Reconstructing tumor history in breast cancer: signatures of mutational processes and response to neoadjuvant chemotherapy⋆.

BACKGROUND: Different endogenous and exogenous mutational processes act over the evolutionary history of a malignant tumor, driven by abnormal DNA editing, mutagens or age-related DNA alterations, among others, to generate the specific mutational landscape of each individual tumor. The signatures of these mutational processes can be identified in large genomic datasets. We investigated the hypothesis that genomic patterns of mutational signatures are associated with the clinical behavior of breast cancer, in particular chemotherapy response and survival, with a particular focus on therapy-resistant disease. PATIENTS AND METHODS: Whole exome sequencing was carried out in 405 pretherapeutic samples from the prospective neoadjuvant multicenter GeparSepto study. We analyzed 11 mutational signatures including biological processes such as APOBEC-mutagenesis, homologous recombination deficiency (HRD), mismatch repair deficiency and also age-related or tobacco-induced alterations. RESULTS: Different subgroups of breast carcinomas were defined mainly by differences in HRD-related and APOBEC-related mutational signatures and significant differences between hormone-receptor (HR)-negative and HR-positive tumors as well as correlations with age, Ki-67 and immunological parameters were observed. We could identify mutational processes that were linked to increased pathological complete response rates to neoadjuvant chemotherapy with high significance. In univariate analyses for HR-positive tumors signatures, S3 (HRD, P < 0.001) and S13 (APOBEC, P = 0.001) as well as exonic mutation rate (P = 0.002) were significantly correlated with increased pathological complete response rates. The signatures S3 (HRD, P = 0.006) and S4 (tobacco, P = 0.011) were prognostic for reduced disease-free survival of patients with chemotherapy-resistant tumors. CONCLUSION: The results of this investigation suggest that the clinical behavior of a tumor, in particular, response to neoadjuvant chemotherapy and disease-free survival of therapy-resistant tumors, could be predicted by the composition of mutational signatures as an indicator of the individual genomic history of a tumor. After additional validations, mutational signatures might be used to identify tumors with an increased response rate to neoadjuvant chemotherapy and to define therapy-resistant subgroups for future therapeutic interventions.

Tumor mutational burden and immune infiltration as independent predictors of response to neoadjuvant immune checkpoint inhibition in early TNBC in GeparNuevo.

BACKGROUND: The predictive value of tumor mutational burden (TMB), alone or in combination with an immune gene expression profile (GEP), for response to neoadjuvant therapy in early triple negative breast cancer (TNBC) is currently not known, either for immune checkpoint blockade (ICB) or conventional chemotherapy. PATIENTS AND METHODS: We obtained both whole exome sequencing and RNA-Seq data from pretreatment samples of 149 TNBC of the recent neoadjuvant ICB trial, GeparNuevo. In a predefined analysis, we assessed the predictive value of TMB and a previously developed immune GEP for pathological complete remission (pCR). RESULTS: Median TMB was 1.52 mut/Mb (range 0.02-7.65) and was significantly higher in patients with pCR (median 1.87 versus 1.39; P = 0.005). In multivariate analysis, odds ratios for pCR per mut/Mb were 2.06 [95% confidence intervals (CI) 1.33-3.20, P = 0.001] among all patients, 1.77 (95% CI 1.00-3.13, P = 0.049) in the durvalumab treatment arm, and 2.82 (95% CI 1.21-6.54, P = 0.016) in the placebo treatment arm, respectively. We also found that both continuous TMB and immune GEP (or tumor infiltrating lymphocytes) independently predicted pCR. When we stratified patients in groups based on the upper tertile of TMB and median GEP, we observed a pCR rate of 82% (95% CI 60% to 95%) in the group with both high TMB and GEP in contrast to only 28% (95% CI 16% to 43%) in the group with both low TMB and GEP. CONCLUSIONS: TMB and immune GEP add independent value for pCR prediction. Our results recommend further analysis of TMB in combination with immune parameters to individually tailor therapies in breast cancer.

Survival analysis of carboplatin added to an anthracycline/taxane-based neoadjuvant chemotherapy and HRD score as predictor of response-final results from GeparSixto.

