Atsushi Osoegawa

Mutations of the EGFR, K-ras, EML4–ALK, and BRAF genes in resected pathological stage I lung adenocarcinoma

Background and purposeThe EGFR, K-ras, EML4–ALK, and BRAF genes are oncogenic drivers of lung adenocarcinoma. We conducted this study to analyze the mutations of these genes in stage I adenocarcinoma.MethodsThe subjects of this retrospective study were 256 patients with resected stage I lung adenocarcinoma. We analyzed mutations of the EGFR, K-ras, and BRAF genes, and the EML4–ALK fusion gene. We also assessed disease-free survival (DFS) to evaluate the prognostic value and overall survival (OS) to evaluate the predictive value of treatment after recurrence.ResultsMutations of the EGFR, K-ras, EML4–ALK, and BRAF genes were detected in 120 (46.8xa0%), 14 (5.5xa0%), 6 (2.3xa0%), and 2 (0.8xa0%) of the 256 tumors. Two tumors had double mutations (0.8xa0%). The incidence of EGFR mutations was significantly higher in women than in men. The EML4–ALK fusion gene was detected only in younger patients. The DFS and OS of the K-ras mutant group were significantly worse than those of the EGFR mutant group, the EML4–ALK fusion gene group, and the wild-type group. Six of the seven patients with the EML4–ALK fusion gene are still alive without recurrent disease.ConclusionsIn patients with stage I adenocarcinoma, mutation of the K-ras gene was a poor prognostic factor for recurrence. The presence of a mutation of the EGFR or EML4–ALK gene was not a prognostic factor.

Clinicopathological features of younger (aged ≤ 50 years) lung adenocarcinoma patients harboring the EML4-ALK fusion gene

The EML4‐ALK fusion gene has recently been identified as a driver mutation in a subset of non‐small cell lung cancers. In subsequent studies, EML4‐ALK has been detected in a low percentage of patients, and was associated with a lack of EGFR or KRAS mutations, younger age, and adenocarcinoma with acinar histology. Cases with the EML4‐ALK fusion gene were examined to clarify the clinicopathological characteristics of young adenocarcinoma patients.

Prognostic Factors for Survival after Resection of Pulmonary Metastases from Colorectal Carcinoma

PURPOSEnAs chemotherapy has improved, the survival of patients with metastatic colorectal carcinoma has reached up to 2.5 years. Many of these patients experience pulmonary metastases; however, the prognosis after pulmonary metastasectomy is not satisfying. In this study, we analyzed the prognostic factors for survival in patients who underwent pulmonary metastasectomy.nnnMETHODSnEighty-seven patients with colorectal carcinoma received pulmonary metastasectomy. The pathological status of the primary tumor, outcome of the pulmonary metastasectomy, disease-free interval, perioperative carcinoembryonic antigen (CEA) level and history of liver metastases were assessed.nnnRESULTSnThe five-year survival was 42.5% after pulmonary metastasectomy. A univariate analyses revealed that the CEA level (p = 0.043) and the number of pulmonary metastases (p = 0.047) were prognostic factors for survival. The CEA level was an independent prognostic factor in a multivariate analysis (relative risk = 2.01, p = 0.037). Among cases with elevated preoperative CEA levels, those whose CEA level normalized after metastasectomy had a better prognosis compared with those whose CEA level decreased but was still high, or whose level increased after metastasectomy (median survival time of 41.8 months compared with 28.1 or 15.7 months, respectively p = 0.021).nnnCONCLUSIONnThe CEA level can be a predictive marker for the prognosis in patients with pulmonary metastases from colorectal carcinoma.

