Prediction of outcome of early ER+ breast cancer is improved using a biomarker panel, which includes Ki-67 and p53.

BACKGROUND: The aim of this study is to determine whether immunohistochemical (IHC) assessment of Ki67 and p53 improves prognostication of oestrogen receptor-positive (ER+) breast cancer after breast-conserving therapy (BCT). In all, 498 patients with invasive breast cancer from a randomised trial of BCT with or without tumour bed radiation boost were assessed using IHC. METHODS: The ER+ tumours were classified as 'luminal A' (LA): ER+ and/or PR+, Ki-67 low, p53-, HER2- or 'luminal B' (LB): ER+ and/or PR+and/or Ki-67 high and/or p53+ and/or HER2+. Kaplan-Meier and Cox proportional hazards methodology were used to ascertain relationships to ispilateral breast tumour recurrence (IBTR), locoregional recurrence (LRR), distant metastasis-free survival (DMFS) and breast cancer-specific survival (BCSS). RESULTS: In all, 73 patients previously LA were re-classified as LB: a greater than four-fold increase (4.6-19.3%) compared with ER, PR, HER2 alone. In multivariate analysis, the LB signature independently predicted LRR (hazard ratio (HR) 3.612, 95% CI 1.555-8.340, P=0.003), DMFS (HR 3.023, 95% CI 1.501-6.087, P=0.002) and BCSS (HR 3.617, 95% CI 1.629-8.031, P=0.002) but not IBTR. CONCLUSION: The prognostic evaluation of ER+ breast cancer is improved using a marker panel, which includes Ki-67 and p53. This may help better define a group of poor prognosis ER+ patients with a greater probability of failure with endocrine therapy.

Radiotherapy for stage 2 testicular seminoma: the prognostic influence of tumor bulk.

Forty-nine consecutive patients with stage 2 testicular seminoma were treated with primary radiotherapy from 1968 to 1985. Overall diseases-free survival (DFS) for patients with 36 months minimum follow-up was 82% at 3 years. This figure did not decline further with time. Infradiaphragmatic bulk disease was found to be a significant prognostic factor for local and distant relapse as well as for ultimate survival. Patients with either stage 2A or 2B disease (infradiaphragmatic bulk less than or equal to 10 cm size) had a 3-year DFS of 89% compared with a 64% 3-year DFS rate for patients with stage 2C disease (infradiaphragmatic bulk greater than or equal to 10 cm size). The (local plus distant) relapse rate was 4.0% for patients with stage 2A disease, 16.7% for patients with stage 2B disease, and 33.3% for patients with stage 2C disease. The majority of distant relapses were multifocal and prophylactic mediastinal irradiation did not appear to influence either relapse rate nor overall survival. Of seven patients who relapsed, four died of progressive malignancy, two deaths were related to salvage chemotherapy, and only one patient is alive and well following successful chemotherapeutic salvage. On the basis of our experience, we recommend radiotherapy with the use of modern imaging techniques as initial treatment for patients with retroperitoneal masses less than 10 cm size. Aggressive cisplatin-based chemotherapy should be seriously considered for patients with retroperitoneal masses greater than or equal to 10 cm size, or for patients who relapse following radiotherapy.

Paraneoplastic cerebellar disease. Remission with excision of the primary tumor.

We describe two patients with paraneoplastic cerebellar syndromes who gained clinically useful neurologic remissions following radical excision of the primary cancer. In both patients the syndrome was characterized by the rapid onset of gait ataxia, nausea, postural vertigo, central positional nystagmus, and saccadic oscillations. These observations encourage radical treatment of the primary cancer in patients with advanced malignant neoplasms who are disabled by cerebellar dysfunction, and lend support to a current hypothesis that paraneoplastic cerebellar degeneration is due to anticerebellar Purkinje cell antibodies elaborated by the primary cancer.

Adjunctive radiotherapy with strontium-90 in the treatment of conjunctival squamous cell carcinoma.