Background: In the neoadjuvant GeparSixto study, adding carboplatin to taxane- and anthracycline-based chemotherapy improved pathological complete response (pCR) rates in patients with triple-negative breast cancer (TNBC). Here, we present survival data and the potential prognostic and predictive role of homologous recombination deficiency (HRD). Patients and methods: Patients were randomized to paclitaxel plus nonpegylated liposomal doxorubicin (Myocet®) (PM) or PM plus carboplatin (PMCb). The secondary study end points disease-free survival (DFS) and overall survival (OS) were analyzed. Median follow-up was 47.3 months. HRD was among the exploratory analyses in GeparSixto and was successfully measured in formalin-fixed, paraffin-embedded tumor samples of 193/315 (61.3%) participants with TNBC. Homologous recombination (HR) deficiency was defined as HRD score ≥42 and/or presence of tumor BRCA mutations (tmBRCA). Results: A significantly better DFS (hazard ratio 0.56, 95% CI 0.34-0.93; P = 0.022) was observed in patients with TNBC when treated with PMCb. The improvement of OS with PMCb was not statistically significant. Additional carboplatin did not improve DFS or OS in patients with HER2-positive tumors. HR deficiency was detected in 136 (70.5%) of 193 triple-negative tumors, of which 82 (60.3%) showed high HRD score without tmBRCA. HR deficiency independently predicted pCR (ypT0 ypN0) [odds ratio (OR) 2.60, 95% CI 1.26-5.37, P = 0.008]. Adding carboplatin to PM significantly increased the pCR rate from 33.9% to 63.5% in HR deficient tumors (P = 0.001), but only marginally in HR nondeficient tumors (from 20.0% to 29.6%, P = 0.540; test for interaction P = 0.327). pCR rates with carboplatin were also higher (63.2%) than without carboplatin (31.7%; OR 3.69, 1.46-9.37, P = 0.005) in patients with high HRD score but no tmBRCA. DFS rates were improved with addition of carboplatin, both in HR nondeficient (hazard ratio 0.44, 0.17-1.17, P = 0.086) and HR deficient tumors (hazard ratio 0.49, 0.23-1.04, P = 0.059). Conclusions: The addition of carboplatin to neoadjuvant PM improved DFS significantly in TNBC. Long-term survival analyses support the neoadjuvant use of carboplatin in TNBC. HR deficiency in TNBC and HRD score in non-tmBRCA TNBC are predictors of response. HRD does not predict for carboplatin benefit.

BRCA1-like profile is not significantly associated with survival benefit of non-myeloablative intensified chemotherapy in the GAIN randomized controlled trial.

PURPOSE: The BRCA1-like profile identifies tumors with a defect in homologous recombination due to inactivation of BRCA1. This profile has been shown to predict which stage III breast cancer patients benefit from myeloablative, DNA double-strand-break-inducing chemotherapy. We tested the predictive potential of the BRCA1-like profile for adjuvant non-myeloablative, intensified dose-dense chemotherapy in the GAIN trial. METHODS: Lymph node positive breast cancer patients were randomized to 3 × 3 dose-dense cycles of intensified epirubicin, paclitaxel, and cyclophosphamide (ETC) or 4 cycles concurrent epirubicin and cyclophosphamide followed by 10 cycles of weekly paclitaxel combined with 4 cycles capecitabine (EC-TX). Only triple negative breast cancer patients (TNBC) for whom tissue was available were included in these planned analyses. BRCA1-like or non-BRCA1-like copy number profiles were derived from low coverage sequencing data. RESULTS: 119 out of 163 TNBC patients (73%) had a BRCA1-like profile. After median follow-up of 83 months, disease free survival (DFS) was not significantly different between BRCA1-like and non-BRCA1-like patients [adjusted hazard ratio (adj.HR) 1.02; 95% confidence interval (CI) 0.55-1.86], neither was overall survival (OS; adj.HR 1.26; 95% CI 0.58-2.71). When split by BRCA1-like status, DFS and OS were not significantly different between treatments. However, EC-TX seemed to result in a trend to an improvement in DFS in patients with a BRCA1-like tumor, while the reverse accounted for ETC treatment in patients with a non-BRCA1-like tumor (p for interaction = 0.094). CONCLUSIONS: The BRCA1-like profile is not associated with survival benefit for a non-myeloablative, intensified regimen in this study population. Considering the limited cohort size, capecitabine might have additional benefit for TNBC patients.

