Tobias Grob

HNSCC cell lines positive for HPV and p16 possess higher cellular radiosensitivity due to an impaired DSB repair capacity

BACKGROUND AND PURPOSE When treated by radiotherapy, patients with squamous cell carcinomas of the head and neck (HNSCC) positive for HPV and p16(INK4a) possess a clearly favorable prognosis as compared to those with HPV-negative HNSCC. The aim of this work was to study whether the better outcomes might be caused by an enhanced cellular radiosensitivity. MATERIALS AND METHODS The radiation response of five HPV/p16(INK4a)-positive and five HPV-negative cell lines was characterized with regard to cellular radiosensitivity by colony formation assay. Furthermore G1- and G2-arrest, apoptosis and residual DNA double-strand breaks (DSB) were analyzed by the colcemid-based G1-efflux assay, propidium iodide staining, the detection of PARP cleavage, the fluorescence-based detection of caspase activity and the immunofluorescence staining of γH2AX and 53BP1 foci. RESULTS On average, the cellular radiosensitivity of the HNSCC cell lines positive for HPV and p16(INK4a) was higher as compared to the sensitivity of a panel of five HPV-negative HNSCC cell lines (SF3=0.2827 vs. 0.4455). The higher sensitivity does not result from increased apoptosis or the execution of a permanent G1-arrest, but is rather associated with both, elevated levels of residual DSBs and extensive G2-arrest. CONCLUSIONS Increased cellular radiosensitivity due to compromised DNA repair capacity is likely to contribute to the improved outcome of patients with HPV/p16(INK4a)-positive tumors when treated by radiotherapy.

Reliability of Human Epidermal Growth Factor Receptor 2 Immunohistochemistry in Breast Core Needle Biopsies

PURPOSE Core needle biopsies (CNBs) are widely used to determine human epidermal growth factor receptor 2 (HER2) status in breast cancer. Recent publications reported up to 20% false-positive results on CNBs if immunohistochemistry (IHC) is compared with fluorescent in situ hybridization (FISH). To clarify, if confirmation of IHC positivity by FISH is generally required, we analyzed the reliability of IHC positivity on CNBs versus surgical specimens in a multi-institutional study. PATIENTS AND METHODS Five pathologic laboratories contributed to this study by performing IHC on 500 CNBs and the corresponding surgical specimens overall. If IHC revealed score 2+ or 3+, HER2 status was confirmed by FISH in a central laboratory. We compared evaluation according to US Food and Drug Administration-approved scoring criteria and recently published American Society of Clinical Oncology (ASCO)-College of American Pathologists (CAP) guidelines. RESULTS CNBs scored 3+ revealed five false-positive results if scoring followed the US Food and Drug Administration criteria (five of 40; 12.5%) and two false-positives in terms of the ASCO-CAP criteria (two of 33; 6.1%). IHC was false negative in one CNB only. By contrast, IHC on surgical specimens revealed five false-negative results, but only one false-positive result (one of 35; 2.9%) if scored following US Food and Drug Administration-approved criteria. With the aid of the ASCO-CAP criteria, false-positive IHC results were obtained in only one of the five participating institutions. CONCLUSION IHC 3+ scores on CNBs proved to be reliable in four of the five participating institutions if scoring followed the ASCO-CAP criteria. Therefore, accurate determination of HER2 status in breast cancer is possible on CNB using the common strategy to screen all cases by IHC and retest only 2+ scores by FISH. Prerequisites are quality assurance and the application of the new ASCO-CAP criteria.

HPV status in patients with head and neck of carcinoma of unknown primary site: HPV, tobacco smoking, and outcome.

OBJECTIVES Infection with human papillomavirus (HPV) is linked to oropharyngeal cancer. This analysis investigated possible associations between HPV status, smoking history and survival outcome in patients with neck metastasis and carcinoma of unknown primary (CUP). MATERIALS AND METHODS Registries at the Universities of Hamburg and Kiel were searched for patients with CUP diagnosed from 2002 to 2011 who had formalin-fixed and paraffin-embedded metastatic lymph node samples available. All patients underwent routine diagnostic procedures to establish the primary site and received radiotherapy (60Gy using conventional fractionation) with or without concurrent cisplatin-based chemotherapy depending on disease extent. Genotyping was performed using polymerase chain reaction; p16([INK4a]) expression was assessed using immunohistochemistry. RESULTS Sixty-three patients were included; 23 (37%) had HPV DNA/p16+ samples and 40 (63%) were negative for either/both markers. A high proportion of patients had a history of tobacco smoking; significantly fewer patients with HPV+/p16+ samples were smokers than those who were negative for either/both markers (61% vs. 90%, respectively; p = 0.0067). There were no statistically significant differences between overall or recurrence-free survival in HPV+/p16+ patients vs. those negative for either/both markers. Overall survival appeared to be superior in patients with <10 pack-years smoking history and HPV+/p16+ disease. CONCLUSIONS This study, the largest to date investigating HPV status in head and neck CUP, identified HPV and p16 overexpression in over one-third of patients. Tobacco smoking history appeared to affect survival in HPV+/p16+ patients. Smoking status should be considered as a prognostic factor in patients with CUP, along with HPV DNA status.

