Doxorubicin Exposure and Breast Cancer Risk in Survivors of Adolescent and Adult Hodgkin Lymphoma.

PURPOSE: Female Hodgkin lymphoma (HL) survivors treated with chest radiotherapy (RT) at a young age have a strongly increased risk of breast cancer (BC). Studies in childhood cancer survivors have shown that doxorubicin exposure may also increase BC risk. Although doxorubicin is the cornerstone of HL chemotherapy, the association between doxorubicin and BC risk has not been examined in HL survivors treated at adult ages. METHODS: We assessed BC risk in a cohort of 1,964 female 5-year HL survivors, treated at age 15-50 years in 20 Dutch hospitals between 1975 and 2008. We calculated standardized incidence ratios, absolute excess risks, and cumulative incidences. Doxorubicin exposure was analyzed using multivariable Cox regression analyses. RESULTS: After a median follow-up of 21.6 years (IQR, 15.8-27.1 years), 252 women had developed invasive BC or ductal carcinoma in situ. The 30-year cumulative incidence was 20.8% (95% CI, 18.2 to 23.4). Survivors treated with a cumulative doxorubicin dose of >200 mg/m2 had a 1.5-fold increased BC risk (95% CI, 1.08 to 2.1), compared with survivors not treated with doxorubicin. BC risk increased 1.18-fold (95% CI, 1.05 to 1.32) per additional 100 mg/m2 doxorubicin (Ptrend = .004). The risk increase associated with doxorubicin (yes v no) was not modified by age at first treatment (hazard ratio [HR]age <21 years, 1.5 [95% CI, 0.9 to 2.6]; HRage ≥21 years, 1.3 [95% CI, 0.9 to 1.9) or chest RT (HRwithout mantle/axillary field RT, 1.9 [95% CI, 1.06 to 3.3]; HRwith mantle/axillary field RT, 1.2 [95% CI, 0.8 to 1.8]). CONCLUSION: This study shows that treatment with doxorubicin is associated with increased BC risk in both adolescent and adult HL survivors. Our results have implications for BC surveillance guidelines for HL survivors and treatment strategies for patients with newly diagnosed HL.

Risk of male breast cancer after Hodgkin lymphoma.

Female survivors of Hodgkin lymphoma (HL) treated with chest radiotherapy have a strongly increased risk of breast cancer (BC), but the treatment-specific BC risk in male survivors of HL has not been evaluated. We assessed BC risk in a cohort of 3077 male survivors of 5-year HL treated at age ≤51 years in 20 Dutch hospitals between 1965 and 2013. We estimated standardized incidence ratios (SIRs), absolute excess risks per 10 000 person-years, and cumulative BC incidences. After a 20-year median follow-up, we observed 8 cases of male with BC. Male survivors of HL experienced a 23-fold (95% confidence interval [CI], 10.1-46.0) increased BC risk compared with the general population, representing 1.6 (95% CI, 0.7-3.3) excess BC incidences per 10 000 person-years. The 20- and 40-year cumulative BC incidences after HL treatment were 0.1% (95% CI, 0.02-0.3) and 0.7% (95% CI, 0.3-1.4), respectively. Treatment with chest radiotherapy without alkylating chemotherapy yielded a strongly increased SIR (20.7; 95% CI, 2.5-74.8), which was not significantly different for chest radiotherapy and alkylating chemotherapy (41.1; 95% CI, 13.4-96.0). Males treated with chest radiotherapy and anthracyclines had an SIR of 48.1 (95% CI, 13.1-123.1). Two patients died from BC (median follow-up, 4.7 years). To ensure early diagnosis and treatment, clinicians should be alert to BC symptoms in male survivors of HL.

Prognostic value of the proliferation marker Ki-67 in laryngeal carcinoma: results of the accelerated radiotherapy with carbogen breathing and nicotinamide phase III randomized trial.