Squamous cell carcinoma of the ocular conjunctiva is a relatively rare malignancy which is attended by a high rate of local recurrence following simple surgical excision. To date, the management of conjunctival squamous cell cancer has been controversial. From 1950 to 1985, 146 consecutive patients with superficial conjunctival squamous cell cancer were treated at the Queensland Radium Institute. All patients were treated by simple surgical excision of the visible conjunctival lesion followed by adjunctive radiotherapy. Of 140 patients with histologically confirmed squamous cell cancer, 123 were treated with a strontium-90 source, 10 with a radon "ring," and 7 with superficial X ray therapy. Standard policy since 1960 has been to deliver an incident dose of 30 Gy in a single fraction within the first 48 post-operative hours to the surgical bed using a strontium-90 source on a stand-off eye applicator. This report will largely focus on the 123 patients who were treated with a strontium-90 source, of whom 107 received 30 Gy, 14 received 40 Gy (pre 1960) and one patient each received 20 and 25 Gy incident dose. Of 131 evaluable patients, there were only 3 who developed local recurrence. All 3 local recurrences developed in elderly men who had presented with extensive superficial primary tumors. Two of the three recurrences occurred in the two patients who were treated with doses less than 30 Gy. Both early and late radiation-induced complications following ablative surgery and treatment with strontium-90 were very uncommon. Three patients developed unsightly conjunctival telangiectasia, 2 patients developed a persistent scleral ulcer and 2 patients developed clinically significant cataracts. This negligible degree of treatment-related side effects contrasts with the experience of 10 patients who had previously been treated with a radon ring, 8 of whom developed serious complications, although none developed local recurrence. On the basis of our excellent local control rates with minimal morbidity we would continue to advocate the use of simple surgical excision followed by 30 Gy beta radiation from a strontium-90 source as the definitive treatment for superficial conjunctival squamous cell cancer.

The importance of postoperative radiation therapy in the treatment of Merkel cell carcinoma.

PURPOSE: To determine the role of postoperative radiation therapy in the treatment of Merkel cell carcinoma (MCC). METHODS AND MATERIALS: Eighty patients with MCC of the skin were treated with curative intent at the Queensland Radium Institute between 1981 and 1991. Fifty-one patients (63.7%) were referred after initial biopsy for further treatment and 29 patients (36.3%) were referred with locally recurrent disease following primary surgery elsewhere. Thirteen patients (16.3%) presented with nodal disease without a clinically definable primary skin lesion. RESULTS: Of the 80 patients, 38 had undergone surgery (S) alone, 34 surgery plus radiotherapy (S + RT), 7 RT after incomplete S, and 1 patient had chemotherapy (CT) plus RT. Overall survival at 36 months for all patients was 68%. All of the 38 patients treated with S alone relapsed. The median time to recurrence was 5.5 months. Ten of the 34 patients treated with S + RT relapsed. The median time to recurrence was 16.5 months. Of the 80 patients, 55 have relapsed after primary treatment, 25 have developed systemic metastases, and 26 patients have died as a direct result of MCC. CONCLUSION: Our large series confirms earlier reports from this Institute and highlights the importance of S + RT over S alone in preventing local recurrence of this highly malignant skin cancer.

Radiotherapy for superficial skin cancer at the Queensland Radium Institute: famine in the land of plenty.

Superficial skin cancer is the most common malignancy in man, and radiotherapy has played an important curative role since the early part of the 20th century. We present an overview of the changing pattern of care for patients with superficial skin cancer at the Queensland Radium Institute (QRI), Brisbane--the skin cancer "capital" of the world. Although some 90,000 clinically-diagnosed skin cancers have been treated by radiotherapy at the QRI during the period 1944-1985, we document a dramatic decline in radiotherapy usage for superficial skin cancers over the past 10-15 years. We identify and discuss the major reasons for this changing pattern of care: (a) policy changes initiated by radiation oncologists because of unsightly radiation scars caused by the Queensland climate, (b) improvements in the availability and technical aspects of surgery and dermatology, and (c) surgical preference.

