Neutrophils promote aerogenous spread of lung adenocarcinoma with bronchioloalveolar carcinoma features.

PURPOSE: Adenocarcinoma with bronchioloalveolar carcinoma (BAC) features is a subtype of non-small cell lung cancers characterized by an intense inflammatory reaction composed of macrophages and neutrophils and by a distinct natural history with intrapulmonary spread leading to death due to respiratory failure. We hypothesized that neutrophils could promote aerogenous spread of lung adenocarcinoma with BAC features. EXPERIMENTAL DESIGN: We examined the effect of neutrophils on A549 cell line detachment in vitro and we quantified desquamation of tumor cells on tumor tissue (n = 25) and on matched bronchioloalveolar lavage (n = 17) in vivo in a series of patients with adenocarcinoma with BAC features. RESULTS: Neutrophils induced A549 detachment mediated by signals through cell-to-cell contact. Detached A549 cells were still viable and able to proliferate in vitro. Neutralization studies identified several membrane-bound molecules involved in detachment (i.e., intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, tumor necrosis factor alpha/tumor necrosis factor alpha receptor inhibitor, interleukin-1alpha /interleukin-1alpha receptor, and neutrophil elastase). In tumor tissue, shedding was detected in all samples, with a median shedding score of 42% (range, 4-95%). Micropapillary clusters were detected in 23 of the 25 tumor tissue samples, with a median micropapillary score of 1.40 (range, 0-2.1), and tumor cells were detected in 7 of 17 lavages. The micropapillary score was associated with a high neutrophil count in bronchioloalveolar lavage (P = 0.051). The shedding cell percentage was a significant factor in shorter survival (P = 0.034, univariate Cox analysis). CONCLUSIONS: Tumor shedding is induced by neutrophils. It is a significant factor of shorter survival and may be an important event in adenocarcinoma progression.

Subsequent brain metastasis responses to epidermal growth factor receptor tyrosine kinase inhibitors in a patient with non-small-cell lung cancer.

In response to the paper by Popat et al. "Recurrent responses to non-small-cell lung cancer brain metastases with erlotinib", we wish to report a similar case and to provide comments. A 32-year-old Chinese never-smoker female presented a primary lung adenocarcinoma with brain metastasis and three subsequent responses to EGFR tyrosine kinase inhibitors (gefitinib and erlotinib). Direct sequencing of epidermal growth factor receptor (EGFR) gene exons 18 to 21 and K-ras gene was performed on tissue obtained from initial biopsies and post-chemotherapy surgical specimens. An EGFR exon 21 L858R point mutation was identified on pre- and post-chemotherapy samples. K-ras mutations and EGFR exon 20 T790M point mutations were not detected. Moreover, EGFR protein overexpression was observed by immunohistochemistry as well as EGFR gene high polysomy by fluorescent in situ hybridization. These case suggest that re-challenging patients with NSCLC several times with EGFR-TKI should be considered when progressive disease is observed under chemotherapy. However, we do not yet know whether this option should be considered in light of tumor molecular evaluation, or whether it should be proposed to patients who experienced a clinical response after a first administration.

Pro-tumoural CXCL10/CXCR3-A autocrine loop in invasive mucinous lung adenocarcinoma.

