Age-Related Biology of Early-Stage Operable Breast Cancer and Its Impact on Clinical Outcome.

As age advances, breast cancer (BC) tends to change its biological characteristics. This study aimed to explore the natural progression of such changes. The study included 2383 women with clinically T0-2N0-1M0 BC, managed by primary surgery and optimal adjuvant therapy in a dedicated BC facility. Tissue micro-arrays were constructed from their surgical specimens and indirect immunohistochemistry was used for analysis of a large panel (n = 16) of relevant biomarkers. There were significant changes in the pattern of expression of biomarkers related to luminal (oestrogen receptor (ER), progesterone receptors (PgR), human epidermal growth factor receptor (HER-2), E-cadherin, MUC1, bcl2 CK7/8, CK18 and bcl2) and basal (CK5/6, CK14, p53 and Ki67) phenotypes, lymph node stage, histological grade and pathological size when decade-wise comparison was made (p < 0.05). The ages of 40 years and 70 years appeared to be the milestones marking a change of the pattern. There were significantly higher metastasis free and breast cancer specific survival rates among older women with ER positive tumours while there was no significant difference in the ER negative group according to age. Biological characteristics of BC show a pattern of change with advancing age, where 40 years and 70 years appear as important milestones. The pattern suggests <40 years as the phase with aggressive phenotypes, >70 years as the less aggressive phase and 40-70 years being the transitional phase.

Liver Kinase B1-A Potential Therapeutic Target in Hormone-Sensitive Breast Cancer in Older Women.

Background: The role of liver kinase B1 (LKB1), a serine/threonine kinase, has been described in the development of PeutzJagher's syndrome, where a proportion (~45%) of patients have developed breast cancer in their lifetime. Cell line studies have linked LKB1 with oestrogen receptors (ER) and with the Adenosine monophosphate-activated protein kinase (AMPK) pathway for energy metabolism. However, limited studies have investigated protein expression of LKB1 in tumour tissues and its intracellular relationships. This study aimed to investigate the intracellular molecular relationships of LKB1 in older women with early operable primary breast cancer and its correlation with long-term clinical outcome. Methods: Between 1973 and 2010, a consecutive series of 1758 older (≥70 years) women with T0-2N0-1M0 breast carcinoma were managed in a dedicated facility. Of these, 813 patients underwent primary surgery, and 575 had good quality tumour samples available for tissue microarray construction. LKB1 was assessed in 407 cases by indirect immunohistochemistry (IHC). Tumours with 30% or more of cells with cytoplasmic LKB1 expression were considered positive. LKB1 expression was compared with tumour size, histological grade, axillary lymph node stage, ER, PgR, EGFR, HER2, HER3, HER4, BRCA1&2, p53, Ki67, Bcl2, Muc1, E-Cadherin, CD44, basal (CK5, CK5/6, CK14 and CK17) and luminal (CK7/8, CK18 and CK19) cytokeratins, MDM2 and MDM4, and correlated with long-term clinical outcome. Results: Positive LKB1 expression was seen in 318 (78.1%) patients, and was significantly associated with high tumour grade, high Ki67, over-expression of HER2, VEGF, HER4, BRCA2, MDM2 and negative expression of CD44 (p < 0.05). There was no significant correlation with tumour size, axillary lymph node status, ER, PgR, p53, basal or luminal cytokeratins, Bcl2, Muc1, EGFR, HER3, MDM4, E-cadherin and BRCA1. LKB1 did not show any significant influence on survival in the overall population; however, in those patients receiving adjuvant endocrine therapy for ER positive tumours, those with positive LKB1 had significantly better 5-year breast cancer specific survival when compared to those without such expression (93% versus 74%, p = 0.03). Conclusion: LKB1 expression has shown association with poor prognostic factors in older women with breast cancer. However, LKB1 expression appears to be associated with better survival outcome among those patients receiving adjuvant endocrine therapy. Further research is required to explore its potential role as a therapeutic target.

Breast conservation in ductal carcinoma in situ (DCIS): what defines optimal margins?

