Yuko Tanabe

Utility of 10 Immunohistochemical Markers Including Novel Markers (Desmocollin-3, Glypican 3, S100A2, S100A7, and Sox-2) for Differential Diagnosis of Squamous Cell Carcinoma from Adenocarcinoma of the Lung

Introduction: Recent clinical trials revealed that accurate histologic typing of non-small cell lung cancer, especially squamous cell carcinoma (SCC), is essential. Patients and Methods: We analyzed 10 antibodies expression in 150 SCC cases (53 well-, 51 moderately, and 46 poorly differentiated cases) and 159 adenocarcinoma (AC) cases (49 well-, 52 moderately, and 58 poorly differentiated cases). Results: In all SCC and AC cases, p63 was the most sensitive marker for SCC (98.7%), followed by high-molecular-weight (HM) cytokeratin (CK) (97.3%), CK5/6 (93.3%), Sox2 (80%), thrombomodulin (79.3%), desmocollin-3 (72.7%), S100A7 (70.7%), S100A2 (63.3%), and glypican-3 (46.7%). Desmocollin-3 was the most specific marker for SCC (100%), followed by CK5/6 (98%), Sox2 (95.5%), glypican-3 (92.4%), S100A7 (86.8%), thrombomodulin (79.9%), S100A2 (64.6%), p63 (51.6%), and HMCK (33.3%). Thyroid transcription factor-1 (TTF-1) expression was observed in 87.4% of AC cases and 2.0% of SCC cases. When analyzing only poorly differentiated tumors, HMCK was the most sensitive marker for SCC (100%), followed by p63 (97.8%), CK5/6 (87.0%), Sox2 (71.7%), thrombomodulin (58.7%), desmocollin-3 (52.2%), S100A2 (50%), glypican-3 (45.7%), and S100A7 (45.7%). Desmocollin-3 was the most specific marker for poorly differentiated SCC (100%), followed by CK5/6 (98.3%), glypican-3 (94.8%), Sox2 (94.8%), S100A2 (81%), S100A7 (75.9%), thrombomodulin (72.4%), p63 (48.3%), and HMCK (36.8%). The 10-fold crossvalidated classification and regression tree analysis revealed that the combination of CK5/6 and TTF-1 was the best immunohistochemical marker panel for the differentiation between SCC and AC. Conclusion: CK5/6 is the best marker for differentiating SCC and AC, irrespective of the histological grade of the tumor. Thus, the combination of CK5/6 and TTF-1 is the most recommended combination of immunohistochemical markers.

Transmission of circulating cell-free AA amyloid oligomers in exosomes vectors via a prion-like mechanism

Recent studies clearly demonstrated that several types of pathogenic amyloid proteins acted as agents that could transmit amyloidosis by means of a prion-like mechanism. Systemic AA amyloidosis is one of the most severe complications of chronic inflammatory disorders, particularly rheumatoid arthritis. It is well known that, similar to an infectious prion protein, amyloid-enhancing factor (AEF) acts as a transmissible agent in AA amyloidosis. However, how AEF transmits AA amyloidosis in vivo remained to be fully elucidated. In the present study, we focused on finding cell-free forms of AEF and its carriers in circulation by using the murine transfer model of AA amyloidosis. We first determined that circulating cell-free AEF existed in blood and plasma in mice with systemic AA amyloidosis. Second, we established that plasma exosomes containing AA amyloid oligomers derived from serum amyloid A had AEF activity and could transmit systemic AA amyloidosis via a prion-like mechanism. These novel findings should provide insights into the transmission mechanism of systemic amyloidoses.

Phase I study of palbociclib, a cyclin-dependent kinase 4/6 inhibitor, in Japanese patients.