Invasive mucinous adenocarcinoma (IMA) is a mucinous variant of lepidic predominant lung adenocarcinoma (LPA) and associated with a worse prognosis. We postulated that cytokine expression would enable us to differentiate IMA from LPA in terms of prognosis and acquisition of pro-tumoural capacities. A 30-cytokine panel was assessed in bronchoalveolar lavage fluids (BALF) from IMA (n=38), LPA (n=25) and control samples (n=7). We investigated the expression of differentially expressed cytokines and splice variants of their receptors in surgical samples. The presence of EGFR and KRAS mutations were determined. We also examined the expression of cytokines and splice variants of their receptors in different cell lines, exploring their functional impact on signalling pathways, proliferation and migration. Only C-X-C motif chemokine 10 (CXCL10) was differentially expressed, namely overexpressed in IMA BALF compared with LPA. CXCL10 overexpression in BALF was linked to a worse prognosis. In surgical samples, CXCL10 and its receptor C-X-C motif chemokine receptor 3 (CXCR3) were overexpressed in IMA compared to LPA. A pro-tumoural CXCR3-A splice variant was overexpressed in IMA, suggesting a CXCL10/CXCR3-A autocrine loop in IMA. CXCL10 and CXCR3 expression were not correlated with EGFR or KRAS status. CXCL10 up-regulated CXCR3-A expression, Erk1/2 phosphorylation and enhanced migration in the mucinous H2228 cell line. CXCL10/CXCR3-A may play a pro-tumoural role in IMA via an autocrine mechanism.

Lepidic predominant adenocarcinoma and invasive mucinous adenocarcinoma of the lung exhibit specific mucin expression in relation with oncogenic drivers.

OBJECTIVES: To evaluate MUC1, MUC2, MUC5B, MUC5AC, and MUC6 expression in invasive lepidic predominant adenocarcinoma (LPA) and invasive mucinous adenocarcinoma (IMA) of the lung, and the impact of oncogenic drivers. MATERIALS AND METHODS: MUC1, MUC2, MUC5B, MUC5AC, MUC6, TTF1 and Hnf4α immunohistochemistry was performed on surgical samples from 52 patients with IMA (n=25) or LPA (n=27). We searched for EGFR, KRAS, BRAF, and HER2 mutations and ALK, ROS1, and NRG1 rearrangements. RESULTS: MUC1, MUC2, MUC5B, MUC5AC, and MUC6 expression was detected in tumor cells in 77%, 2%, 63%, 36%, and 21% of cases, respectively. MUC1 was significantly more overexpressed in LPA. MUC5B, MUC5AC, and MUC6 were typically detected in goblet cells and overexpressed in IMA. Hnf4α-positive IMA (n=11) were TTF1-negative and typically did not expressed MUC1 and expressed MUC5AC and MUC6. Hnf4α-negative IMA (n=14) showed a reverse profile of mucins expression, with MUC1 expression and a lack of MUC5AC and MUC6 expression. EGFR-positive status was significantly associated with LPA, MUC1 expression, and no MUC5B, MUC5AC, or MUC6 expression. KRAS-positive status was significantly associated with IMA and MUC5B and MUC5AC expression. CONCLUSIONS: LPA and IMA exhibit specific mucin expression profiles, with MUC1 being associated with LPA, while MUC5B, MUC5AC, and MUC6 were associated with IMA. Hnf4α expression and EGFR and KRAS mutations may play a role in mucin expression profiles of these lung adenocarcinoma subtypes.

BRAF V600E detection in cytological thyroid samples: A key component of the decision tree for surgical treatment of papillary thyroid carcinoma.

BACKGROUND: Whether preoperative knowledge of the BRAF mutation status would help to determine the extent of surgery for thyroid nodules is still under investigation. METHODS: We developed a method to state the V600E mutation before surgery on fine-needle aspiration (FNA) stained smears checked to contain tumor cells. We evaluated the interest of the preoperative assessment of the mutation for surgical strategy of nodules, diagnosed as malignant, suspicious for malignancy or follicular neoplasms. RESULTS: The mutation was found in 81% (79 of 97) malignant, 59% (20 of 34) suspicious nodules, and in none of follicular neoplasms (n = 29). Overall, the mutation was detected in 82% of papillary carcinomas. The sensitivity, specificity, and positive and negative predictive values for the diagnosis of malignancy were 75%, 100%, 100%, and 46%, respectively. CONCLUSION: The preoperative knowledge of the V600E mutation status is fundamental to plan total thyroidectomy with certainty and should be part of the decision tree for the management of thyroid nodules. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1017-1021, 2016.