Miliary never-smoking adenocarcinoma of the lung: strong association with epidermal growth factor receptor exon 19 deletion.

Miliary pattern of pulmonary metastases is a rarity in patients with lung cancer. We report five cases of patients with a never-smoking adenocarcinoma of the lung with such a pattern of metastases. In the tumor cells of all five patients, epidermal growth factor receptor (EGFR) mutation gene sequencing identified a deletion in exon 19 of the EGFR gene, and all five patients had a dramatic response to EGFR tyrosine kinase inhibitors. No echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) translocation was detected. We believe that the miliary never-soking adenocarcinoma of the lung is a distinct clinically relevant subgroup of the never-smoking non-small cell lung cancer. Physician should recognize this subgroup of patients with lung cancer when facing a picture of miliary pulmonary metastases in chest x-ray or computed tomography scan in patients with a history of never smoking and consider upfront therapy with EGFR tyrosine kinase inhibitors.

Heterogeneity of ERBB2 amplification in adenocarcinoma, squamous cell carcinoma and large cell undifferentiated carcinoma of the lung

The HER2 protein, encoded by the ERBB2 gene, is a molecular target for the treatment of breast and gastric cancer by monoclonal antibodies or tyrosine kinase inhibitors. While intratumoral heterogeneity of ERBB2 amplification is rare in breast cancer it is reported to be frequent in bladder and colorectal cancer. To address the potential heterogeneity of the HER2 status in adenocarcinomas, squamous cell carcinomas and large cell undifferentiated carcinomas of the lung, 590 tumors were analyzed for HER2 overexpression and ERBB2 amplification using FDA-approved reagents for immunohistochemistry and fluorescence in-situ hybridization (FISH). Moderate and strong immunostaining (2+, 3+) was seen in 10% of the tumors. ERBB2 amplification was found in 17 (3%) lung cancer patients including 10 cases (2%) with high-level amplification forming gene clusters. ERBB2 amplification was significantly related to histologic subtype and tumor grade, resulting in 12% ERBB2 amplified tumors in the subgroup of high-grade adenocarcinomas. Heterogeneity was analyzed in all highly amplified tumors. For this purpose, all available tumor tissue blocks from these patients were evaluated. Heterogeneity of ERBB2 amplification was found in 4 of 10 tumors as assessed by FISH. These included two tumors with a mixture of low-level and high-level amplification and two tumors with non-amplified tumor areas next to regions with high-level ERBB2 amplification. High-level ERBB2 amplification occurs in a small fraction of lung cancers with a strong propensity to high-grade adenocarcinomas. Heterogeneity of amplification may limit the utility of anti-HER2 therapy in some of these tumors. Further attempts to assess the utility of HER2-targeting therapy in homogeneously amplified lung cancers appear to be justified.

Oestrogen receptor gene (ESR1) amplification is frequent in endometrial carcinoma and its precursor lesions.

Oestrogen receptor alpha (ER) plays a critical, diverse and not fully understood role in endometrial carcinoma. Most endometrial carcinomas express ER and some of these tumours respond favourably to anti‐oestrogen therapy. On the other hand, tamoxifen therapy constitutes a major risk factor for endometrial carcinoma development. Amplification of the ESR1 gene encoding ER was recently shown to constitute a mechanism for ER over‐expression in breast carcinoma. This study was designed to determine the potential role of ESR1 amplifications in endometrial carcinoma. Tissue microarrays of 368 endometrial carcinomas and large sections of 43 cases of endometrial hyperplasia were analysed for ESR1 gene amplification and ER protein expression by means of fluorescence in situ hybridization (FISH) and immunohistochemistry. FISH revealed ESR1 amplification in 40/176 (23%) cancers, 6/19 (32%) atypical complex hyperplasias, 3/10 (30%) complex hyperplasias without atypia and 2/14 (14%) simple hyperplasias without atypia. Strong ER protein expression was significantly linked to ESR1 amplification in endometrial carcinoma (p = 0.0036). These data indicate that ESR1 amplification might be one mechanism for ER over‐expression in endometrial carcinoma, and suggest an early role for ESR1 amplification in the development of a significant fraction of endometrial carcinoma. Given the predictive role of ESR1 amplification for tamoxifen response in breast carcinoma, it will be interesting to investigate the response of ESR1‐amplified endometrial cancers to anti‐oestrogenic drugs. Copyright

Radiosensitization of NSCLC cells by EGFR inhibition is the result of an enhanced p53-dependent G1 arrest.