BACKGROUND: The prognostic and predictive value of the proliferation marker Ki-67 was investigated in a randomized trial comparing accelerated radiotherapy with carbogen breathing and nicotinamide (ARCON) to accelerated radiotherapy in laryngeal carcinoma. METHODS: Labeling index of Ki-67 (Li Ki-67) in immunohistochemically stained biopsies and the colocalization with carbonic anhydrase IX (CAIX) were related to tumor control and patient survival. RESULTS: On average, node-positive patients had a higher Li Ki-67 (median 14% vs 8%; p < .01). In patients with a high Li Ki-67, the 5-year regional control and metastases-free survival were 79% versus 96% (p < .01) and 71% versus 88% (p = .05) for accelerated radiotherapy and ARCON, respectively. The 5-year local control and disease-specific survival were not significantly different. Patients with low Ki-67 expression had an excellent outcome with accelerated radiotherapy alone. CONCLUSION: Patients with laryngeal carcinomas with high proliferative activity are at increased risk of regional and distant metastases formation. This risk can be reduced by treatment with ARCON.

Hypoxia, metabolism, and growth factor signaling in head and neck squamous cell carcinoma: correlation between primary and xenograft tumors.

BACKGROUND: Hypoxia, metabolism, and growth factor signaling are important prognostic features in most solid tumors. The purpose of this study was to determine whether head and neck squamous cell carcinoma (HNSCC) xenografts show similar biological and molecular characteristics as the primary tumor they originate from. METHODS: Eighteen HNSCC primary tumor-xenograft pairs were immunofluorescently stained for pimonidazole (hypoxia), carbonic anhydrase IX (CAIX), glucose transporter-1 (GLUT-1), monocarboxylate transporter-1 (MCT-1), monocarboxylate transporter-4 (MCT-4), epidermal growth factor receptor (EGFR), and phosphorylated protein kinase B (pAKT). RESULTS: Although no correlation was found for the amount of hypoxia, significant correlations between primary tumors and xenografts were observed for both the percentage of cells positive for expression and the hypoxia-related expression pattern of CAIX, GLUT-1, and MCT-1. For EGFR and MCT-4, the intensity of expression was correlated. No correlation was observed for pAKT. CONCLUSION: Xenografts did not always resemble the primary tumor they originate from, but the xenografts did represent the variability in expression levels and patterns observed in the primary tumors.

Improved recurrence-free survival with ARCON for anemic patients with laryngeal cancer.

PURPOSE: Anemia is associated with poor tumor control. It was previously observed that accelerated radiotherapy combined with carbogen breathing and nicotinamide (ARCON) can correct this adverse outcome in patients with head and neck cancer. The purpose of this study was to validate this observation based on data from a randomized trial. EXPERIMENTAL DESIGN: Of 345 patients with cT2-4 laryngeal cancer, 174 were randomly assigned to accelerated radiotherapy and 171 to ARCON. Hemoglobin levels, measured before treatment, were defined as low when <7.5 mmol/L for women and <8.5 mmol/L for men. The hypoxia marker pimonidazole was used to assess the oxygenation status in tumor biopsies. Data were analyzed 2 years after inclusion of the last patient. RESULTS: Pretreatment hemoglobin levels were available and below normal in 27 of 173 (16%) accelerated radiotherapy and 27 of 167 (16%) ARCON patients. In patients with normal pretreatment, hemoglobin levels treatment with ARCON had no significant effect on 5-year loco-regional control (LRC, 79% versus 75%; P = 0.44) and disease-free survival (DFS, 75% vs. 70%; P = 0.46) compared with accelerated radiotherapy. However, in patients with low pretreatment, hemoglobin levels ARCON significantly improved 5-year LRC (79% vs. 53%; P = 0.03) and DFS (68% vs. 45%; P = 0.04). In multivariate analysis including other prognostic factors, pretreatment hemoglobin remained prognostic for LRC and DFS in the accelerated radiotherapy treatment arm. No correlation between pretreatment hemoglobin levels and pimonidazole uptake was observed. CONCLUSION: Results from the randomized phase III trial support previous observations that ARCON has the potential to correct the poor outcome of cancer patients with anemia (ClinicalTrials.gov number, NCT00147732).

Pattern of CAIX expression is prognostic for outcome and predicts response to ARCON in patients with laryngeal cancer treated in a phase III randomized trial.