NRG1 fusion in a French cohort of invasive mucinous lung adenocarcinoma.

Invasive mucinous lung adenocarcinoma (IMA) is a rare subtype of lung adenocarcinoma with no effective treatment option in advanced disease. KRAS mutations occur in 28-87% of the cases. NRG1 fusions were recently discovered in KRAS-negative IMA cases and otherwise negative for known driver oncogenes and could represent an attractive therapeutic target. Published data suggest that NRG1 fusions occur essentially in nonsmoking Asian women. From an IMA cohort of 25 French patients of known ethnicity, driver oncogenes EGFR, KRAS, BRAF, ERBB2 mutations, and ALK and ROS1 rearrangements presence were analyzed. In the IMA samples remaining negative for these driver oncogenes, an NRG1 rearrangement detection was performed by FISH. A driver oncogene was identified in 14/25 IMA, namely 12 KRAS mutations (48%), one ROS1 rearrangement (4%), and one ALK rearrangement (4%). The detection of NRG1 rearrangement by FISH was conducted in the 11 pan-negative IMA. One sample was NRG1FISH-positive and 100% of the tumor nuclei analyzed were positive. This NRG1-positive patient was a 61-year-old nonsmoking woman of Vietnamese ethnicity and was the sole patient of Asian ethnicity of the cohort. She died 6 months after the diagnosis with a pulmonary multifocal disease. NRG1FISH detection should be considered in patients with IMA pan-negative for known driver oncogenes. These results might suggest that NRG1 fusion is more frequent in IMA from Asian patient. Larger studies are needed.

EGFR and KRAS mutation status in non-small-cell lung cancer occurring in HIV-infected patients.

Non-small-cell lung cancer (NSCLC) is the most common non-acquired immune deficiency syndrome-related malignancy responsible for death. Mutational status is crucial for choosing treatment of advanced NSCLC, yet no data is available on the frequency of epidermal growth factor receptor (EGFR) and Kirsten ras (KRAS) mutations and their impact on NSCLC in human immunodeficiency virus (HIV)-infected patients (HIV-NSCLC). All consecutive HIV-NSCLC patients diagnosed between June 1996 and August 2013 at two Paris university hospitals were reviewed, with tumor samples analyzed for EGFR and KRAS mutational status. Overall, 63 tumor samples were analyzed out of 73 HIV-NSCLC cases, with 63% of advanced NSCLC. There were 60 non-squamous and nine squamous cell carcinomas, with EGFR and KRAS mutations identified in two (3.3%) and seven (11.5%) tumors, respectively. The proportion of KRAS mutations was 29% if solely the more sensitive molecular techniques were considered. The two patients with advanced adenocarcinoma harboring EGFR mutations exhibited lasting partial response to EGFR-tyrosine kinase inhibitors. Overall survival for patients with advanced NSCLC were >30 months for those with EGFR mutations, <3 months for KRAS mutations (n=2), and the median was 9 months [4.1-14.3] for wild-type (n=34). In multivariate analysis, KRAS mutation and CD4<200 cells/µL were associated with poor prognosis (hazard ratio (HR): 24 [4.1-140.2], p=0.0004; HR: 3.1 [1.3-7.5], p=0.01, respectively). EGFR mutation must be investigated in HIV-NSCLC cases due to its predictive and prognostic impact, whereas KRAS mutation is of poor prognostic value. Clinicians should search for drugs dedicated to this target population.

Is there a specific phenotype associated with the different subtypes of KRAS mutations in patients with advanced non-small-cell lung cancers?

