Jaakko Pulkkinen

Signaling via ErbB2 and ErbB3 Associates with Resistance and Epidermal Growth Factor Receptor (EGFR) Amplification with Sensitivity to EGFR Inhibitor Gefitinib in Head and Neck Squamous Cell Carcinoma Cells

Purpose: The epidermal growth factor receptor (EGFR) inhibitor gefitinib (Iressa) has shown antitumor activity in clinical trials against cancers, such as non–small cell lung cancer and head and neck squamous cell carcinoma (HNSCC). Research on non–small cell lung cancer has elucidated factors that may predict response to gefitinib. Less is known about molecular markers that may predict response to gefitinib in HNSCC patients. Experimental Design: We analyzed possible associations of responsiveness to gefitinib with molecular markers of the EGFR/ErbB receptor family signaling pathway using 10 established HNSCC lines in vitro. IC50 of gefitinib sensitivity was determined using clonogenic survival assays. ErbB signaling was assessed by Western and real-time reverse transcription-PCR analyses of EGFR, ErbB2, ErbB3, and ErbB4 expression levels as well as by phosphorylation analysis of pEGFR, pErbB2, pErbB3, pAkt, and pErk. EGFR sequences encoding kinase domain and EGFR gene copy numbers were determined by cDNA sequencing and real-time PCR, respectively. Finally, responsiveness to gefitinib was compared with responsiveness to the anti-EGFR antibody cetuximab (Erbitux). Results: Expression levels of pErbB2 (P = 0.02) and total ErbB3 protein (P = 0.02) associated with resistance to gefitinib. Combining gefitinib with pertuzumab (Omnitarg), an antibody targeting ErbB2 heterodimerization, provided additional growth-inhibitory effect over gefitinib alone on relatively gefitinib-resistant HNSCC cell lines. The same markers did not predict resistance to cetuximab. In contrast, a similar trend suggesting association between EGFR gene copy number and drug sensitivity was observed for both gefitinib (P = 0.0498) and cetuximab (P = 0.053). No activating EGFR mutations were identified. Conclusions:EGFR amplification may predict sensitivity to gefitinib in HNSCC. However, other EGFR/ErbB receptor family members than EGFR may contribute to resistance to gefitinib. ErbB2 and ErbB3 may have potential as predictive markers and as therapeutic targets for combination therapy in treatment of HNSCC with gefitinib.

Syndecan-1: A New Prognostic Marker in Laryngeal Cancer

The cell adhesion molecule syndecan-1 expression is induced during keratinocyte differentiation and reduced during the formation of squamous cell carcinomas (SCCs). A significant correlation between decreased syndecan-1 expression in head and neck SCC measured from frozen sections with immuohistochemical methods and clinical outcome are reported. The clinical relevance of the cellular proliferation marker Ki-67 is controversial in SCC of the head and neck. The purpose of this study was to determine the expression of syndecan-1 and Ki-67 in SCC of the larynx and correlate the results with known prognostic factors and clinical outcome. Paraffin-embedded samples of 100 patients with laryngeal SCC (44 glottic, 36 supraglottic, 20 transglottic) treated at Turku University Central Hospital were re-examined and divided into four histological grades of differentiation, four grades of keratinisation, and four grades of 104-9 (syndecan-1) immunostaining. The mitotic index was analysed as the number of mitoses per volume corrected high power fields. The relative number of Ki-67 positive cells was evaluated. The patients mean age was 64 years and the 5-year survival was 69%. In univariate analysis, intermediate or strong staining for syndecan-1 was associated with higher overall survival than those tumours with no or little syndecan-1 expression (p = 0.048). Nodal status (p = 0.0001), tumour size (p = 0.0004) and localisation (p = 0.0008), general condition (p = 0.0001), histological grade (p = 0.02) and patient age (p = 0.03) correlated with overall survival whereas the Ki-67 index (p = 0.093), mitotic index (p = 0.23) and grade of keratinisation (p = 0.90) failed to do so. The results suggest that syndecan-1 could be a useful prognostic factor in SCC of the larynx.

Bioactive glass S53P4 in the filling of cavities in the mastoid cell area in surgery for chronic otitis media.

Objectives Chronic infection of the middle ear and cholesteatoma can be treated surgically by exenteration of the mastoid air cells behind the ear. After a procedure with the canal wall–down technique, a cavity remains that is sometimes difficult to clean, collects crust, and becomes repeatedly infected. Such problematic mastoid cavities can be eliminated by filling the created cavity surgically after thorough removal of mucous membranes and cleaning of the bone. Methods We treated 7 patients with cavities after canal wall–down surgery for the treatment of chronic suppurative otitis media or cholesteatoma by filling the difficult-to-clean cavity in the mastoid cell area with granules of bioactive glass (BAG) S53P4 to avoid further retraction formation. The area with BAG was carefully closed with a musculoperiosteal flap. Results After the canal wall–down tympanomastoidectomy, the mastoid cavities were successfully filled in all 7 patients. No biomaterial-associated infection was seen, and no disadvantages for the patients due to the BAG were observed. The cavity in the mastoid cell area decreased in size in all patients treated. Conclusions This BAG seems to be a promising material for filling mastoid cavities after canal wall–down tympanomastoidectomy.