This phase I study in Japanese patients evaluated the safety, pharmacokinetics, and preliminary efficacy of palbociclib, a highly selective and reversible oral cyclin‐dependent kinase 4/6 inhibitor, as monotherapy for solid tumors (part 1) and combined with letrozole as first‐line treatment of postmenopausal patients with estrogen receptor‐positive, human epidermal growth factor receptor 2‐negative advanced breast cancer (part 2). Part 1 evaluated palbociclib 100 and 125 mg once daily (3 weeks on/1 week off; n = 6 each group) to determine the maximum tolerated dose. Part 2 evaluated palbociclib maximum tolerated dose (125 mg) plus letrozole 2.5 mg (n = 6). The most common treatment‐related adverse event was neutropenia (all grades/grade 3/4): 100 mg, 83%/67%; 125 mg, 67%/33%; and palbociclib plus letrozole, 100%/83%. Heavier pretreatment with chemotherapy may have resulted in higher neutropenia rates observed with the 100‐mg dose. Palbociclib exposure was higher with 125 vs 100 mg (mean area under the plasma concentration–time curve over dosing interval [τ]: 1322 vs 547.5 ng·h/mL [single dose], 2838 vs 1276 ng·h/mL [multiple dose]; mean maximum plasma concentration: 104.1 vs 41.4 ng/mL [single dose], 185.5 vs 77.4 ng/mL [multiple dose]). Half‐life was 23–26 h. No drug–drug interactions between palbociclib and letrozole occurred. Four patients had stable disease (≥24 weeks in one patient with rectal cancer [100 mg] and one with esophageal cancer [125 mg]) in part 1; two patients had partial response and two had stable disease (both ≥24 weeks) in part 2. Palbociclib at the 125‐mg dose (schedule 3/1) was tolerated and is the recommended dose for monotherapy and letrozole combination therapy in Japanese patients. The trials are registered with www.ClinicalTrials.gov: A5481010 and NCT01684215.

Comprehensive screening of target molecules by next-generation sequencing in patients with malignant solid tumors: guiding entry into phase I clinical trials

It is still controversial whether comprehensive genome screening of target molecules by next generation sequencing (NGS) is needed to increase clinical efficacy of investigational drugs or accelerate drug development, although several studies are being carried out. Therefore, we performed a prospective study to evaluate the feasibility of comprehensive gene screening in this setting. Our findings indicate that actionable alterations were identified in 45% of the analyzed patients, most frequently in those with breast cancer. Common actionable alterations were found in PIK3CA mutation, BRCA2 mutation, ERBB2 amplification, and CCND1 amplification. In total, 22% of the analyzed patients could be entered into phase I clinical trials, and 8% of them were treated with “matched” drugs. Among patients who received matched therapies, response and disease control rates were 33 and 78%, respectively. On the other hand, in the patients who received “non-matched” therapy, the objective response rate was 6%. We believe this data indicates that NGS-based molecular pre-screening is a potent platform for use before patient entry into phase I trials.

Tremelimumab-associated tumor regression following after initial progression: two case reports

The human IgG2 monoclonal antibody tremelimumab is an immune checkpoint inhibitor that blocks cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). The therapeutic response of anti-CTLA-4 monoclonal antibodies possess unique kinetics, in that antitumor responses are often observed after initial short-term disease progression, in some cases as long as 6-12 months after anti-CTLA-4 treatment initiation. Here, we report two cases: one of bile duct cancer and the other of squamous cell carcinoma of unknown primary, both of which demonstrated initial rapid disease progression followed by dramatic tumor shrinkage after one or two doses of tremelimumab, without any immune-related adverse events. This delayed, yet dramatic antitumor response suggests that tremelimumab may hold promise in the treatment of solid tumors.