OBJECTIVES: KRAS mutations occur in 20 to 25% of non-small-cell lung cancers (NSCLC) and seem to predict a poor prognosis. There is heterogeneousness in the frequency and spectrum of KRAS mutations, which can be categorized in transitions and transversions. We wondered if subtypes of KRAS mutation were associated with specific clinical phenotypes and specific survival. MATERIALS AND METHODS: Between July 2007 and May 2012, patients with advanced NSCLC and KRAS mutation diagnosed in two university hospitals were included. Clinical and histological characteristics, therapeutics and survival data were collected. RESULTS: Among 635 patients screened for KRAS mutations, 90 were found to be mutated and were included. Median age was 59 years (range: 54-69). Most were males (60%), current or former smokers (63% and 33%, respectively) and had an adenocarcinoma (ADC) (80%). Eighty patients were stage IV and 10 were stage IIIB. Eighty percent of the KRAS mutations were transversions and 20% were transitions. In uni- and multivariate analyses, there was a trend for fewer smokers among patients with transitions than among those with transversions (Odds Ratio [OR]=0.28, 95% CI [0.079-0.999], p=0.05). No significant difference was noted between transitions and transversions for other clinical characteristics. Patients with transitions had more frequently squamous-cell carcinoma (SCC) compared to those with transversions, who had more frequently adenocarcinomas (OR=16.7, 95% CI [2.76-100.8], p=0.002). Seventy-nine patients (86%) had received first-line chemotherapy. No significant difference was seen for disease-control rate, median progression-free survival or overall survival between transitions and transversions. CONCLUSION: A higher proportion of non-smokers and SCC subtypes were observed in the transitions compared to transversions. This confirms the heterogeneity of KRAS mutations and could suggest to expand KRAS testing in SCC to assess impact of RAS in SCC, which remains poorly investigated.

The impact of intracytoplasmic mucin in lung adenocarcinoma with pneumonic radiological presentation.

OBJECTIVES: This study investigated the clinical and prognostic impact of intracytoplasmic mucin in lung adenocarcinoma with "pneumonic" radiological presentation, formerly known as bronchioloalveolar carcinoma (BAC). PATIENTS AND METHODS: Between 1986 and 2011, clinical and pathological data from 120 consecutive patients with lung adenocarcinoma with "pneumonic" radiological presentation were reviewed. Intracytoplasmic mucin was assessed using a diastase-resistant periodic acid-Schiff staining. The presence of EGFR or K-Ras mutations and ALK rearrangement were determined in surgical samples. RESULTS: The two predominant histological patterns were invasive mucinous adenocarcinoma (40%) and lepidic predominant adenocarcinoma (32%). Intracytoplasmic mucin was detected in 71 patients (59.2%) who were more likely to be non-smokers (p=0.04) and have bronchorrhea (p=0.006), crepitant rales (p=0.02), or neutrophil alveolitis (p=0.0004). In mucin-producing tumors, EGFR mutation was not detected, K-Ras mutations and ALK rearrangement were present in 32% and 3% of cases, respectively. In non-mucin-producing tumors, EGFR and K-Ras mutations were detected in 17% and 10% of cases, respectively, no ALK rearrangement was detected. In univariate analysis, performance status>0, crepitant rales, bronchorrhea, neutrophil alveolitis, bilateral extension, intracytoplasmic mucin and no surgery were associated with worse survival. In multivariate analyses, intracytoplasmic mucin, neutrophil alveolitis, and no surgery were independent factors for worse survival. CONCLUSION: Intracytoplasmic mucin is associated with specific clinical characteristics and is an independent factor for worse survival in lung adenocarcinoma formerly known as BAC.

Blood vessel invasion is a major feature and a factor of poor prognosis in sarcomatoid carcinoma of the lung.