Radiation sensitivity of Merkel cell carcinoma cell lines.

PURPOSE: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. METHODS AND MATERIALS: Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after gamma irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. RESULTS: We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to gamma irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. CONCLUSION: Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution.

The relationship between early and late gastrointestinal complications of radiation therapy for carcinoma of the cervix.

In a retrospective analysis of 1,390 consecutive patients with carcinoma of the cervix treated by high dose radiation therapy alone at the Queensland Radium Institute, we report a quantitative relationship between the early and late gastrointestinal complications arising from such treatment. Of these 1,390 patients, 157 (11.3%) experienced early, serious complications. For geographic reasons, it was only possible to evaluate 784 patients for late post-irradiation complications. Twenty-eight (3.6%) developed one or more late bowel complications, which included adhesions, fistulae, strictures, perforation, colitis and vascular occlusion. Factors affecting the relative risk of developing either an early or late complication were analyzed and are discussed. There was an 8.2% incidence of late complications developing in those patients who had experienced early complications, compared with a 3.0% incidence of late complications developing in patients without early complications. Thus, the risk of developing a late complication was greater by a factor of 2.7 in those patients developing an early one (p less than 0.05). However, of the 28 patients developing late complications, 21 (75%) did not experience a severe acute one.

Absence of ATM truncations in patients with severe acute radiation reactions.

PURPOSE: Severe acute toxicity limits the effective use of radiotherapy in patients who are radiosensitive, and it is not usually possible to identify these radiohypersensitive (R-H) individuals before treatment commences. Five such R-H patients were detected over a 3-year period. We undertook this study to determine whether the severe acute radiohypersensitivity of these five individuals showed any correlation with cellular and molecular parameters known to be abnormal in radiosensitivity-related syndromes such as ataxia-telangiectasia (A-T). METHODS AND MATERIALS: Lymphoblastoid cells were isolated from fresh blood from the 5 R-H individuals who had previously demonstrated clinical R-H at least 9 months prior to sampling. Lymphoblastoid cell lines (LCLs) were established to determine the extent of postradiation chromosomal aberrations, cell cycle delay, cell proliferation, and tumor suppressor p53 protein stabilization. The polymerase chain reaction (PCR) and protein truncation (PTT) assays were used to test for the possibility of mutations in the gene mutated in A-T, termed ATM. RESULTS: LCLs derived from R-H subjects retained a significantly higher degree of radiation-induced chromosomal aberrations when compared to normal control LCLs. p53 stabilization by ionizing radiation appeared normal in all but one R-H subject. There was no evidence of A-T gene truncation mutations in any of the R-H subjects tested. CONCLUSIONS: All R-H subjects in this study had their cellular radiosensitivity confirmed by the chromosomal aberration assay. Delayed p53 stabilization at 4 hours postirradiation in one R-H subject suggested that different etiologies may apply in the radiohypersensitivity investigated in this study.

Chromosomes 1, 11, and 13 are frequently involved in karyotypic abnormalities in metastatic Merkel cell carcinoma.

We report a cytogenetic study of six Merkel cell carcinomas (MCC) in which rearrangement of chromosome 1 was noted in four cases: two cases were trisomic, in one case there was a reciprocal translocation between chromosomes 1 and 5 [t(1;5)(p36;p13)], and in the fourth case all cells had a normal chromosome 1 and three derivatives, a del(1)(p22) and del(1)(q21), and a translocation involving material of unknown origin to the long arm, t(1;?)(q21;?). Four cases demonstrated loss of chromosome 13; in two of these, both copies were lost, and the survival for these two patients was much longer than is common for MCC patients. Partial trisomy of chromosome 11 was noted in two cases, and two patients demonstrated loss of chromosome 22 in all cells examined. Although no consistent chromosome change was noted in our cases, our data and those of previously published reports, show that abnormalities of chromosomes 1, 11, and 13 occur in 30-47% of cytogenetic reports of this rare malignancy.