Loss of BAP1 protein expression in the first metastatic site predicts prognosis in patients with clear cell renal cell carcinoma

OBJECTIVES To investigate the intratumoral heterogeneity of BAP1 and PBRM1 expression at the primary site and metastatic sites and to evaluate whether BAP1 and PBRM1 expression in metastatic sites of clear cell renal cell carcinoma (ccRCC) has prognostic value. METHODS AND MATERIALS We collected paired samples from the primary site and the first metastatic site in 41 patients with ccRCC. Immunohistochemistry analyses were performed for the expression of BAP1 and PBRM1 proteins. We retrospectively analyzed the associations between the expression of BAP1 and PBRM1 and overall survival (OS). RESULTS The most common first metastatic sites were lung (68.3%) and lymph node (12.2%). BAP1 protein expression was negative in 8 (19.5%) primary sites and in 11 (26.8%) metastatic sites. PBRM1 protein expression was negative in 9 (22.0%) primary sites and in 11 (26.8%) metastatic sites. The incidences of intratumoral heterogeneity for BAP1 and PBRM1 protein expression in primary/metastatic sites were 9.8%/2.4% and 24.4%/7.3%, respectively. The concordance rates between primary and metastatic sites for BAP1 and PBRM1 protein expression were 82.9% and 63.4%, respectively. Median OS from the first occurrence of metastasis in patients with BAP1-positive and BAP1-negative metastatic sites were 97 months (95% CI: 58-136) and 51 months (95% CI: 13-82), respectively (P = 0.0077). Median OS in patients with PBRM1-positive and PBRM1-negative metastatic sites were 82 (95% CI: 42-97) and 120 (95% CI: 52-120) months, respectively (P = 0.25). CONCLUSION Intratumoral heterogeneity of BAP1 protein expression is more frequent in primary tumor than in metastatic sites. The loss of BAP1 protein expression in metastatic sites predicts poor prognosis in patients with ccRCC.

Prognostic value of Programmed death-ligand 1 expression in patients with stage III colorectal cancer

The programmed death‐1/programmed death‐ligand 1 (PD‐L1) pathway is a negative feedback pathway that suppresses the activity of T cells. Previous studies reported that high PD‐L1 expression on tumor cells (TC) was associated with poor survival in patients with colorectal cancer; however, the prognostic evaluation of these studies was limited because they included patients at various disease stages. The purpose of the present study was to evaluate the relationship between PD‐L1 status in the immune microenvironment and the clinicopathological features of stage III colorectal cancer. Two hundred and thirty‐five patients were included in the analysis. PD‐L1 expression on TC and tumor‐infiltrating mononuclear cells (TIMC) was evaluated by immunohistochemistry. The median follow‐up of thisi study was 52.9 months. A total of 8.1% of stage III colorectal cancer showed high PD‐L1 expression on TC and 15.3% showed high PD‐L1 expression on TIMC. Patients with high PD‐L1 expression on TC had significantly shorter disease‐free survival (DFS) than patients with low expression (hazard ratio [HR] 2.36; 95% confidence interval [CI], 1.21–4.62; P = 0.012). In addition, patients with high PD‐L1 expression on TIMC were associated with longer DFS than patients with low expression (HR 0.40; 95% CI, 0.16–0.98; P = 0.046). These findings suggest that PD‐L1 expression status may be a new predictor of recurrence for stage III colorectal cancer patients and highlight the necessity of evaluating PD‐L1 expression on TC and TIMC separately in the tumor microenvironment.

Pathological features of triple‐negative breast cancers that showed progressive disease during neoadjuvant chemotherapy

Clinical progressive disease (cPD) occurs during neoadjuvant chemotherapy (NAC) in 3%–5% of triple‐negative breast cancer (TNBC) patients. We aimed to identify the histopathological and immunohistochemical parameters that are correlated with the TNBC that showed cPD. We identified 22 TNBCs that showed cPD during NAC (cPD group) and 80 TNBCs that did not receive NAC (control group). Using surgically resected tumor specimens, we performed histopathologic examinations and immunohistochemical analysis of 11 molecules that appeared relevant to epithelial‐mesenchymal transition (EMT), and basal‐like, molecular apocrine and other features. Metaplastic carcinomas (MPCs) and high proliferation (≥50 mitoses per 10 high‐power fields or ≥50% Ki‐67 score) were more frequent in the cPD than in the control (41% vs 3%, P < 0.001, and 86% vs 50%, P = 0.0049, respectively). Positive cytokeratin 5/6, ZEB1, TWISTNB, vimentin, and HMGB1 expressions and negative androgen receptor were more frequent in the cPD than in the control. By an unsupervised hierarchical cluster analysis incorporating these 11 molecules, the 102 TNBCs were divided into two major clusters and seven subclusters that appeared to correspond to intrinsic subtype, cPD status, histological type, and clinical outcome. In 27% of cPD cases, the MPC component appeared only in the post‐NAC specimens. The combinations of high proliferation, metaplastic features, and immunohistochemical statuses of some EMT and basal‐like markers and androgen receptor appeared to be able to characterize the TNBCs that showed cPD after NAC.