Pemetrexed-induced scleroderma-like conditions in the lower legs of a patient with non-small cell lung carcinoma

Pemetrexed, which is used for the treatment of non‐small cell lung carcinoma and malignant mesothelioma, induces cutaneous adverse reactions in approximately 20% of patients. There are also reports of the induction of fibrosing disorders. We describe a case of pemetrexed‐induced scleroderma‐like conditions in the lower legs of a patient whose pulmonary carcinoma has been relatively well controlled, with prolongation of the dose interval, in spite of the discomfort in both his legs. Skin biopsy revealed dermal fibrosis and dilated lymph vessels in the dermis, but lymphocytic infiltration around the lymph vessels, in contrast to the blood vessels, was minimal. Immunohistochemical staining revealed that the major subsets of T cells that had infiltrated around blood vessels were CD3 and CD45Ro, but no B cells were detected. High serum levels of interleukin (IL)‐4 and IL‐6 suggested that T cells, which secrete these cytokines, may be involved in the pathogenesis of this condition. Magnetic resonance imaging of the lower extremities revealed muscular and fascial involvement. Several chemotherapeutic agents, such as taxanes, gemcitabine and bleomycin, are known to induce scleroderma‐like changes, and we should also keep the side‐effects of pemetrexed in mind when we encounter patients with fibrosing conditions.

Intratumoral heterogeneity of copy number variation in lung cancer harboring L858R via immunohistochemical heterogeneous staining

BACKGROUNDnAlthough intratumoral heterogeneity is commonly observed in several cancers, few studies have shown its presence in EGFR-mutated lung cancer. We performed immunohistochemistry to analyze the intratumoral heterogeneity in EGFR-mutated (L858R) lung cancer and performed targeted sequencing in specific cases. We discuss the effects of intratumoral heterogeneity and acquired resistance to EGFR-TKI.nnnMETHODSnTwenty resected primary lung cancers known to harbor EGFR L858R were analyzed. IHC was performed using an L858R mutant-specific rabbit monoclonal antibody and the samples were scored by staining intensity (0-3) and proportion. For cases with heterogeneous L858R protein expression, the nucleic acids were extracted from each differently stained lesion, and targeted sequencing was performed. Single nucleotide variations (SNVs) and copy number variations (CNVs) were then analyzed. The cell proliferation and apoptosis were also evaluated by the ki-67 labeling index and TUNEL staining.nnnRESULTSnAmong 20 cases, 3 showed heterogeneous staining. Genetic analyses for cases with heterogeneous staining revealed an increase in the copy number of EGFR in the IHC-positive part compared to the negative part, and an increase in the copy number of CCNE1 was observed in the IHC-positive part compared to the negative part in one case (case 1). In another case (case 2), an increase in the copy number of EGFR was observed in the IHC-positive part compared to the negative part, and an increase in the copy number of MDM2 was observed in the IHC-positive part compared to the negative part. In three cases, no SNV changes were observed. An increase in the ki-67 labeling index in the L858R-positive part in case 1 and increased apoptosis in the L858R-positive part in case 2 were observed, suggesting the functional significance of CNV changes.nnnCONCLUSIONnThese cases exhibiting L858R IHC intratumoral heterogeneity suggest a heterogeneous effect on the cell activity due to CNV heterogeneity.

Acquired resistance to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) in an uncommon G719S EGFR mutation

SummaryBackground Acquired resistance (AR) to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a common event, and several underlying mechanisms, including T790xa0M, MET amplification and PTEN downregulation, have been reported for the common EGFR mutations. EGFR G719X is an uncommon mutation that has been reported to show sensitivity to EGFR-TKIs. However, no established cell lines harboring the EGFR G719X have been reported in the literature. Materials and Methods G719S-GR cells were established from malignant pleural effusion of a patient whose tumor developed AR from gefitinib treatment. G719S-GR cells were then genotyped and tested for drug sensitivities. Multiplex ligation-dependent probe amplification (MLPA) was used to compare the clinical tumor samples with G719S-GR. Results G719S-GR cells were resistant to EGFR-TKIs with an LC50 of around 10xa0μM. A genomic analysis showed that G719S-GR cells harbor the EGFR G719S mutation as well as the amplification of EGFR locus. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. On comparing the copy number of tumor suppressor genes using MLPA, G719S-GR cells were found to lack one copy of PTEN, which was not observed in a tumor obtained before gefitinib treatment. Loss of PTEN may result in AKT activation. The mTORC1/2 inhibitor Torin-1 was able to inhibit the downstream signaling when combined with osimertinib. Discussion The newly established G719S-GR cell line may be useful for investigating the mechanism underlying the development of AR in the G719X mutation; the loss of PTEN may be one such mechanism.