The EndoPredict score provides prognostic information on late distant metastases in ER+/HER2- breast cancer patients.

BACKGROUND: ER+/HER2- breast cancers have a proclivity for late recurrence. A personalised estimate of relapse risk after 5 years of endocrine treatment can improve patient selection for extended hormonal therapy. METHODS: A total of 1702 postmenopausal ER+/HER2- breast cancer patients from two adjuvant phase III trials (ABCSG6, ABCSG8) treated with 5 years of endocrine therapy participated in this study. The multigene test EndoPredict (EP) and the EPclin score (which combines EP with tumour size and nodal status) were predefined in independent training cohorts. All patients were retrospectively assigned to risk categories based on gene expression and on clinical parameters. The primary end point was distant metastasis (DM). Kaplan-Meier method and Cox regression analysis were used in an early (0-5 years) and late time interval (>5 years post diagnosis). RESULTS: EP is a significant, independent, prognostic parameter in the early and late time interval. The expression levels of proliferative and ER signalling genes contribute differentially to the underlying biology of early and late DM. The EPclin stratified 64% of patients at risk after 5 years into a low-risk subgroup with an absolute 1.8% of late DM at 10 years of follow-up. CONCLUSION: The EP test provides additional prognostic information for the identification of early and late DM beyond what can be achieved by combining the commonly used clinical parameters. The EPclin reliably identified a subgroup of patients who have an excellent long-term prognosis after 5 years of endocrine therapy. The side effects of extended therapy should be weighed against this projected outcome.

EndoPredict improves the prognostic classification derived from common clinical guidelines in ER-positive, HER2-negative early breast cancer.

BACKGROUND: In early estrogen receptor (ER)-positive/HER2-negative breast cancer, the decision to administer chemotherapy is largely based on prognostic criteria. The combined molecular/clinical EndoPredict test (EPclin) has been validated to accurately assess prognosis in this population. In this study, the clinical relevance of EPclin in relation to well-established clinical guidelines is assessed. PATIENTS AND METHODS: We assigned risk groups to 1702 ER-positive/HER2-negative postmenopausal women from two large phase III trials treated only with endocrine therapy. Prognosis was assigned according to National Comprehensive Cancer Center Network-, German S3-, St Gallen guidelines and the EPclin. Prognostic groups were compared using the Kaplan-Meier survival analysis. RESULTS: After 10 years, absolute risk reductions (ARR) between the high- and low-risk groups ranged from 6.9% to 11.2% if assigned according to guidelines. It was at 18.7% for EPclin. EPclin reassigned 58%-61% of women classified as high-/intermediate-risk (according to clinical guidelines) to low risk. Women reclassified to low risk showed a 5% rate of distant metastasis at 10 years. CONCLUSION: The EPclin score is able to predict favorable prognosis in a majority of patients that clinical guidelines would assign to intermediate or high risk. EPclin may reduce the indications for chemotherapy in ER-positive postmenopausal women with a limited number of clinical risk factors.

Increased selection response in larger populations. I. Selection for wing-tip height in Drosophila melanogaster at three population sizes.

The effect of population size on selection response was investigated with replicated selection lines of 40, 200 and 1000 selected parents, using Drosophila melanogaster homozygous for the mutant raised. Selection for increased wing-tip height was carried out for 55 generations, with an average selection intensity of 0.6 standard deviation. The rank order of responses in the seven individual lines was significantly in order of population size, and the variance of response among lines showed a significant effect of population size. The final mean responses (selected - controls, +/- standard errors) in the three treatments, in order of increasing population size, were 8.6 +/- 1.8 mils (three small lines), 15.1 +/- 1.3 mils (two medium lines), and 19.8 +/- 1.5 mils (two large lines). The differences between treatments seem to have emerged too rapidly to be the result of mutations, and are probably due mainly to the utilization of existing variation with greater efficiency by selection in larger populations.