Loss of allelic heterozygosity on distal chromosome 1p in Merkel cell carcinoma. A marker of neural crest origins?

The location of genes involved in tumor evolution has been inferred from experiments in which loss of constitutional heterozygosity has been detected in tumor DNA at high frequency in specific chromosome regions. For example, cytogenetic and molecular abnormalities on chromosome 1p have been reported in tumors such as malignant melanoma and neuroblastoma which arise in cells derived from embryonic neural crest tissue. To extend these observations, we have examined tumor DNA from three cases of Merkel cell carcinoma for evidence of loss of constitutional heterozygosity on the short arm of chromosome 1. In all three cases, heterozygous allelic deletions of varying extent on distal chromosome 1p were detected in tumor DNA. Comparisons with neural crest tumors suggest that loss of heterozygosity on distal chromosome 1p in Merkel cell tumors may be a marker of neural crest origin.

A comparison of epidermal growth factor receptor (EGFR) and c-erbB-2 oncogene expression in head and neck squamous cell carcinomas.

The proto-oncogenes c-erbB-2 and epidermal growth factor (EGF) receptor which encode 2 closely homologous transmembrane glycoproteins have been found amplified and/or overexpressed in a range of epithelial malignancies. In a series of 46 head and neck squamous cell cancers (SCCs), immunohistochemical reactivity for the EGF receptor was detected in all cases, particularly at the invading edge of cellular islands of SCC and in the basal cells of normal adjacent squamous epithelium. Southern blot analysis demonstrated EGF receptor gene amplification in 3 cases. In contrast, strong membrane staining for the c-erbB-2 oncoprotein was not detected in any sample, and there were no cases of c-erbB-2 gene amplification. Despite a close structural and (presumed) functional homology between these 2 receptor-oncoproteins in the development of malignancy, we report that their expression in SCCs is markedly different. Furthermore, unlike the situation for breast cancer, quantitation of the c-erbB-2 or EGF receptor oncoproteins is unlikely to yield important prognostic information in this group of patients.

HPV DNA in oropharyngeal squamous cell cancers: comparison of results from four DNA detection methods.

Oropharyngeal squamous cell cancers (SCCs) were examined for human papillomavirus (HPV) related DNA sequences. The techniques employed were Southern blotting under stringent and non stringent conditions, dot blotting, primer directed gene amplification using the polymerase chain reaction (PCR), and in-situ hybridization. HPV 16 DNA was found in 4 of 30 tumor samples using PCR. HPV 16 DNA was found in 2 further tumors using in-situ hybridization. No HPV DNA could be found by Southern blot or dot blot in any tumor sample. The Southern blot assays were sensitive enough to detect clonally integrated HPV 16 DNA of length greater than 250 bp in the tumors. While HPV DNA is present in some oropharyngeal SCCs, there is no molecular evidence to support a causal association of HPV 16 gene products with continued tumor growth in oropharyngeal cancer.

nm23 protein expression and p53 immunoreactivity in cutaneous fibrohistiocytic tumors.

Recent data indicate that reduced expression of the 17-kD protein encoded by the nm23 gene may be important in the pathogenesis of several types of human tumors. Immunohistochemistry was performed using a murine monoclonal antibody, NCL-nm23 (Novocastra, 1:150 dilution) to investigate nm23 protein immunoreactivity in a group of locally aggressive cutaneous fibrohistiocytic tumors; dermatofibrosarcoma protuberans (DFSP) (n = 14) and atypical fibroxanthoma (AFX) (n = 7). Cases of dermatofibroma (DF) (n = 17) formed the benign control group. Comparison with p53 protein immunoreactivity in the same cases studied previously was made. Strong immunohistological expression of the nm23 protein was seen in most of the cases of DF (n = 15; 88%) in the form of strong cytoplasmic immunolabelling without nuclear staining. However, strong nm23 immunoreactivity was observed in only a minority of the cases of DFSP (n = 5; 36%) and AFX (n = 2; 29%). Statistically significant differences in nm23 immunoreactivity were found between DFSP and DF (p = 0.008, chi 2 test with continuity correction) and between AFX and DF (p = 0.015; chi 2 test with continuity correction). No significant difference was seen between DFSP and AFX (p = 0.87, chi 2 test with continuity correction). There was inverse correlation between nm23 and p53 immunoreactivity (r = 0.331; r2 = 0.109; p = 0.046; simple regression analysis). In summary, nm23 protein immunoreactivity is reduced in DFSP and AFX but not in dermatofibroma suggesting that reduced expression of the protein may be important in influencing the behavior of fibrohistiocytic tumors, although this is not well characterised. nm23 protein expression is also found to be inversely related to p53 immunohistological expression in these tumors.