The introduction of mammographic screening has resulted in a rise in the detection rate of ductal carcinoma in situ (DCIS), currently accounting for one-fifth of screen-detected breast cancers. Although 60-70% of DCIS are treated with breast-conserving surgery (BCS) with or without radiotherapy, the frequency of subsequent surgery to re-excise positive margins in order to reduce the probability of recurrences remains high. DCIS recurrence is associated not only with financial, health and psychological implications; approximately half these recurrences are invasive disease. An appropriate margin width for patients undergoing BCS for invasive breast cancer has been largely agreed. Although there is a perception that such recommendations may be applicable to DCIS, major differences exist which may affect this application. Importantly, DCIS patients often do not receive systemic adjuvant (endocrine) therapy and not all receive radiotherapy in routine practice. There is evidence that wide margins (i.e. >10 mm) confer better protection against recurrence than positive (i.e. 0 mm) margins; however, there remains a debate concerning the optimum margin width between 0 and 10 mm. Previous studies have demonstrated that radiation therapy may not compensate for lack of re-excision in those patients with positive or close margins, while wide margins will inevitably compromise cosmesis and patients' body image perception. This review aims to address the clinical question of the minimal margin width in DCIS treated with BCS that is associated with the lowest recurrence rate and when, therefore, further surgical intervention for re-excision can be safely avoided. A range of clinical circumstances that might affect this are considered.

Prognostic Factors for Local Control in Breast Cancer After Long-term Follow-up in the EORTC Boost vs No Boost Trial: A Randomized Clinical Trial.

IMPORTANCE: Prognostic factors of ipsilateral breast tumor recurrence (IBTR) may change over time following breast-conserving therapy. OBJECTIVE: The EORTC "boost no boost" trial showed that young age and high-grade invasive carcinoma were the most important risk factors for IBTR. This study reanalyses pathological prognostic factors related to IBTR using long-term follow-up. DESIGN, SETTING, AND PARTICIPANTS: Participants included 5569 early-stage breast cancer patients, treated with breast-conserving surgery (BCS) and whole-breast irradiation (WBI), who were randomized between no boost and a 16-Gy boost in the EORTC phase III "boost no boost" trial (1989-1996). A total of 1616 patients with a microscopically complete resection (according to local pathologists), included in the central pathology review, have been analyzed in this study. Median follow-up was 18.2 years. INTERVENTIONS: No further treatment or 16-Gy boost, after BCS and 50-Gy WBI. MAIN OUTCOMES AND MEASURES: Time to ipsilateral breast tumor recurrence (IBTR) as first event. RESULTS: The 20-year cumulative incidence of IBTR in 1616 patients (160 events observed) was 15% (95% CI, 12%-17%). Young age (P < .001) and presence of ductal carcinoma in situ (DCIS) (HR, 2.15; 95% CI, 1.36-3.38; P = .001) were associated with an increased risk of IBTR in multivariable analysis. The cumulative incidence of IBTR at 20 years was 34% (95% CI, 25%-41%), 14% (95% CI, 10%-18%), and 11% (95% CI, 8%-15%), in patients 40 years or younger, 41 to 50 years and 50 years or older, respectively (P < .001). This incidence was 18% (95% CI, 14%-22%) and 9% (95% CI, 6%-12%) for tumors with and without DCIS (P < .001). High-grade tumors relapsed more frequently early during follow-up but the relative effect of age and presence of DCIS seemed stable over time. The boost reduced the 20-year IBTR incidence from 31% (95% CI, 22%-39%) to 15% (95% CI, 8%-21%) (HR, 0.37; 95% CI, 0.22-0.62; P < .001) in high-risk patients (≤50 years with DCIS present). CONCLUSIONS AND RELEVANCE: The association of high-grade invasive tumor with IBTR diminished during follow-up, while the effect of DCIS adjacent to invasive tumor seemed to remain stable. Therefore, patients with high-grade invasive tumors should be monitored closely, especially in the first 5 years, while additional DCIS is an indication for longer follow-up, emphasizing the importance of long-term trial follow-up to estimate absolute effects accurately. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02295033.

Education and Training Needs in Radiation Oncology in India: Opportunities for Indo-US Collaborations.