Large genetic change at small fitness cost in large populations of Drosophila melanogaster selected for wind tunnel flight: rethinking fitness surfaces.

The fitness effects of extreme genetic change by selection were studied in large populations subjected to prolonged, intense selection. Two replicate populations of Drosophila melanogaster, with estimated effective sizes 500 < or = Ne < or = 1000, were selected for increased performance in a wind tunnel, selecting on average the fastest 4.5% of flies. The mean apparent flying speed of both lines increased from approximately 2 to 170 cm/sec and continued to respond at diminishing rates, without reaching a plateau, for 100 generations. Competitive fitness tests in generations 50 and 85 showed minimal or no fitness loss in selected lines compared to controls. Sublines relaxed in generations 65 and 85 showed minimal or no regression in apparent flying speed. Hybrid lines, from a cross of selected x control lines in generation 75, responded to reselection saltationally, showing that the chromosomes of the selected lines had been assembled from alleles at many loci, from many different chromosomes in the base population. Thus, major genetic change was achieved, but without the costs usually associated with strong directional selection. Large population size has been interpreted, in opposing models, as either a brake or an accelerator in its effects on long-term change by selection. These results favor the second model, and challenge the concept of rugged fitness surfaces underlying the first model.

Selection on wing allometry in Drosophila melanogaster.

Five bivariate distributions of wing dimensions of Drosophila melanogaster were measured, in flies 1) subjected to four defined environmental regimes during development, 2) taken directly from nature in seven U.S. states, 3) selected in ten populations for change in wing form, and 4) sampled from 21 long inbred wild-type lines. Environmental stresses during development altered both wing size and the ratios of wing dimensions, but regardless of treatment all wing dimensions fell near a common allometric baseline in each bivariate distribution. The wings of wild-caught flies from seven widely separated localities, and of their laboratory-reared offspring, also fell along the same baselines. However, when flies were selected divergently for lateral offset from these developmental baselines, response to selection was rapid in every case. The mean divergence in offset between oppositely selected lines was 14.68 SD of the base population offset, after only 15 generations of selection at 20%. Measurements of 21 isofemale lines, founded from wild-caught flies and maintained in small populations for at least 22 years, showed large reductions in phenotypic variance of offsets within lines, but a large increase in the variance among lines. The variance of means of isofemale lines within collection localities was ten times the variance of means among localities of newly established wild lines. These observations show that much additive genetic variance exists for individual dimensions within the wing, such that bivariate developmental patterns can be changed in any direction by selection or by drift. The relative invariance of the allometric baselines of wing morphology in nature is most easily explained as the result of continuous natural selection around a local optimum of functional design.

How small are the smallest selectable domains of form?

Two lines of Drosophila melanogaster from the same base population were selected in opposite directions to produce simultaneous antagonistic changes in two very small (less than 0.2 mm) and closely adjacent (less than 0.3 mm) dimensions within the base of the wing. Wing dimensions near the targeted area became differentiated by large positive and negative percentage differences, while only small homogeneous percentage changes occurred in the remainder of the wing. If very small regions of morphology (less than 100 cells across) can respond to selection almost independently, even in small population samples, then the control of developmental detail must involve many genes, and the diversity of possible outcomes in development and adaptation must be large.

Increased selection response in larger populations. II. Selection for ethanol vapor resistance in Drosophila melanogaster at two population sizes.

The effect of large population size on selection response was investigated using Drosophila melanogaster, with four "small" lines of 160 selected parents/generation compared to two "large" lines of 1,600 selected parents/generation. All lines were selected under similar conditions at a selection intensity of approximately 0.55 standard deviations, for 65 generations, for increased ethanol vapor resistance (measured in minutes required to become anesthetized). Two unselected control lines of 320 parents/generation were also maintained. A significant effect of population size was found. The final treatment means and standard errors were: 27.91 +/- 1.28 min (two "large" lines); 19.40 +/- 1.54 min (four "small" lines); and 4.98 +/- 0.35 min (two control lines). To estimate the mutation rate for the trait, two isogenic lines of about 400 selected parents were selected for 29 generations. The mean increase in additive genetic variance per generation was 0.0009 times the initial environmental variance of the outbred lines. This is comparable to other reported mutation rates. Mutation can explain part of the difference in evolved resistance between treatments, but it appears that even at rather large population sizes, a large difference in long-term response can be obtained in larger outbred lines, from more complete utilization of the initial genetic variation.