Paclitaxel-induced apoptotic changes followed by time-lapse video microscopy in cell lines established from head and neck cancer

Paclitaxel (Taxol) is a potent chemotherapeutic drug for squamous-cell carcinoma (SCC) of the head and neckin vitro with microtubule-stabilizing activity that arrests cells in G2-M. To study the mechanism of its cytotoxic effect on SCCin vitro, we exposed five laryngeal SCC cell lines to 10 nM paclitaxel. The cell lines were studied by time-lapse video microscopy for 96 h, and by agarose gel electrophoresis. Paclitaxel blocked the cells in the premitotic phase for 6–24 h, after which the cells died morphologically by apoptosis. Mitotically arrested cells were seen within a few minutes after exposure to paclitaxel. No mitoses were seen in the paclitaxel-treated cells. A few apoptoses were also seen in the control cultures grown without paclitaxel, but they represented only 6%–20% of the frequency of apoptoses seen in the paclitaxel-treated group. In some paclitaxel-treated cultures the cells escaped the mitotic arrest without cytokinesis and formed multinucleated cells that eventually died. Agarose gel electrophoresis showed oligonucleosomal DNA fragmentation characteristic of apoptosis. We conclude that time-lapse video microscopy is an efficient method of observing drug-induced morphological changes in cell culture. Paclitaxel at a 10 nM concentration rapidly induces a premitotic block, which usually leads to apoptotic cell death. In some cases multinucleated cells are formed that morphologically also eventually die by apoptosis.

Cervical Metastasis of Unknown Origin: A Series of 72 Patients

Cervical metastasis of unknown origin is still a challenging problem because of its relatively poor prognosis and the uncertainty regarding the primary site. We analyzed retrospectively all 72 patients with cervical metastases of unknown origin, diagnosed and treated between 1985 and 1995 in the five university hospitals of Finland in order to analyze survival rates and some prognostic and clinical factors of the disease. The most common sites where the primary tumor was found during follow-up or at autopsy were the lung (8%), the oral and pharyngeal region (7%) and the skin (6%). When the lower neck nodes (regions IV-VI) were affected, the primary tumor was significantly ( p <0.001) more often found from the subclavicular sites. The disease-specific 5-year survival rate was 32%. In multivariate analysis, nodal stage N2c or N3 [adjusted relative hazard of death (HR) 2.43], other metastases found at the time of treatment (HR 2.15) and age>65 years (HR 2.12) were significantly associated with a poor prognosis. Median survival tended to be longer for patients treated with surgery combined with radiotherapy (39.9 months) compared with those treated with radiotherapy alone (16.8 months), but this difference was not statistically significant ( p =0.153).

Early percutaneous endoscopic gastrostomy nutrition in head and neck cancer patients

Objective Many head and neck cancer patients suffer from poor nutrition. Nutrition is a problem during and after therapy, especially when it consists of extensive surgery, intensive (chemo)radiotherapy or their combination. Additional enteral nutrition has been provided by means of either nasogastric tube feeding, surgical gastrostomy, radiologic percutaneous gastrostomy or percutaneous endoscopic gastrostomy (PEG). Because of the straightforward, easy technique involved and its low complication rate, PEG has become established as the primary route of nutrition in these patients. Previously, the aim of assisted enteral nutrition was to compensate for already existing malnutrition; nowadays, an additional purpose is to diminish or prevent the development of malnutrition. The main objective of this study was to evaluate the safety of pre-treatment PEG in a sample of patients with an upper aerodigestive tract area malignancy treated in a tertiary referral centre. Material and methods A total of 79 patients with an upper aerodigestive tract area malignancy were treated with a total of 80 PEGs during the period 1997–2001. Results Most of the PEGs (62/80; 77.5%) were performed by an otolaryngologist. An open gastrostomy was needed in five cases because of unsuccessful gastroscopy due to oesophageal stricture (n=4) or severe trismus (n=1). Both acute and late complications were minor and the respective complication rates (1/80; 1.3% and 12/80; 15%) were low. In addition, all complications were easily managed and did not seriously affect the actual treatment. Conclusions A major advantage of having the PEG performed by the otorhinolaryngologist was the possibility to combine it easily with other necessary procedures, such as panendoscopy, tracheostomy and additional biopsy. In addition, the timing of the procedure was easy to schedule.