Rapid radiation-induction of ATM protein levels in situ.

Ataxia-telangiectasia (A-T) is characterised by hypersensitivity to ionising radiation (IR), immunodeficiency, neurodegeneration and predisposition to malignancy. Mutations in the A-T gene (ATM) often result in reduced levels of ATM protein and/or compromise ATM function. IR induced DNA damage is known to rapidly upregulate ATM kinase activity/phosphorylation events in the control of cell cycle progression and other processes. Variable expression of ATM levels in different tissues and its upregulation during cellular proliferation indicate that the level of ATM is also regulated by mechanisms other than gene mutation. Here, we report on the IR induction of ATM protein levels within a number of different cell types and tissues. Induction had begun within 5 min and peaked within 2 h of exposure to 2 Gy of IR, suggesting a rapid post-translational mechanism. Low basal levels of ATM protein were more responsive to IR induction compared to high ATM levels in the same cell type. Irradiation of fresh skin biopsies led to an average three-fold increase in ATM levels while immunohistochemical analyses indicated "low expressing" cells within the basal layer with ten-fold increases in ATM levels following IR. ATM "high expressing" lymphoblastoid cell lines (LCLs) which were initially resistant to the radiation-induction of ATM levels also became responsive to IR after ATM antisense expression was used to reduce the basal levels of the protein. These results demonstrate that ATM is present in variable amounts in different tissue/cell types and where basal levels are low ATM levels can be rapidly induced by IR to saturable levels specific for different cell types. ATM radiation-induction is a sensitive and rapid radioprotective response that complements the IR mediated activation of ATM.

Production and verification of an anti-ovine IgE monoclonal antibody.

Recombinant mouse/sheep IgE was used in the production of an anti-IgE monoclonal using conventional hybridoma techniques. The specificity of hybridomas secreting anti sheep IgE monoclonal antibodies was verified using a number of assays including competitive ELISAs, ability to induce mediator release from mast cells, and IgE binding using western blotting. Immunohistochemical staining demonstrated the binding of putative anti-IgE monoclonals to the sheep mast cell surface. The isotype of the antibody was IgG1:K.

PI3K/Akt/mTOR pathway inhibitors enhance radiosensitivity in radioresistant prostate cancer cells through inducing apoptosis, reducing autophagy, suppressing NHEJ and HR repair pathways.

The PI3K/Akt/mTOR pathway has a central role in cancer metastasis and radiotherapy. To develop effective therapeutics to improve radiosensitivity, understanding the possible pathways of radioresistance involved and the effects of a combination of the PI3K/Akt/mTOR inhibitors with radiotherapy on prostate cancer (CaP) radioresistant cells is needed. We found that compared with parent CaP cells, CaP-radioresistant cells demonstrated G0/G1 and S phase arrest, activation of cell cycle check point, autophagy and DNA repair pathway proteins, and inactivation of apoptotic proteins. We also demonstrated that compared with combination of single PI3K or mTOR inhibitors (BKM120 or Rapamycin) and radiation, low-dose of dual PI3K/mTOR inhibitors (BEZ235 or PI103) combined with radiation greatly improved treatment efficacy by repressing colony formation, inducing more apoptosis, leading to the arrest of the G2/M phase, increased double-strand break levels and less inactivation of cell cycle check point, autophagy and non-homologous end joining (NHEJ)/homologous recombination (HR) repair pathway proteins in CaP-radioresistant cells. This study describes the possible pathways associated with CaP radioresistance and demonstrates the putative mechanisms of the radiosensitization effect in CaP-resistant cells in the combination treatment. The findings from this study suggest that the combination of dual PI3K/Akt/mTOR inhibitors (BEZ235 or PI103) with radiotherapy is a promising modality for the treatment of CaP to overcome radioresistance.