PURPOSE How EGF receptor (EGFR) inhibition induces cellular radiosensitization and with that increase in tumor control is still a matter of discussion. Since EGFR predominantly regulates cell cycle and proliferation, we studied whether a G1-arrest caused by EGFR inhibition may contribute to these effects. MATERIALS AND METHODS We analyzed human non-small cell lung cancer (NSCLC) cell lines either wild type (wt) or mutated in p53 (A549, H460, vs. H1299, H3122) and HCT116 cells (p21 wt and negative). EGFR was inhibited by BIBX1382BS, erlotinib or cetuximab; p21 was knocked down by siRNA. Functional endpoints analyzed were cell signaling, proliferation, G1-arrest, cell survival as well as tumor control using an A549 tumor model. RESULTS When combined with IR, EGFR inhibition enhances the radiation-induced permanent G1 arrest, though solely in cells with intact p53/p21 signaling. This increase in G1-arrest was always associated with enhanced cellular radiosensitivity. Strikingly, this effect was abrogated when cells were re-stimulated, suggesting the initiation of dormancy. In line with this, only a small non-significant increase in tumor control was observed for A549 tumors treated with fractionated RT and EGFR inhibition. CONCLUSION For NSCLC cells increase in radiosensitivity by EGFR inhibition results from enhanced G1-arrest. However, this effect does not lead to improved tumor control because cells can be released from this arrest by re-stimulation.

Epidermal growth factor receptor (EGFR) in salivary gland carcinomas: Potentials as therapeutic target

AIMS Epidermal growth factor (EGFR) is involved in angiogenesis, cell differentiation, proliferation and progression of many cancers and is an important therapy target in lung and colorectal cancer. To determine the potential applicability of EGFR targeted therapies, EGFR status of over 800 salivary gland tumors of different entities were analyzed on DNA and protein level by FISH and IHC. MATERIALS AND METHODS A tissue microarray was constructed from 721 carcinomas and 205 adenomas of the salivary gland. EGFR expression and EGFR gene copy number was assessed by means of immunohistochemistry and fluorescence in situ hybridization (FISH). EGFR mutation analysis of exon 19 and 21 was performed in a subset of 107 carcinomas. RESULTS Positive immunohistochemical staining (definition?) for EGFR was shown in 324 of 663 (48.9%) salivary gland carcinomas. The frequency was dependent on the tumor entity and ranged from 17.9% (30 of 168 cases) positive immunostaining in acinic cell adenocarcinomas to 85.7% (42 of 49 cases) in Warthin tumors. No EGFR amplification was found by FISH. EGFR mutation analysis of Exon 19 and 21 in 107 salivary gland carcinomas revealed mutations in two acinic cell adenocarcinomas. CONCLUSION EGFR protein expression is common in salivary gland tumors but is not associated with gene amplification. Activating mutations of EGFR are rare. Nonetheless, selected cases of patients with salivary gland carcinomas might potentially benefit of anti-EGFR therapy.

Sorafenib sensitizes head and neck squamous cell carcinoma cells to ionizing radiation

BACKGROUND AND PURPOSE There is a great need to improve the outcome of locoregionally advanced squamous cell carcinomas of the head and neck (HNSCC). Standard treatment includes a combination of surgery, radio- and chemotherapy. The addition of molecular targeting agents to conventional treatment may improve outcomes. In this study the Raf inhibitor sorafenib was used to increase the radiosensitivity of HNSCC cell lines. MATERIAL AND METHODS In a panel of six cell lines (A549, FaDu, UTSCC 60A, UTSCC 42A, UTSCC 42B, UTSCC 29) radiosensitivity was measured by colony formation assay and apoptosis and cell cycle analysis were performed by flow cytometry. DNA repair was analyzed by 53BP1 immunohistochemistry. RESULTS Sorafenib added prior to irradiation resulted in an increased cellular radiosensitivity (DEF0.5=1.11-1.84). Radiosensitization was not caused by an enhanced rate of apoptosis or cell cycle effects. In contrast, sorafenib was shown for the first time to block the repair of DNA double-strand breaks (DSB). CONCLUSION Our data suggest that sorafenib may be used to overcome the radioresistance of HNSCC through the inhibition of DSB repair.

Frequent intratumoral heterogeneity of EGFR gene copy gain in non-small cell lung cancer

Next to EGFR mutation, EGFR gene copy number evaluated by fluorescence in situ hybridization (FISH) emerged as a potential predictive marker for sensitivity to EGFR tyrosine kinase inhibitors, although controversial data exist. As the diagnostic accuracy of predictive biomarkers can be substantially limited by regional differences within tumors, heterogeneity of EGFR gene copy gain in NSCLC was assessed in this study. For this purpose, a novel tissue microarray (TMA) based analysis platform was developed. TMAs were constructed containing 8 different tissue cylinders from 144 primary NSCLCs. From 62 of these patients additional nodal metastases were sampled. EGFR gene copy number and EGFR expression was analyzed by FISH and immunohistochemistry according to the suggested guidelines. 13 (9.0%) of the 144 evaluated tumors showed EGFR amplification and 37 (25.7%) tumors high polysomy in at least one tumor area. In 7 (53.8%) of 13 amplified cases the analysis of different tumor areas revealed subclones without EGFR gene copy gain next to subclones with amplification. All of the 36 evaluable tumors with high polysomy showed heterogeneity of EGFR gene copy number with areas negative for gene copy gain within the individual tumors. Heterogeneity of EGFR gene copy gain in lung cancer challenges the concept of using small biopsies for the analysis of EGFR FISH status. EGFR gene copy number is highly heterogeneous within individual NSCLCs and this finding might well be a reason for the controversial clinical data existing regarding responsiveness to anti-EGFR therapy.