Abstract 2227: Positive correlation between the gamma-H2AX and PD-L1 expressions in lung squamous cell carcinoma

Lung cancer is a leading cause of cancer death worldwide. Recently, molecular targeting therapy has been developed and it has shown promising results against advanced lung cancer. Among them, lung squamous cell carcinoma has fewer treatment options because it is not driven by oncogenic mutations, but by alterations in tumor suppressor genes and subsequent chromosomal instability. Programmed death-ligand 1 (PD-L1) is one of the immune checkpoint molecules that is expressed on the surface of tumor cells, where it inhibits activities of cytotoxic T cells. Recent studies revealed that the PD-1/PD-L1 blockade could improve the overall survival in lung squamous cell carcinoma, potentially because it carries high mutation burdens and neoantigens which could be targeted by cytotoxic T cells. We hypothesized that DNA damage could accumulate in tumors with high mutation burdens, thereby inducing the PD-L1 expression on tumor cells, sensitizing them to anti-PD-1 therapy. An immunohistochemical analysis of 41 consecutive lung squamous cell carcinoma cases, which received surgery at our institution between April 2013 and March 2014, revealed that a high PD-L1 expression was observed in 15 patients (37%) that was associated related with a poor recurrence-free survival (p = 0.028). The PD-L1 expression level was also positively associated with the γH2AX expression (a direct marker for DNA damage) (p = 0.02). The γH2AX expression in tumor cell nuclei was confirmed by immunofluorescent staining. It forms foci in tumor cell nuclei, indicating the incidence of DNA double strand breaks. Our findings demonstrate for the first time that the nuclear γH2AX expression in lung squamous cell carcinoma is positively associated with the PD-L1 expression. Further studies are warranted to investigate whether γH2AX could be a biomarker for PD-1 targeting therapy and whether combination therapy with PD-1 targeting therapy and a DNA-damaging agent has synergistic effects. Citation Format: Atsushi Osoegawa, Takafumi Hashimoto, Yohei Takumi, Miyuki Abe, Hitomi Hiraishi, Shuji Suehiro, Michiyo Miyawaki, Kenji Sugio. Positive correlation between the gamma-H2AX and PD-L1 expressions in lung squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2227.

The clinical manifestations and treatment of male breast cancer: a report of three cases

Male breast cancer is an extremely rare malignancy. We treated three male breast cancer patients. All three patients showed clinical N0 and received sentinel lymph node biopsy. Because the sentinel lymph node was positive for metastasis in one patient, a total mastectomy with axillary lymph node dissection was performed. The other two patients were negative for sentinel lymph node metastasis, and a simple mastectomy was performed. Two of the patients were postoperatively treated with tamoxifen; another patient was treated with adjuvant chemotherapy using taxotere and cyclophosphamide before tamoxifen. There was no recurrence in any of the three patients during an average follow-up period of 56.7xa0months (range 11.8–80.3). A sentinel lymph node biopsy is recommended for node staging in both male and female breast cancer patients as it is associated with a lower incidence of complications.

Abstract B13: Non-small cell lung cancer with double driver mutations.