OBJECTIVES: Pulmonary sarcomatoid carcinomas (SC) are highly disseminated types of non-small-cell lung carcinoma. Their prognosis is poor. New therapeutic targets are needed to improve disease management. MATERIALS AND METHODS: From 1995 to 2013, clinical and survival data from all consecutive patients with surgically treated SC were collected. Pathological and biomarker analyses were performed: TTF1, P63, c-MET and ALK expression (immunohistochemistry), PAS staining, ALK rearrangement (FISH), and EGFR, KRAS, HER2, BRAF, PIK3CA, and MET genes mutations (PCR). RESULTS: Seventy-seven patients were included. Median age was 61 years (53-69). Histological subtypes were pleomorphic carcinoma (78%), carcinosarcoma (12%), and giant-cell and/or spindle-cell carcinoma (10%). Blood vessel invasion (BVI) was present in 90% of cases. Morphology and immunohistochemistry were indicative of an adenocarcinoma, squamous, and adenosquamous origin in 41.5%, 17% and 11.5%, respectively, 30% remained not-otherwise-specified. KRAS, PIK3CA, EGFR, and MET mutations were found in 31%, 8%, 3%, and 3%, respectively. No tumors had HER2 or BRAF mutations, or ALK rearrangement, whereas 34% had a c-MET positive score. Five-year overall survival (OS) was 29% for the whole population. At multivariate analysis, tumor size <50mm (HR=1.96 [1.04-3.73], p=0.011), no lymph-node metastasis (HR=3.25 [1.68-6.31], p<0.0001), no parietal pleural invasion (HR=1.16 [1.06-1.28], p=0.002), no BVI (HR=1.22 [1.06-1.40], p=0.005), and no squamous component (HR=3.17 [1.48-6.79], p=0.01) were associated with longer OS. Biomarkers did not influence OS. CONCLUSION: Dedifferentiation in NSCLC could lead to SC and an epithelial subtype component could influence outcome. BVI was present in almost all SCs and was an independent factor of poor prognosis.

Impact of systematic EGFR and KRAS mutation evaluation on progression-free survival and overall survival in patients with advanced non-small-cell lung cancer treated by erlotinib in a French prospective cohort (ERMETIC project--part 2).

BACKGROUND: Epidermal growth factor and v-Ki-ras2 Kirsten ras sarcoma (KRAS) mutation status, although associated with EGFR- tyrosine kinase inhibitor (TKI) efficacy, has not been used in clinical practice until recently. The prospective Evaluation of the EGFR Mutation status for the administration of EGFR-TKIs in non small cell lung Carcinoma (ERMETIC) study aimed to implement these biomarkers in France. METHODS: Between March 2007 and April 2008, EGFR and KRAS were studied by sequencing DNA tumor specimens from 522 consecutive advanced non-small-cell lung cancer patients treated with EGFR-TKI, mostly in second- or third-line settings. Cox models were used to investigate the impact of patient characteristics and mutations on progression-free survival (PFS) and overall survival (OS). Added value from mutation status was evaluated using likelihood ratio (LR) tests. Classification and regression tree analysis aimed to identify homogeneous groups in terms of survival. RESULTS: Among the 522 patients, 87% were white, 32% were women, and 18% were never-smokers, with 65% presenting with adenocarcinoma. Biological data were available for 307 patients, showing 44 EGFR mutations (14%) and 42 KRAS (14%) mutations. Median PFS was 2.4 months (interquartile range, 1.4-4.6) and median OS 5.6 months (interquartile range, 2.2-14.0). Factors independently associated with PFS were performance status 1 or 2 to 3 (hazards ratio [HR] = 1.5, 95% confidence interval [CI] 1.1-1.9; and HR = 2.3, CI 1.7-3.1, respectively; p < 0.001); former or current smoker status (HR = 1.8, CI 1.4-2.4 and 2.0,CI 1.4-2.8, respectively; p < 0.001); nonadenocarcinoma histology (squamous cell: HR = 0.9 CI 0.7-1.2]; others: HR = 1.6, 1.3-2.1; p < 0.001); at least two metastatic sites (HR = 1.3, CI 1.1-1.6 and 1.6, CI 1.3-2.1, respectively; p < 0.001); prior taxane-based chemotherapy (HR = 1.3, CI 1.0-1.3, p = 0.01); non-white (HR = 0.7, CI 0.5-0.9, p = 0.009). Similar results were found for OS. In addition, EGFR and KRAS mutations were significantly associated with PFS (HR = 0.5, CI 0.3-0.7 and HR = 1.2, CI 0.8-1.8, respectively, versus no mutation; LR p = 0.001). In the OS model, adjusted HR was 0.7 (0.4-1.0) for EGFR mutation and 1.7 (1.1-2.4) for KRAS (LR p = 0.004). Classification and regression tree analysis revealed EGFR mutation to be the primary factor for identifying homogeneous patient subgroups in terms of PFS. CONCLUSIONS: EGFR and KRAS status independently impacts outcomes in advanced non-small-cell lung cancer patients treated with EGFR-TKI. However, EGFR status impacts both PFS and OS whereas KRAS only impacts OS. These findings support the nationwide use of EGFR status for patient selection before EGFR-TKI therapy. The role of KRAS mutations remains to be elucidated.