Acquisition of epithelial-mesenchymal transition and cancer stem cell phenotypes is associated with activation of the PI3K/Akt/mTOR pathway in prostate cancer radioresistance.

Radioresistance is a major challenge in prostate cancer (CaP) radiotherapy (RT). In this study, we investigated the role and association of epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and the PI3K/Akt/mTOR signaling pathway in CaP radioresistance. We developed three novel CaP radioresistant (RR) cell lines (PC-3RR, DU145RR and LNCaPRR) by radiation treatment and confirmed their radioresistance using a clonogenic survival assay. Compared with untreated CaP-control cells, the CaP-RR cells had increased colony formation, invasion ability and spheroid formation capability (P<0.05). In addition, enhanced EMT/CSC phenotypes and activation of the checkpoint proteins (Chk1 and Chk2) and the PI3K/Akt/mTOR signaling pathway proteins were also found in CaP-RR cells using immunofluorescence, western blotting and quantitative real-time PCR (qRT-PCR). Furthermore, combination of a dual PI3K/mTOR inhibitor (BEZ235) with RT effectively increased radiosensitivity and induced more apoptosis in CaP-RR cells, concomitantly correlated with the reduced expression of EMT/CSC markers and the PI3K/Akt/mTOR signaling pathway proteins compared with RT alone. Our findings indicate that CaP radioresistance is associated with EMT and enhanced CSC phenotypes via activation of the PI3K/Akt/mTOR signaling pathway, and that the combination of BEZ235 with RT is a promising modality to overcome radioresistance in the treatment of CaP. This combination approach warrants future in vivo animal study and clinical trials.

Radioresistant malignant myoepithelioma of the breast with high level of ataxia telangiectasia mutated protein.

Malignant myoepithelioma of the breast (MMB) is a rare and often aggressive disease with poor prognosis. Little is known regarding its optimal treatment and progression. We describe the clinical history of a woman following excision of a benign adenomyoepithelioma which recurred years later as a radioresistant malignant myoepithelioma with high levels of ataxia telangiectasia mutated protein and mutant p53 (Cys135Phe). MMB requires close follow-up and aggressive treatment. If adjuvant radiotherapy is adopted to improve local control, minimal postoperative delay and higher doses than for standard post-mastectomy radiation are recommended.

High-dose radiotherapy for localized prostatic cancer. An analysis of treatment results and early complications.

Between 1970 and 1983, 477 consecutive patients with clinically localized prostatic adenocarcinoma were treated with primary external-beam radiotherapy. With a minimum follow-up period of 60 months, the five- and 10-year survival rates were 59% and 33%, respectively. The five-year survival rate was 89% for patients with stage-A disease, 59% for stage-B disease and 25% for stage-C disease; it was 79% for patients with well-differentiated carcinomas, but only 37% for patients with high-grade tumours. The over-all local in-field control rate was 88%. Local failure occurred in 6% of patients with stage-A disease, 11% with stage-B disease, and 18% with stage-C disease. All patients experienced some radiation-induced reactions, but these were significant in only 14.2% of cases. The role of local- versus extended-field radiotherapy for curative treatment of prostatic cancer is discussed in some detail. Our unexpectedly low over-all survival figures emphasize the need to exclude the presence of distant metastases as fully as possible before commencing radical radiotherapy. High-dose radiotherapy to localized prostatic cancer offers significant advantages over radical surgery and is associated with an excellent local control rate, which can be achieved with an acceptable degree of early morbidity.