Issues in natural resources management in developing countries.

PIP: The use and management of public and private natural resources is greatly affected by institutional, politicoeconomic, and socioeconomic factors. These factors operate in tandem at the household, regional, national, and international levels in affecting resource management. Any policy that focuses only on one dimension of the problem, such as population growth, and ignores such issues as poverty, environmentally unsuitable cropping systems, and the unavailability of nonfarm employment opportunities will be inadequate. National policies must consider structural factors: resource use, property rights regimes, poverty, unemployment, illiteracy, and population growth. A comprehensive strategy should account for these structural features, be tailored to regional circumstances, and have the input of local governments. Local governments and communities must be in a position to implement and monitor resource management. Hardin identified common property as a major contributor to land degradation and declining forest resources in developing countries. Only with a common agreement by the community to invest in planting trees and managing pastures can communal lands benefit multiple users in the long and short run. Social forestry projects have failed due to poor community participation, fragmented communities, poor social organization, and disputes over the distribution of profits. Traditional communal hunting and gathering activities adhered to institutional regulation; with the inception of urbanization, industrialization, and monetization of agriculture, the destruction of forests and pasture began. There was an increased need for food for the urban population; a small and powerful group of politicians, social leaders, and administrators retained access to public or "crown" lands and disenfranchised small and marginal farmers and the landless poor from previously accessible lands. Land redistribution schemes have not effectively distributed land resources. Commercial business and forestry managers are all responsible for deforestation. Land management is affected by personal, perceptual, attitudinal, social, economic, and institutional factors.^ieng

Population and environment in the hills of Nepal.

PIP: This article examines how the people in the hills of Nepal, in spite of their physical and economic isolation from the national mainstream, could manage their economic activities for such a long time without any heavy environmental stress. The major proportion of Nepal's population lives in the hills. In the early 1900s, the population in the hills was small in absolute numbers. The population grew during the 1900s, despite continuous out-migration. Under present circumstances, the hills of Nepal do not allow any further intensification of cropping because of the zone's high elevation, steep slopes, poor soils, and high level of precipitation. The hills were mostly bypassed in terms of policy and planning until the beginning of the 5th Five Year Plan period (1976-1980), when development plans were drawn up and budget allocations were made on a regional basis. Because of concentration on agriculture in the plains, nonfarm employment opportunities barely exist there. Strategies the hill people have used to cope with the limited carrying capacity of land resources and few alternative employment opportunities include 1) expansion of agricultural lands into marginally suited areas, 2) enlarging the size of their livestock herds, 3) out-migration, and 4) collection of fuel wood. These practices have led to the deterioration of the ecological balance. The hill people are reluctant to adopt family planning because a small family size puts heavier pressure on the household economy and its available labor force. To stabilize the economic and environmental systems and to control accelerated population growth in the hills, a comprehensive development program must be worked out, focusing on alleviating pressure on the fragile resource system. The strategy would include 1) agricultural development, 2) implementation of an integrated agrarian and land reform program, 3) promotion of non-farm employment opportunities, 4) population control, and 5) people's participation in decision-making and planning. Watersheds are the appropriate spatial planning units for regional development planning processes.^ieng

Resettlement experiences and alternatives in Thailand.

Problems concerning the resettlement of those made homeless by the construction of dams to create reservoirs in Thailand are considered. The authors attempt "firstly, to examine the rationale for spontaneous and planned resettlement in Thailand. Secondly, the determinants of the success or failure of resettlers are explored with reference to selected planned resettlement projects. Finally, resettlement alternatives for prospective reservoir evacuees are proposed."

Reflection of Confucianism, Hinduism, and Buddhism on gender relations and gender specific occupation in Thai society.