BACKGROUND AND PURPOSE: In a phase III trial in patients with advanced stage laryngeal carcinoma comparing ARCON (accelerated radiotherapy with carbogen breathing and nicotinamide) to accelerated radiotherapy alone (AR) the prognostic and predictive value of CAIX, a hypoxia-associated protein, was investigated. MATERIAL AND METHODS: 261 Paraffin embedded tumor biopsies and 79 fresh frozen biopsies from patients entered in the trial were immunohistochemically stained for CAIX. CAIX-fraction and CAIX expression pattern were related to tumor control and patient survival. RESULTS: Low CAIX-fraction was prognostic for worse regional control and overall survival in patients treated with AR. Patients with a low CAIX-fraction treated with ARCON had better regional control and metastasis-free survival compared to AR (RC 97% vs 71%, p < 0.01 and MFS 92% vs 69%, p = 0.06). Patients with a perinecrotic CAIX staining pattern had a significantly worse local control, metastasis-free and overall survival compared to patients with a diffuse pattern (65% vs 84%, p = 0.01, 70% vs 96%, p < 0.01 and 42% vs 71%, p < 0.01 respectively), and this could not be improved with ARCON. After multivariate analysis CAIX pattern and N-stage emerged as significant predictors for metastasis-free survival and overall survival. CONCLUSIONS: ARCON improves regional control and metastasis-free survival only in patients with low CAIX expression. The different patterns of CAIX expression suggest different mechanisms of upregulation and have important prognostic value.

Accelerated radiotherapy with carbogen and nicotinamide for laryngeal cancer: results of a phase III randomized trial.

PURPOSE: To report the results from a randomized trial comparing accelerated radiotherapy (AR) with accelerated radiotherapy plus carbogen inhalation and nicotinamide (ARCON) in laryngeal cancer. PATIENTS AND METHODS: Patients with cT2-4 squamous cell laryngeal cancer were randomly assigned to AR (68 Gy within 36 to 38 days) or ARCON. To limit the risk of laryngeal necrosis, ARCON patients received 64 Gy on the laryngeal cartilage. The primary end point was local control. Secondary end points were regional control, larynx preservation, toxicity, disease-free survival, and overall survival. In a translational side study, the hypoxia marker pimonidazole was used to assess the oxygenation status in tumor biopsies. RESULTS: From April 2001 to February 2008, 345 patients were accrued. After a median follow-up of 44 months, local tumor control rate at 5 years was 78% for AR versus 79% for ARCON (P = .80), with larynx preservation rates of 84% and 87%, respectively (P = .48). The 5-year regional control was significantly better with ARCON (93%) compared with AR (86%, P = .04). The improvement in regional control was specifically observed in patients with hypoxic tumors and not in patients with well-oxygenated tumors (100% v 55%, respectively; P = .01). AR and ARCON produced equal levels of toxicity. CONCLUSION: Despite lack of benefit in local tumor control for advanced laryngeal cancers, a significant gain in regional control rate, with equal levels of toxicity, was observed in favor of ARCON. The poor regional control of patients with hypoxic tumors is specifically countered by ARCON treatment.

Differences in metabolism between adeno- and squamous cell non-small cell lung carcinomas: spatial distribution and prognostic value of GLUT1 and MCT4.

BACKGROUND: Hypoxia leads to changes in tumor cell metabolism such as increased glycolysis. In this study, we examined the spatial distribution of the glycolysis and hypoxia related markers glucose transporter 1 (GLUT1) and monocarboxylate transporter 4 (MCT4) expression in relation to the vasculature in stage I, II and resectable stage IIIA NSCLC. Furthermore, associations of these markers with survival were investigated. METHODS: GLUT1 and MCT4 expression were determined in 90 NSCLC fresh frozen biopsies using immunohistochemical techniques and a computerized image analysis system. Markers were analyzed for adenocarcinomas (n=41) and squamous cell carcinomas (n=34) separately. Eighty-four patients were retrospectively evaluated for relapse and survival. RESULTS: Squamous cell carcinomas demonstrated higher GLUT1 expression, relative to adenocarcinomas. Also, in squamous cell carcinomas, GLUT1 and MCT4 expression increased with increasing distance from the vasculature, whereas in adenocarcinomas upregulation of MCT4 was already found at closer distance from vessels. In adenocarcinomas, high GLUT1 expression correlated with a poor differentiation grade and positive lymph nodes at diagnosis. High GLUT1 plus high MCT4 expression was associated with a poor disease-specific survival in only adenocarcinomas (p=0.032). CONCLUSION: Analysis of GLUT1 and MCT4 expression on the histological level suggested a different metabolism for adenocarcinomas and squamous cell carcinomas. Likely, adenocarcinomas rely mainly on aerobic glycolysis for ATP production, whereas the behavior of squamous cell carcinomas is more physiologically, i.e. mitochondrial oxidation with anaerobic glycolysis under hypoxic conditions. High GLUT1 plus high MCT4 expression indicated an aggressive tumor behavior in adenocarcinomas. This subgroup of tumors may benefit from new treatment approaches, such as MCT4 inhibitors. Since this study has an exploratory character, our results warrant further investigation and need independent validation.