Cross-validation study for epidermal growth factor receptor and KRAS mutation detection in 74 blinded non-small cell lung carcinoma samples: a total of 5550 exons sequenced by 15 molecular French laboratories (evaluation of the EGFR mutation status for the administration of EGFR-TKIs in non-small cell lung carcinoma [ERMETIC] project--part 1).

INTRODUCTION: The Evaluation of the epidermal growth factor receptor (EGFR) Mutation status for the administration of EGFR-Tyrosine Kinase Inhibitors in non-small cell lung Carcinoma (NSCLC) (ERMETIC) project part 1 assessed the accuracy of EGFR and KRAS mutations detection in NSCLC among 15 French centers. METHODS: The 15 ERMETIC centers selected 74 NSCLC surgical specimens from previously untreated patients. Paraffin and paired frozen DNA were sequenced for EGFR exons 18 to 21 and KRAS exon 2 by an external molecular laboratory, yielding a gold standard. The 74 blinded paraffin DNAs were redistributed to the 15 ERMETIC laboratories for sequencing of a total of 5550 exons. Results were compared with the gold standard and between centers by discordance rates and kappa statistics. RESULTS: The gold standard included 39 mutated samples with 22 EGFR and 17 KRAS mutated samples. Kappa statistics showed that 10, 6, and 6 of the 15 ERMETIC centers had a moderate to good kappa score, when compared with external laboratory for EGFR exon 19, EGFR exon 21, and KRAS exon 2, respectively. Kappa statistics showed moderate score between centers which increased to good for EGFR exon 19 mutation when removing 16 poor-quality samples with high nonamplificable rates. CONCLUSIONS: Paraffin-embedded specimens may represent a suitable source of DNA for sequencing analyses in ERMETIC centers. EGFR exon 19 deletions were most accurately detected by ERMETIC centers. Ease and accuracy of results, depended more on the quality of sample than on the difference in molecular sequencing procedures between centers, emphasize the need of preanalytical quality control programs.

Non-mucinous and mucinous subtypes of adenocarcinoma with bronchioloalveolar carcinoma features differ by biomarker expression and in the response to gefitinib.

There is no optimal established therapy for treating advanced or recurrent adenocarcinoma with bronchioloalveolar carcinoma features (ADC-BAC), and it remains unclear whether chemotherapy achieves therapeutic results comparable to those seen in the more common non-small lung carcinoma subtypes. In order to improve the decisions made during the treatment of advanced ADC-BAC, we attempted to better characterize the mucinous and non-mucinous ADC-BAC subtypes. Fifty pathological samples were obtained from 62 patients included in a multicenter prospective phase II trial (IFCT0401) conducted to evaluate gefitinib as a first-line therapy for non-resectable ADC-BAC. These samples were centrally reviewed and re-classified as non-mucinous (n=25) or mucinous (n=25) subtypes. We demonstrated that demographic data, clinical characteristics and stage at presentation (extrathoracic versus lung metastasis, as well as TNM staging) did not distinguish between the two subtypes. In contrast, three biological markers (PAS staining, TTF-1 expression and EGFR genomic gain combined with mutation analysis) enabled us to independently segregate all but 2 of the 50 patients into the mucinous and non-mucinous ADC-BAC subtypes. Finally, only mucinous tumors appeared to be resistant to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Additional prospective studies are required to better approach therapeutic strategy in mucinous tumors, which are a distinct entity from non-mucinous tumors.