PURPOSE: To conduct a survey of radiation oncologists in India, to better understand specific educational needs of radiation oncology in India and define areas of collaboration with US institutions. METHODS AND MATERIALS: A 20-question survey was distributed to members of the Association of Indian Radiation Oncologists and the Indian Brachytherapy Society between November 2013 and May 2014. RESULTS: We received a total of 132 responses. Over 50% of the physicians treat more than 200 patients per day, use 2-dimensional or 3-dimensional treatment planning techniques, and approximately 50% use image guided techniques. For education needs, most respondents agreed that further education in intensity modulated radiation therapy, image guided radiation therapy, stereotactic radiation therapy, biostatistics, and research methods for medical residents would be useful areas of collaboration with institutions in the United States. Other areas of collaboration include developing a structured training module for nursing, physics training, and developing a second-opinion clinic for difficult cases with faculty in the United States. CONCLUSION: Various areas of potential collaboration in radiation oncology education were identified through this survey. These include the following: establishing education programs focused on current technology, facilitating exchange programs for trainees in India to the United States, promoting training in research methods, establishing training modules for physicists and oncology nurses, and creating an Indo-US. Tumor Board. It would require collaboration between the Association of Indian Radiation Oncologists and the American Society for Radiation Oncology to develop these educational initiatives.

The potential value of comprehensive geriatric assessment in evaluating older women with primary operable breast cancer undergoing surgery or non-operative treatment--a pilot study.

OBJECTIVES: Breast cancer in older women raises a number of discrete issues, including how healthcare professionals can best decide which patients are candidates for surgery. A pilot study involving women aged ≥70years newly diagnosed with early operable primary breast cancer was conducted aiming to explore the potential value of comprehensive geriatric assessment (CGA). MATERIALS AND METHODS: Decision of primary treatment followed consultation with the clinical team and was not guided by any aspect of this study. CGA, using a validated cancer-specific tool, was conducted within 6weeks and 6months after diagnosis, complemented by formal measures of quality of life (QOL) (using EORTC QLQ-C30 and QLQ-BR23) and semi-structured interviews. A total of 47 female patients with a new diagnosis of clinically early (stage 1 or 2; cT0-2N0-1M0) operable primary breast cancer proven histologically, were recruited. RESULTS: CGA determined that increasing age (≥80years) (p=0.001), greater (≥4) comorbidity (p=0.022), greater number (≥4) of daily medications (p=0.002), and slower (≥19s) timed up and go (TUG) (p=0.016) score were significantly related to non-surgical treatment at 6weeks after diagnosis. Baseline QOL scores were generally good and they remained stable at 6months follow-up. As opposed to CGA, there was no correlation between QOL scores and the treatment modality identified. Semi-structured interviews identified themes consistent with findings from QOL assessment. CONCLUSION: The pilot study confirmed the feasibility of conducting CGA in a research setting which appeared to have value in assessing this patient population. More data will be required to definitively identify the components for geriatric assessment in this setting. The study has now extended into two more centres.

Involvement of metformin and AMPK in the radioresponse and prognosis of luminal versus basal-like breast cancer treated with radiotherapy.

Metformin is under evaluation as a potential anticancer agent. Expression of total and phospho(Thr172)-adenosine monophosphate-activated kinase-α (AMPKα and pAMPKα(Thr172) respectively), a main metformin target, was examined in radiotherapy treated breast cancers and metformin's ability to modulate Trx system expression and breast cancer radiosensitivity evaluated in vitro. AMPKα and pAMPKα(Thr172) expression was assessed using a discovery (n=166) and validation cohort (n=609). Metformin's role in regulating radioresponse, and Trx family expression, was examined via clonogenic assays and Western blots. Intracellular reactive oxygen species (ROS) levels, cell cycle progression and apoptosis were assessed by flow cytometry. High AMPKα expression associated with improved local recurrence-free (P=0.019), relapse-free (P=0.016) and breast cancer-specific survival (P=0.000065) and was, from multivariate analysis, an independent prognostic factor from the discovery cohort. From the validation cases AMPKα expression associated with relapse-free and breast cancer-specific survival in luminal breast cancers. Metformin substantially increased radiosensitivity, intracellular ROS levels and reduced Trx expression, in luminal breast cancer cells, but had little effect on basal phenotype cells. In conclusion, high AMPKα expression associates with improved prognosis, especially in luminal breast cancer. Metformin preferentially radiosensitises luminal breast cancer cells, potentially due to alterations to intracellular ROS levels via modulation of Trx family protein expression.

Biological characteristics and clinical outcome of triple negative primary breast cancer in older women - comparison with their younger counterparts.

