in patients with a salmon roe allergy.

Objectives: Fish roe is a common allergen in Japan. We have previously reported that although immunoglobulin (IgE) from patients with salmon roe (SR) or pollock roe (PR) allergies cross-react, 70% of patients with SR allergy can consume PR without developing any symptoms. However, a correlation between clinical cross-reactivity and serological cross-reactivity remains to be demonstrated. Methods: Serum samples were collected from 15 patients with SR allergy who had consumed cooked PR previously. Among these volunteers, four had experienced immediate symptoms after consuming cooked PR, while the others had exhibited no symptoms of PR allergy. A competitive enzyme-linked immunosorbent assay (ELISA) was performed to analyze the serological cross-reactivity with SR and PR. Immunoblotting inhibition assays were performed using serum samples that had been pre-incubated with SR or PR extracts. Results: In ELISAs, binding to SR was inhibited by >50% when the serum samples from patients with both SR and PR allergies were pre-incubated with PR extract (p=0.0256). In immunoblots, pre-incubation of serum samples with PR extract inhibited detection of the 16-kDa protein, which likely corresponds to the major SR allergen beta' component, significantly more for samples from patients with both SR and PR allergies (100%) than for samples from those with only an SR allergy (18.2%) (p=0.011). Conclusions: The superior competitive binding of the sera from patients with both SR and PR allergies to PR compared with that to SR may induce clinical cross-reactivity between SR and PR.

Combination of epidermal growth factor receptor mutation and the presence of high-grade patterns is associated with recurrence in resected stage I lung adenocarcinoma.

OBJECTIVES: This study aimed to evaluate the prognostic impact of the combination of epidermal growth factor receptor (EGFR) mutation and the presence of high-grade patterns (solid or micropapillary component) in resected stage I lung adenocarcinoma. METHODS: Patients who underwent curative resection for pathological stage I lung adenocarcinoma and EGFR mutation analysis were included in this study. The impact of the combination of EGFR mutation and the presence of >5% high-grade patterns on recurrence-free survival (RFS) was retrospectively analysed using Cox proportional hazards model and propensity score-matched analysis. RESULTS: Among the included 721 patients, EGFR mutations were positive in 380 (52.7%). In the EGFR-mutated group, cases with high-grade patterns showed poorer RFS than those without (5-year RFS, 77.7% vs 92.5%, P < 0.001), whereas there were no significant prognostic differences in the EGFR wild-type group (5-year RFS, 89.8% vs 88.2%, P = 0.807). Multivariable analyses revealed that the combination of EGFR mutations and the presence of high-grade patterns was associated with poor RFS (hazard ratio = 1.655, P = 0.035). Furthermore, EGFR mutation was associated with poor RFS in the group with high-grade patterns (hazard ratio = 2.108, P = 0.008). After propensity score matching, EGFR-mutated cases with high-grade patterns showed poorer RFS (P = 0.028). CONCLUSIONS: The combination of EGFR mutation and the presence of high-grade patterns was associated with recurrence in resected stage I lung adenocarcinoma. Histological subtypes, including minor components, should be considered when evaluating the risk of recurrence in patients with EGFR-mutated lung adenocarcinoma.

Investigation of the sensitization rate for gibberellin-regulated protein in patients with Japanese cedar pollinosis.

BACKGROUND: Pollen-food allergy syndrome (PFAS) usually manifests as an itching sensation in the mouth and throat immediately after eating fresh fruits and vegetables. However, some patients with PFAS experience systemic symptoms including anaphylaxis. In Europe, cypress gibberellin-regulated protein (GRP) has been noted to cause allergenicity and exhibit cross-reactivity with peach GRP. Japanese cedar (Cryptomeria japonica), classified in the cypress family, is the primary causative substance among all environmental allergens in Japan. However, studies on the prevalence of GRP sensitization in patients with cedar pollinosis are lacking. OBJECTIVE: This study examined the prevalence of GRP sensitization in patients with Japanese cedar pollinosis. METHODS: We enrolled 52 patients who had requested sublingual immunotherapy treatment with mild-to-severe rhinitis during spring, and had a JCP-specific immunoglobulin E (IgE) levels of >0.7 UA/mL. Peach GRP was purified using affinity chromatography with a monoclonal antibody column. Specific IgE levels to peach GRP were measured using an enzyme-linked immunosorbent assay. Samples exhibiting absorbance at 450 nm of over mean plus three standard deviations of the negative control value were defined as positive. Sera from three patients with severe peach allergy were used as positive controls. RESULTS: Eleven sera from 52 patients with JCP-induced allergic rhinitis were positive for peach GRP. CONCLUSION: Twenty percent of patients with cedar pollinosis were sensitized to peach GRP. Well-powered studies are needed to clarify whether these patients are at an increased risk for systemic symptoms or whether they primarily demonstrate only localized symptoms.

Investigation of the sensitization rate for gibberellin-regulated protein in patients with Japanese cedar pollinosis

Background Pollen-food allergy syndrome (PFAS) usually manifests as an itching sensation in the mouth and throat immediately after eating fresh fruits and vegetables. However, some patients with PFAS experience systemic symptoms including anaphylaxis. In Europe, cypress gibberellin-regulated protein (GRP) has been noted to cause allergenicity and exhibit cross-reactivity with peach GRP. Japanese cedar (Cryptomeria japonica), classified in the cypress family, is the primary causative substance among all environmental allergens in Japan. However, studies on the prevalence of GRP sensitization in patients with cedar pollinosis are lacking.Objective This study examined the prevalence of GRP sensitization in patients with Japanese cedar pollinosis.Methods We enrolled 52 patients who had requested sublingual immunotherapy treatment with mild-to-severe rhinitis during spring, and had a JCP-specific immunoglobulin E (IgE) levels of >0.7 UA/mL. Peach GRP was purified using affinity chromatography with a monoclonal antibody column. Specific IgE levels to peach GRP were measured using an enzyme-linked immunosorbent assay. Samples exhibiting absorbance at 450 nm of over mean plus three standard deviations of the negative control value were defined as positive. Sera from three patients with severe peach allergy were used as positive controls.Results Eleven sera from 52 patients with JCP-induced allergic rhinitis were positive for peach GRP.Conclusion Twenty percent of patients with cedar pollinosis were sensitized to peach GRP. Well-powered studies are needed to clarify whether these patients are at an increased risk for systemic symptoms or whether they primarily demonstrate only localized symptoms (AU)