PIP: It is posited that present-day Thai society is the product of different settlement patterns among Chinese Confucian followers and those ascribing to matrilocal practices. Chinese settled in urban areas and maintained Confucian dogma that barred women from high ranking positions and dictated a women's role of subservience to men. Matrilocal systems proliferated in rural areas. The village kinship system was egalitarian, until a class-state society was instituted and patriarchal systems dominated. At that time, women's status was reduced. Massive Chinese immigration occurred during the mid-19th century until World War II. Some Indians also migrated during this time period. The dominant use of the Thai language forced the Chinese to assimilate into Thai culture. Thai Buddhist practices were open and similar enough to Confucian ideology that religious assimilation also occurred. A small group of Chinese immigrants retained their ties to Chinese customs. The pattern of foreigners' involvement in the Thai economy was promoted by official policy. In the last several decades Thai policy shifted to an increasingly Thai-influenced economy. Other influences on gender patterns and Thai culture were the Hinduism of Indians who settled in Central Siam, the Mon aristocracy, and Brahmin cults. After the Khmers took over control of this region, the Indian caste system and the Hindu belief in Manu were integrated into Khmer culture. Women were considered the weaker and inferior sex and dependent upon men. The kings of Siam followed Brahmin rituals after the 15th century. Buddhism and the "sangha" became the central Thai religious institution. Even today Buddhist monks are given 3 months time off with pay for time spent as a monk, while maternity leave for women is limited to 45 days. The status of women is traced during the Sukothai period (1250-1350), the Ayudhaya period (1350-1767), and the Ratanakosin period (since 1782). Present occupational patterns reflect women's dominance in business trades and men's dominance in bureaucratic and political affairs. Current patterns reflect gender occupation specialization that is tied to historical patterns linked to religion and ideology.^ieng

Achieving sustainable agriculture through recognizing gender roles: some salient points.

PIP: This paper assesses existing gender relations in agriculture with the view that the gender issue must be explicitly appraised and incorporated into development planning in order to achieve environmentally sustainable agriculture. The paper opens with a discussion of the reasons why women are more affected by poverty than men, a brief review of studies indicating that men are more privileged than women and children in almost all societies, and the emerging understanding that women have always been involved in sustaining food security. The next section considers the importance of agriculture for sustainable development and for economic development in general. The paper continues with a brief review of women's status in various societies as revealed by anthropological, archeological, and historical evidence. Gender analysis is seen as essential for solving agricultural and environmental problems because it exposes inequality, exploitation, or oppression and leads to a proper acknowledgement of women's role in agriculture. After a review of the results of studies of gender differences in agriculture, the paper points to the importance of considering the managerial role of women in agricultural production and their use of natural resources as well as their role as laborers. In conclusion, it is recommended that a research agenda be composed to: 1) apply a gender analysis to the history of agricultural civilization; 2) emphasize the difficulties associated with measuring, appraising, and recording time expended; 3) focus on the full range of activities, work, and duties in a typical farming community; 4) determine the likely desirable variant of gender diversity in a given culture; and 5) develop focused, comprehensive research designs that will facilitate compatibility assessment, verifiability, and replicability.^ieng

Aerial performance of Drosophila melanogaster from populations selected for upwind flight ability.

A computerized system for three-dimensional tracking of large numbers of individual free-flying insects was used to assess the performance of Drosophila melanogaster from populations that had undergone 160 generations of selection for upwind flight ability. Compared with control lines, the selected lines showed significant increases in mean flight velocity, decreases in angular trajectory and a significant change in the interaction between velocity and angular trajectory. Maximal flight velocity was apparent as a sharply defined upper boundary of the distribution of horizontal and vertical velocity as a function of angular trajectory; this upper bound (0.85 ms-1) differed little between the selected and control lines, although individuals from the selected lines attained maximal performance levels much more frequently. Maximum induced power output was calculated directly from the product of maximum vertical velocity and body weight. This measure (28 W kg-1 muscle) was closely predicted by a scaling relationship derived from the load-lifting limits of larger insects and vertebrates, as well as tethered D. melanogaster stimulated via their optomotor reflex to produce maximal lift. These results indicate that selection for flight performance can readily alter the relative effort and/or the frequency of phenotypes capable of attaining population-wise maximal performance levels, but shows little ability to increase population-wise maximal performance.