Triple negative (ER, PgR and HER2 negative) breast cancers (TNBCs) are often considered as a poor prognostic phenotype. There is dearth of evidence showing the prevalence and biological behaviour of TNBCs in older women. This study aimed to analyse their biological characteristics in comparison with a well characterised younger series from a single centre with long term clinical follow-up. Over 37 years (1973-2010), 1,758 older (≥70 years) women with early operable (<5 cm) primary breast cancer were managed in a dedicated clinic and have complete clinical information available. Of these 813 patients underwent primary surgery and 575 had good quality tumour samples available for tissue microarray analysis using indirect immunohistochemistry. A total of 127 patients (22.1%) had TNBCs and full biological analysis of 15 biomarkers was performed. The results were compared with those of their younger (<70 years) counterparts 342 (18.9%) from a previously characterised, consecutive series of primary breast cancer treated in the same unit (1986-1998). The 127 older patients with TNBCs showed lower rates of Ki67 and CK 7/8 positivity and high rates of bcl2 and CK18 positivity when compared with their younger counterparts (p<0.05). There was no significant difference in the long term clinical outcome between the two age groups, despite the fact that 47% of the younger patients had adjuvant chemotherapy, while none in the older cohort received such treatment. EGFR, axillary stage and pathological size showed prognostic significance in older women with TNBCs on univariate analysis. Despite not having received adjuvant chemotherapy, the older series had clinical outcome similar to the younger patients almost half of whom had chemotherapy. This appears to be related to other biomarkers (in addition to ER/PgR/HER2) eg Ki67, bcl2 and cytokeratins which have different expression patterns influencing prognosis.

11 kGy gamma irradiated demineralized bone matrix enhances osteoclast activity.

BACKGROUND: Demineralized bone matrix (DBM) allografts are widely used in orthopaedic clinics. However, the biological impact on its osteoinductivity after its sterilization process by gamma irradiation is not well studied. Furthermore, little is known about the relationship between residual calcium levels on osteoinductivity. HYPOTHESIS: We hypothesize that low-dose gamma irradiation retains the osteoinducitivity properties of DBM and causes ectopic bone formation. MATERIALS AND METHODS: A randomised animal trial was performed to compare tissue and molecular responses of low-dose (11 kGy) gamma irradiated and non-irradiated human DBM at 6 weeks post-intramuscular implantation using an athymic rat model. In addition, we correlated residual calcium levels and bone formation in gamma irradiated DBM. RESULTS: A modified haematoxylin and eosin stain identified ectopic bony capsules at all implanted sites with no significant difference on the amount of new bone formed between the groups. Statistically significantly lower ratio of alkaline phosphatase expression over tartrate-resistant acid phosphatase and/or cathepsin K expressions was found between the groups. DISCUSSION: This study found that low-dose gamma irradiated DBM, which provides a sterility assurance level of 10(-6) for bone allografts, retained osteoinductivity but exhibited significantly enhanced osteoclastic activity. Furthermore, this is the first study to find a positive correlation between residual calcium levels and bone formation in gamma irradiated DBM.

Outcome of revision total knee arthroplasty with bone allograft in 30 cases.

Revision Total Knee Arthroplasty is often complicated by large bone defects in the distal femur and proximal tibia. These defects can be managed in a variety of ways including the use of allograft bone. The purpose of this study was to retrospectively evaluate the clinical outcome of revision total knee arthroplasty cases where allograft bone was used. Thirty revision TKA's (27 patients) performed between 1994 and 2009 were followed for a mean of 5 years (1-14 years). Preoperative bone defects were classified using the Anderson Orthopaedic Research Institute classification system. Patient follow-up entailed calculation of the Knee Society Score and radiological assessment of the revision joint replacement in addition to review of complications. Kaplan Meier analysis predicted survivorship at 5 years as 93%, with further revision surgery as end point. The average Knee Society Score was 76.4, with 19 (63%) of knees scoring "excellent" results, 4 (14%) "good", 1 (3%) "fair" and 6 (20%) were "poor". The overall complication rate was 233%. Radiological lucency was demonstrated on recent radiographs for one patient. Three knees were re-revised at 1 year, 6 years and 8 years respectively. Our study demonstrates promising short to medium term results with the use of allograft bone in revision total knee replacement presenting with significant bone loss.

Reducing the radiation sterilization dose improves mechanical and biological quality while retaining sterility assurance levels of bone allografts.