Background: Driver mutations, such as an epidermal growth factor receptor (EGFR) mutation or an echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion, generally occur independently in non-small cell lung cancer (NSCLC), as such driver mutations are able to cause cancers by themselves. However, we discovered the first reported lung cancer case that simultaneously harbored both an EML4-ALK fusion and an exon 19 EGFR mutation. We subsequently examined the driver gene mutations in a panel of consecutive, surgically resected, pathological Stage I lung adenocarcinomas to determine the frequency and clinicopathological characteristics in cases with double driver mutations. Patients and Methods: The initial case, which we reported previously (J Thorac Oncol. 2012, 7:e39-41), was a 72-year-old male ex-smoker who underwent right lower lobectomy for a lung peripheral mass of 4.5 cm in diameter. An EGFR mutation and EML4-ALK fusion were found in the tumor by immunohistochemistry and direct sequencing. To further examine the frequency of double driver mutations, a consecutive panel of 256 patients with pathological Stage I lung adenocarcinoma was analyzed for mutations of EGFR, K-ras, EML4-ALK and BRAF. Results: The initial case turned out to have both an exon 19 EGFR mutation and EML4-ALK fusion. Pathologically, the tumor comprised mixed papillary, acinar and lepidic adenocarcinoma, with small cell carcinoma in the central area. An EML4-ALK fusion was detected from the small cell component, while the EGFR mutation was detected from the peripheral lepidic adenocarcinoma component. Next, we analyzed the driver gene mutations in 256 Stage I lung adenocarcinoma cases. Mutations of the EGFR, K-ras, EML4-ALK and BRAF genes were detected in 120 (46.8%), 14 (5.5%), seven (2.7%) and three (1.2%) cases, respectively. Among them, double driver mutations were detected from two cases; one had an EML4-ALK fusion and exon 19 EGFR mutation, and another had a BRAF mutation (V600E) and exon 19 EGFR mutation. Discussion: Although the tumor from the initial case exhibited double driver mutations as a whole, a detailed examination of each histological component enabled us to distinguish one mutation from another. In other words, the driver mutations did not co-exist in a single cancer cell. Accordingly, similar molecular pathological investigations need to be done on newly identified cases with double driver mutations, because such studies may clarify how multiple mutations occur in one tumor. Conclusion: We demonstrated the frequency and the manifestations of rare double driver mutations in NSCLC. Further studies are warranted to clarify the mechanisms underlying the development of double driver mutations. Citation Format: Atsushi Osoegawa, Gouji Toyokawa, Taro Ohba, Michiyo Miyawaki, Masafumi Yamaguchi, Kenichi Taguchi, Takashi Seto, Mitsuhiro Takenoyama, Yukito Ichinose, Kenji Sugio. Non-small cell lung cancer with double driver mutations. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B13.

Abstract 2411: Rapamycin sensitizes cancer cells to growth inhibition by the PARP inhibitor olaparib

Poly (ADP-ribose) polymerase inhibitors (PARPi) have been developed and tested in a context of combining them with drugs that inhibit double-stranded (ds) DNA repair, because PARPi impair single-stranded (ss) DNA break repair, resulting in activation of dsDNA break repair machinery. Rapamycin, an mTOR inhibitor, has been widely prescribed for more than a decade, and recent studies revealed its ability to inhibit dsDNA break repair. The combination of the PARPi, olaparib, and rapamycin synergistically inhibited cell proliferation in non-small cell lung cancer (NSCLC) cells, as well as triple negative breast cancer (TNBC) cells that have BRCA1 mutations. Rad51, which forms a polymer on ssDNA upon dsDNA breaks, plays an essential role in homologous recombination. Olaparib induced Rad51 focus formation and rapamycin successfully inhibited it both in vivo and in vitro, suggesting this combination worked through blocking both ssDNA break repair and dsDNA break repair, resulting in cells that cannot go through G2/M checkpoint. The protein level of PARP was predictive for both PAR activity and Rad51 focus formation with this combination. Collectively, these data suggest that this combination could have a therapeutic potential to inhibit cancer with high PARP expression, or in combination with cytotoxic chemotherapy or radiation. Citation Format: Atsushi Osoegawa, Joell J. Gills, Shigeru Kawabata, Kenji Sugio, Phillip A. Dennis. Rapamycin sensitizes cancer cells to growth inhibition by the PARP inhibitor olaparib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2411. doi:10.1158/1538-7445.AM2014-2411