Long-term survival for patients with non-small-cell lung cancer with intratumoral lymphoid structures.

PURPOSE: It has been established that the immune system plays an important role in tumor rejection. There is also compelling evidence that immune responses can develop independently of secondary lymphoid organs in tertiary lymphoid structures. We studied the presence and the correlation of tertiary lymphoid structures with clinical outcome in non-small-cell lung cancer (NSCLC), as the prognostic value of these structures in patients with cancer had not yet been established. PATIENTS AND METHODS: This retrospective study was performed by immunohistochemistry on paraffin-embedded tissue specimens from 74 patients with early-stage NSCLC. RESULTS: Tertiary lymphoid structures were detected in some tumors but not in nontumoral lungs. Thus we called these structures tumor-induced bronchus-associated lymphoid tissue (Ti-BALT). As in lymph nodes, Ti-BALTs were composed of mature dendritic cell (DC)/T-cell clusters adjacent to B-cell follicles and had features of an ongoing immune response. Because the quantitative counting of Ti-BALT was difficult to achieve, we used mature DCs that homed exclusively in Ti-BALT as a specific marker of these structures. Univariate analysis showed that the density of mature DCs was highly associated with a favorable clinical outcome (overall, disease-specific, and disease-free survival), suggesting that Ti-BALT may participate in antitumoral immunity. The density of tumor-infiltrating lymphocytes, in particular, CD4(+) and T-bet(+) Th1 T cells, was profoundly decreased in tumors weakly infiltrated by mature DCs. CONCLUSION: The density of mature DCs was found to be a better predictor of clinical outcome than the other parameters tested. The number of tumor-infiltrating mature DCs may identify patients with early-stage NSCLC who have a high risk of relapse.

Analysis of the intraclonal diversity of HLA-A0201-restricted T lymphocyte epitopes in follicular lymphoma idiotype.

Lymphoma-derived immunoglobulin idiotype (Id) is a tumour-specific antigen used for antitumour vaccination in follicular lymphoma (FL). However, FL Ids are subject to hypermutation and subclones may escape antitumour cytotoxic T-cell response. To investigate the intraclonal epitope diversity, we sequenced the FL heavy chain gene (consensus Id gene) and subclones of 24 patients. The derived polypeptide sequences were analysed by bioinformatics for human leucocyte antigen (HLA)-A0201-restricted epitope prediction. Epitopes were classified according to BIMAS and SYFPEITHI scores. Surprisingly in these highly mutated polypeptides, the epitopes concentrated in short hotspots in the conserved framework regions (FRs), both in HLA-A0201(+) and HLA-A0201(-) patients. Similar hotspots have been observed by others in chronic lymphocytic leukaemia Ids which in contrast to FL have low mutation frequency. FR3 amino acids 78-88 displayed the best-score epitopes in Ids containing a VH3-family segment, the most represented in FL Ids. Such VH3-FR3(78-88) epitopes were previously demonstrated as immunogenic. Modifications of the epitope pattern in subclones of HLA-A0201(+) patients were generally absent from high-score peptides, including VH3-FR3(78-88) epitopes (83% unmodified). Therefore, no tendency for loss of HLA-A0201-restricted epitopes was evidenced and, given their limited intraclonal diversity, VH3-FR3(78-88) epitopes may provide a useful target for the induction of cytotoxic response in Id-vaccinated FL patients.