Breast cancer subtyping by immunohistochemistry and histological grade outperforms breast cancer intrinsic subtypes in predicting neoadjuvant chemotherapy response.

Intrinsic subtypes are widely accepted for the classification of breast cancer. Lacking gene expression data, surrogate classifications based on immunohistochemistry (IHC) have been proposed. A recent St. Gallen consensus meeting recommends to use this "surrogate intrinsic subtypes" for predicting adjuvant chemotherapy resistance, implying that "Surrogate Luminal A" breast cancers should only receive endocrine therapy. In this study we assessed both gene expression based intrinsic subtypes as well as surrogate intrinsic subtypes regarding their power to predict neoadjuvant chemotherapy benefit. Single institution data of 560 breast cancer patients were reviewed. Gene expression data was available for 247 patients. Subtypes were determined on the basis of IHC, Ki67, histological grade, endocrine responsiveness, and gene expression, and were correlated with chemotherapy response and recurrence-free survival. In ER+/HER2- tumors, a high histological grade was the best predictor for chemotherapy benefit, both in terms of pCR (p = 0.004) and recurrence-free survival (p = 0.002). The gene expression based and surrogate intrinsic subtype based on Ki67 had no predictive or prognostic value in ER+/HER2- tumors. Histological grade, ER, PR, and HER2 were the best predictive factors for chemotherapy response in breast cancer. We propose to continue the conventional use of these markers.

Neoadjuvant chemotherapy in ER+ HER2- breast cancer: response prediction based on immunohistochemical and molecular characteristics.

A pathological complete remission (pCR) is rarely achieved by neoadjuvant chemotherapy in estrogen receptor-positive (ER+) HER2-negative (HER2-) tumors. Therefore, its use might be questionable in specific groups of this tumor type. To select which patients benefit and which could be spared neoadjuvant chemotherapy, we tested standard pathology and molecular markers in ER+ HER2- breast tumors. Pretreatment biopsies were available from 211 ER+ HER2- tumors, who had been treated with neoadjuvant chemotherapy (adriamycin/cyclophosphamide). mRNA expression data were available for 132 tumors. We determined progesterone receptor expression (PR), endocrine sensitivity, HER2 expression, histology, proliferation, and molecular subtypes. We correlated these data to chemotherapy response using pCR rates and the previously published neoadjuvant response index (NRI). PR-negative tumors (n = 65, 30.8%) and luminal B type tumors (n = 43, 20.4%) responded significantly better to chemotherapy than other tumors. These associations remained significant in multivariate analysis. However, even in the subgroup of patients with the lowest response rate, comprising tumors that had both a positive-PR expression and the luminal A subtype (n = 58, 44%), the majority of the patients had downstaging because of chemotherapy. For histology (lobular vs. ductal), endocrine sensitivity, and proliferation, no associations with chemotherapy response were observed. Gene expression array analysis resulted in 28 significant genes (FDR < 0.1). PR expression and luminal B status are associated with a better response to neoadjuvant chemotherapy. However, both markers had only weak response predictive power, and it was not possible to identify a subgroup with no or only minimal chemotherapy benefit. Therefore, the decision to refrain from neoadjuvant chemotherapy to ER+ HER2- breast tumors should not be based on predictive markers, but exclusively on estimates of prognosis.

Photosynthetic response of transgenic soybean plants, containing an Arabidopsis P5CR gene, during heat and drought stress.

The biochemical basis of heat/drought tolerance was investigated by comparing the response of antisense and sense transgenic soybean plants (containing the L-delta1-pyrroline-5-carboxylate reductase gene) with non-transgenic wild-type plants. The plants were subjected to a simultaneous drought and heat stress of 2 days, whereafter they were rewatered at 25 degrees C. During this time the sense plants only showed mild symptoms of stress compared to the antisense plants which were severely stressed. Upon stress, nicotinamide adenine dinucleotide phosphate (NADP+) levels decreased in antisense while it increased in sense plants. Recovery with respect to NADP+ levels was best in sense plants. Sense plants had the highest ability to accumulate proline during stress and to metabolise proline after rewatering. Analyses of the fast phase chlorophyll-a fluorescence transients showed dissociation of the oxygen-evolving complex (OEC) upon stress in all plants tested. In the sense plants, which best resisted the stress, OEC dissociation was bypassed by proline feeding electrons into photosystem 2 (PSII), maintaining an acceptable nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) level, preventing further damage. Upon recovery, NADPH is consumed during oxidation of accumulated proline providing high Levels of NADP+ to act as electron acceptor to PSII, which indirectly may ameliorate the inhibition and/or the effect of uncoupling of the OEC.

Interaction between X-ray and alpha-particle damage in V79 cells.

V79 cells have been exposed to X-rays or 238Pu alpha-particles or to X-rays following priming alpha-particle doses of 0.5, 2 or 2.5 Gy. The survival curve for exposure to alpha-particles was exponential with a D0 of 0.89 Gy. Following exposure to priming alpha-particle doses the resulting X-ray survival curves had the same slope as the single dose X-ray curve, but a reduced shoulder. For alpha-particle priming doses of 0.5 and 2 Gy this reduction was the same as for the same X-ray doses. 2.5 Gy alpha-particles reduced the subsequent X-ray curve Dq to almost zero. alpha-particles do cause damage capable of interacting with X-ray damage.