Metabolic markers in relation to hypoxia; staining patterns and colocalization of pimonidazole, HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4.

BACKGROUND: The cellular response of malignant tumors to hypoxia is diverse. Several important endogenous metabolic markers are upregulated under hypoxic conditions. We examined the staining patterns and co-expression of HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4 with the exogenous hypoxic cell marker pimonidazole and the association of marker expression with clinicopathological characteristics. METHODS: 20 biopsies of advanced head and neck carcinomas were immunohistochemically stained and analyzed. All patients were given the hypoxia marker pimonidazole intravenously 2 h prior to biopsy taking. The tumor area positive for each marker, the colocalization of the different markers and the distribution of the markers in relation to the blood vessels were assessed by semiautomatic quantitative analysis. RESULTS: MCT1 staining was present in hypoxic (pimonidazole stained) as well as non-hypoxic areas in almost equal amounts. MCT1 expression showed a significant overall correlation (r = 0.75, p < 0.001) and strong spatial relationship with CAIX. LDH-5 showed the strongest correlation with pimonidazole (r = 0.66, p = 0.002). MCT4 and GLUT-1 demonstrated a typical diffusion-limited hypoxic pattern and showed a high degree of colocalization. Both MCT4 and CAIX showed a higher expression in the primary tumor in node positive patients (p = 0.09 both). CONCLUSIONS: Colocalization and staining patterns of metabolic and hypoxia-related proteins provides valuable additional information over single protein analyses and can improve the understanding of their functions and environmental influences.

Parametric mapping of immunohistochemically stained tissue sections; a method to quantify the colocalization of tumor markers.

BACKGROUND: Automated analysis of immunohistochemically stained tissue sections is of great importance in cancer research to detect tumor-specific prognostic markers and make therapy decisions. Here, an automated quantitative analysis is presented to assess the colocalization of CAIX, a membrane-bound hypoxic marker and Ki-67, a nuclear proliferation marker. METHODS: Tissue sections of 104 biopsies from 89 patients were stained for CAIX and Ki-67 with diaminobenzidine and haematoxylin counterstain. Image scans of whole tumor sections were recorded and image maps were created with parametric mapping to quantify the markers and assess the colocalization. RESULTS: The fraction of CAIX showed a range of 0-93%. The interobserver correlation and the correlation between manual scores and automated analysis were both very strong (rs=0.96, p<0.0001, and rs=0.97, p<0.0001). The labelling index of Ki-67 exhibited a range of 0-42% with less strong interobserver and manual to automated analysis correlations (rs=0.90, p<0.0001, and rs=0.71, p<0.0008). The relative tumor area positive for both markers varied from 0-76%. CONCLUSION: Parametric mapping of immunohistochemically stained tumor sections is a reliable method to quantitatively analyze membrane-bound proteins and assess the colocalization of various tumor markers in different subcellular compartments.

Molecular aspects of tumour hypoxia.

Hypoxia is an important feature of the microenvironment of a wide range of solid tumours. Its critical role in radio- and chemoresistance and its significance as an adverse prognostic factor have been well established over the last decades. On a cellular level, hypoxia evokes a complex molecular response with a central role for the HIF-1 pathway. The cellular processes under control of HIF-1 contain important prognostic information and comprise potential candidates for directing hypoxia-modifying therapies. This review will provide an overview of the current knowledge on the molecular aspects of tumour hypoxia and the link to clinical practice.