BACKGROUND: Bone allografts carry a risk of infection, so terminal sterilization by gamma irradiation at 25kGy is recommended; but is deleterious to bone quality. Contemporary bone banking significantly reduces initial allograft bioburden, questioning the need to sterilize at 25kGy. METHODS: We inoculated allograft bone with Staphylococcus epidermidis and Bacillus pumilus, then exposed them to gamma irradiation at 0, 5, 10, 15, 20 and 25kGy. Mechanical and biological properties of allografts were also assessed. Our aim was to determine an optimal dose that achieves sterility assurance while minimizing deleterious effects on allograft tissue. RESULTS: 20-25kGy eliminated both organisms at concentrations from 10(1) to 10(3)CFU, while 10-15kGy sterilized bone samples to a bioburden concentration of 10(2)CFU. Irradiation did not generate pro-inflammatory bone surfaces, as evidenced by macrophage activation, nor did it affect attachment or proliferation of osteoblasts. At doses ≥10kGy, the toughness of cortical bone was reduced (P<0.05), and attachment and fusion of osteoclasts onto irradiated bone declined at 20 and 25kGy (P<0.05). There was no change in collagen cross-links, but a significant dose-response increase in denatured collagen (P<0.05). CONCLUSIONS: Our mechanical and cell biological data converge on 15kGy as a threshold for radiation sterilization of bone allografts. Between 5 and 15kGy, bone banks can undertake validation that provides allografts with an acceptable sterility assurance level, improving their strength and biocompatibility significantly. CLINICAL RELEVANCE: The application of radiation sterilization doses between 5 and 15kGy will improve bone allograft mechanical performance and promote integration, while retaining sterility assurance levels. Improved quality of allograft bone will promote superior clinical outcomes.

The use of Gamma-irradiated proximal femoral allografts for bone stock reconstruction in complex revision hip arthroplasty.

We have followed a consecutive series of 49 revision hip arthroplasties, performed for severe femoral bone loss using Gamma-irradiated anatomic-specific proximal femoral allografts longer than five centimetres. The patients were followed for a median 10.2 years, with a five year minimum follow-up. The median preoperative Harris Hip Score (HHS) improved from 42 points to 77 points postoperatively. In four hips the femoral component was further revised for non-union of the allograft and aseptic failure. In one hip the allograft and the femoral component were removed because of infection. In one hip the allograft and the femoral component were re-revised for host step-cut fracture. Junctional-union was observed in 44/49 hips. By defining success as an increase of HHS by 20 points or more, a stable implant and no need for any subsequent re-operations related to the allograft and /or the implant, a success rate of 76% was observed. Kaplan-Meier survivorship analysis predicted 79% rate of survival at 10 years and 75% rate of survival at 17 years, with the need for further revision of the allograft and/or implant as the end point. Three hips underwent re-attachment of the greater trochanter for trochanteric escape. Asymptomatic non-union of the greater trochanter was noticed in another three hips. Moderate allograft resorption was observed in four hips. Two fractures of the host step-cut occurred. There were four dislocations. Good long-term results with the use of large anatomic-specific femoral allografts justify their continued use in cases of revision hip arthroplasty complicated with severe femoral bone loss.

Expression of thioredoxin system and related peroxiredoxin proteins is associated with clinical outcome in radiotherapy treated early stage breast cancer.

BACKGROUND AND PURPOSE: Deregulated redox systems provide cancer cells protection from increased oxidative stress, such as that induced by ionizing radiation. Expression of the thioredoxin system proteins (thioredoxin, thioredoxin reductase and thioredoxin interacting protein) and downstream peroxiredoxins (I-VI), was examined in tumor specimens from early stage breast cancer patients, subsequently treated by breast conserving surgery and locoregional radiotherapy, to determine if redox protein expression is associated with clinical outcome. MATERIAL AND METHODS: Nuclear and cytoplasmic expression was assessed using conventional immunohistochemistry on a tissue microarray of 224 tumors. RESULTS: High expression of cytoplasmic peroxiredoxin-I correlated with a greater risk of local recurrence (p=0.009). When nuclear and cytoplasmic expression patterns were combined, patients with low nuclear but high cytoplasmic expression of peroxiredoxin-I increased significance (p=0.005). Both were independent factors (p=0.006 and 0.003) from multivariate analysis. Associations were obtained between tumor grade and nuclear thioredoxin interacting protein (p=0.01) and with cytoplasmic expression of peroxiredoxin-V (p=0.007) but not with peroxiredoxin-I suggesting that the latter may exert influence via regulation of oxidative stress rather than via altering the tumor phenotype. CONCLUSIONS: Results highlight the potential of using redox protein expression, namely peroxiredoxin-I, to predict clinical outcome and support further studies to validate its usefulness as an independent prognostic, and potentially predictive, marker.