441OIMAGING MASS SPECTROMETRY OF NOVEL DRUG IN HUMAN TUMOR SPECIMENS: DISTRIBUTION OF UNLABELED DRUGS TO SUPPORT EARLY PHASE CLINICAL TRIAL

ABSTRACT Aim: Assessment of drug pharmacokinetics is an important component of early phase drug development. Imaging Mass Spectrometry (IMS) is an innovative technique in the preclinical study that allows for analysis of the distribution of target molecules in tumor tissues. The advantage of imaging technology is the detection of the molecule of interest in tissues without labeling. We performed tumor biopsies in patients with solid tumors who were participating in a phase I trial (NCT01813474) of olaparib. The aim of this study was to examine drug distribution in the tumor biopsy specimens by IMS, which provides a sensitive and label-free approach to imaging drugs. Methods: Patients with solid tumors received the tablet formulation of olaparib in dose escalation (200mg BID; 300mg BID) and expansion (300mg BID) cohorts. The timing of biopsies in consenting patients was during cycle 2 and/or at the time of progression. IMS was performed using an Imaging Mass Microscope (Shimadzu, Japan). The concentrations of olaparib in tissues were validated by using LC-MS/MS. Results: In total, seven tumor biopsies were performed in six patients with solid tumors; three breast cancer including one BRCA1 mutation positive, one ovarian cancer, one peritoneal cancer, one cervical cancer. One patient had biopsies performed at the time of drug administration and progression. One patient received olaparib 200mg BID; the remaining patients received olaparib 300mg BID. IMS signal levels of olaparib correlated well with the concentration of drug in tumor tissues derived from patients using LC-MS/MS, a conventional method used in pharmacokinetic studies. The distribution of Olaparib was in the tumor region and the signal level in areas of necrosis was higher than that observed in living cell areas. Conclusions: The use of IMS has allowed to follow the distribution of an unlabeled olaparib in target tissues. In addition, this technique can also allow further understanding of PK/PD relationships of olaparib in combination with other compound at clinical trial. Disclosure: All authors have declared no conflicts of interest.

Inactive immune pathways in triple negative breast cancers that showed resistance to neoadjuvant chemotherapy as inferred from kinase activity profiles

About 5% of Triple negative breast cancer patients (TNBCs) who receive neoadjuvant chemotherapy (NAC) experience progressive disease (PD). Few reports are published on TNBCs with PD during NAC, whereas TNBCs that respond to NAC have been well-studied. We investigated kinase activity profiles of TNBCs to explore the biological differences underlying the lack of response to NAC. Among 740 TNBCs, 20 non-responders were identified. Seven non-responders and 10 TNBCs that did not receive NAC (control group) were evaluated. No correlation was observed between NAC response and age, menopausal status, tumor size and axillary lymph node status. Tyrosine kinase activity profiles of TNBC primary tissues from NAC non-responders and the controls were determined with a peptide microarray system. Kinase activity measurements showed that 35 peptides had significantly (p < 0.05) lower phosphorylation in non-responders. ZAP70, LCK, SYK and JAK2 were identified as differentially active upstream kinases. Pathway analysis suggested lower activity in immune-related pathways in non-responders. The number of tumor infiltrating lymphocytes (TILs) was significantly lower (p = 0.0053) in non-responders. Kinases related to the immune system are less activated in non-responders. TILs evaluation suggested that the immune system is hardly active in non-responders and is not activated by NAC treatment.