Bioactive Glass S53P4 in Mastoid Obliteration Surgery for Chronic Otitis Media and Cerebrospinal Fluid Leakage

Objectives: We evaluated the results of cases of chronic otitis media treated with mastoid obliteration surgery using bioactive glass S53P4. Methods: Twenty-five patients with chronic otitis media and 1 patient with cerebrospinal fluid leakage without chronic infection were treated with bioactive glass S53P4. Twenty patients had had previous surgery because of chronic otitis media with or without cholesteatoma. A mastoid obliteration was performed with bioactive glass S53P4 granules and a musculoperiosteal flap with or without bone paté. In 2 patients with a bony dehiscence at the middle cranial fossa, a bioactive glass plate was used to support the protruding dura. In addition, in 3 patients, occlusion of a dural fistula was needed. The median follow-up period was 34.5 months (range, 1 to 182 months). Results: Excluding the 2 patients with only 1 month of follow-up at our department, 96% of the patients had a dry, safe ear or only intermittent otorrhea. In 92% of the patients, the objective of achieving a smaller or nonexistent cavity was achieved. Conclusions: Bioactive glass S53P4 is a noteworthy material in mastoid obliteration surgery.

Parotidectomy in the treatment of pleomorphic adenoma – analysis of long-term results

Abstract Conclusion: Parotidectomy is an efficient surgical treatment modality for pleomorphic adenoma of the parotid gland, although some morbidity may occur. In this study, the median time interval between primary surgery and the presentation of the recurrent tumor was observed to be 14.4 years. Objective: Analysis of the long-term results of patients undergoing lateral or total parotidectomy as first-line treatment of parotid pleomorphic adenoma at our institution between the years 1979 and 1996. Methods: The individual patient charts of 230 patients were feasible for retrospective analysis in 2007. Results: In all, 42 patients had dysfunction of the facial nerve after the primary surgery, but only 14 of them had permanent dysfunction. A recurrent tumor occurred in nine cases (3.9%). The time interval between primary surgery and the first recurrence ranged from 7.1 to 24.5 years. Recurrent tumors were treated with surgery, two patients received additional radiotherapy.

Ultrasonically activated scalpel compared with electrocautery in tonsillectomy.

The occurrence of postoperative bleeding, the quantity of operative bleeding and the duration of operation were retrospectively evaluated in 407 patients who underwent tonsillectomy within a 32-month period. They were operated on with either an ultrasonically activated scalpel (UAS), bipolar diathermy (BPD) or blunt dissection with monopolar diathermy (MPD) with the following results. (1) Primary bleeding was more common with MPD: MPD 7.1% vs. BPD 2.4% (p < 0.01) vs. UAS 1.0% (p < 0.001). Secondary bleeding was more common with UAS: UAS 19.6% vs. MPD 14.5% (p < 0.001) vs. BPD 14.5% (p < 0.01). There was no statistical significance in the differences between overall postoperative bleeding rates. (2) There was statistically significantly less operative bleeding with UAS:UAS 24.8 ml vs. MPD 58.7 ml vs. BPD 43.8 ml. (3) On the other hand, the operation time was on average longer with UAS: UAS 32.3 min vs. MPD 18.4 min vs. BPD 22.1 min. Our results suggest that UAS offers no significant advantage over MPD or BPD in tonsillectomy other than minimal operative bleeding possibly due to longer duration of operation.

Concomitant vinorelbine and radiation in head and neck squamous cell carcinoma in vitro.

Concomitant chemoradiotherapy has been used for locally advanced head and neck squamous cell carcinoma (HNSCC) particularily with cisplatin, 5-FU, methotrexate, bleomycin and taxanes. Vinorelbine is a semisynthetic vinca alcaloid, which causes a block in the G2/M phase of the cell cycle. HNSCC cell lines have previously been reported to be sensitive to vinorelbine in nanomolar concentrations. In the current study the effect of vinorelbine as a radiosensitizer in vitro was studied and eight recently established head and neck SCC cell lines of the UT-SCC-series were tested. Vinorelbine concentrations of 0.4–1.6 nM were used, corresponding to the IC70, IC50 and IC30 values of each cell line, resulting in 30%, 50% and 70% inhibition in clonogenic survival. The desired concentrations of vinorelbine were added to the medium and the cells were plated in 96-well culture plates in this solution. The plated cells were irradiated 24 h later with 4MeV photons generated by a linear accelerator and incubated at 37°C with 5% CO2 for 4 weeks. Thereafter, the number of wells containing coherent, living colonies, consisting of 32 cells or more, was counted. The plating efficiency was calculated and the fraction survival data were fitted to the linear quadratic model [F=exp[−(αD+βD2)]]. An additive effect of combining vinorelbine and irradiation could be demonstrated. The dose-dependent decrease in survival was seen at vinorelbine doses of 0.4–1.6 nM in all cell lines tested.