Survival of V79 cells following simultaneous irradiation with X-rays and neutrons in air or hypoxia.

V79 Chinese hamster cells have been irradiated with X-rays and neutrons given simultaneously. The oxygen enhancement ratio and r.b.e. were measured as a function of the proportion of the dose due to the neutrons, which varied from 0 to 100 per cent. These were compared with the values calculated assuming the two types of radiation act independently, following an approach suggested by Curtis. The o.e.r. was less than the predicted value when the neutrons contributed less than about 40 per cent of the total dose. The r.b.e. also did not vary as predicted on the basis of independent action. The 'oxygen gain factor' reached half its maximum value when the proportion of the dose due to neutrons was only about 27 per cent. The results imply that there may be interaction between the damage caused by X-rays and neutrons and that beams having only 20 to 30 per cent of their dose due to high l.e.t. radiation, could be of therapeutic benefit.

The effect of prolonged high dose misonidazole on tumor response to radiation.

WHFIB and SA F tumors were exposed to misonidazole (MISO) concentrations of 2.5 mM or more for up to 4 hours (SA F) or 6 hours (WHFIB). There was no increase in the MISO enhancement ratio (SER) in the SA F for a 4 hour exposure to MISO relative to that following a single injection. In the WHFIB tumor, the ER increased from 2.2 for a single MISO injection to 2.5 for a 4 hour contact with MISO for tumor growth delay, and from 2.1 to 2.3 for a cloning assay. (These differences may not be statistically significant) Prolonged contact with MISO was toxic and reduced the body temperature by 4 to 5 degrees C. For WHFIB cells in vitro, when the contact time (in hypoxia) with 2.5 mM MISO was increased from 0.5 to 2.5 hours, the ER increased from 2.1 to 2.9 at 37 degrees C and from 1.9 to 2.5 at 33 degrees C.

The effect of sequential irradiation with X-rays and fast neutrons on the survival of V79 Chinese hamster cells.

V79 Chinese hamster cells have been exposed to X-rays or fast neutrons or to the two radiations given sequentially. Cells exposed to a priming dose of X-rays and then exposed immediately to a series of neutron doses regard the X-ray dose as equivalent to a neutron dose giving the same surviving fraction (iso-effective). If the cells are exposed to a neutron dose followed by X-rays the resulting survival is higher than would be obtained if the primary dose had been an iso-effective X-ray dose. However, it is lower than would be expected if the two radiations acted independently. The results imply that there is interaction between the damage caused by X-rays and fast neutrons. If the two radiations are given 3 hours apart they act independently.

Enhancement of the action of alkylating agents by single high, or chronic low doses of misonidazole.

Misonidazole (MISO) given as a large single dose enhanced the action of cyclophosphamide (Cy) and melphalan (L-PAM) in two mouse tumours. Below a dose of about 500 mg kg-1 it had no chemosensitizing effect. When MISO was given as a series of small doses by repeat injection over an 8 h period, in order to stimulate human pharmacokinetics, it significantly enhanced the action of Cy in the SA F tumour. It also enhanced the action of Cy and L-PAM in the WHFIB tumour as assayed by tumour cell survival in vitro following treatment in vivo but not when the assay was tumour growth delay. There was no enhancement by MISO of the leukopenia due to Cy or L-PAM. The results suggest that, in some tumours there may be benefit from the combination of clinically relevant MISO doses with alkylating agents. The leucopenia induced by these agents should not be enhanced by the MISO.

Enhancement of the effect of cytotoxic drugs by radiosensitizers.

Misonidazole (MISO) potentiates the action of cyclophosphamide (CY) and melphalan in the WHFIB culture-adapted fibrosarcoma, whether assayed by cell survival or tumour-growth delay. In the case of CY, MISO also inhibited recovery from potentially lethal drug damage. The optimum effect was seen when MISO was given 1 h before CY, though it was also effective when given 6 h before or 1 h after the drug. Other radiosensitizers also potentiated the action of CY. There was only a small effect of MISO on the LD50 of CY and no effect on CY toxicity as assayed by changes in blood counts or damage to bladder epithelium. However, mice bearing multiple lung tumours were less able to cope with the combined treatment than those bearing s.c. tumours.

Radiobiological studies of tumours in situ compared with cell survival.

The dose of radiation needed to eradicate a tumor depends on the radiosensitivity of the individual cells. Direct measurements of tumour cell survival require removal of the cells from their normal environment and may not reflect the course of events in the undisturbed tumour. In situ assays preserve this environment, but only give indirect estimates of tumour cell survival. A major cause of discrepancies between different methods of assaying tumour response is recovery from potentially lethal damage (PLD). For instance, it can affect estimates of the hypoxic fraction and hence of the pattern of reoxygenation. Not all tumours show recovery from PLD. In such tumours, where comparisons have been made between different assays, they have shown differences in the effects of radiation quality and modifying agents and in the capacity for recovery from sublethal damage between tumour cell survival in vivo and in vitro after treatment in vivo. No single assay method can give an adequate description of the response of a tumour to radiation. It is only by comparing different methods of assay that the basic biological properties of tumors that govern their response to therapy will be understood.

Recovery from sublethal damage by acutely hypoxic tumour cells in vivo and in vitro.

The ability of acutely hypoxic tumour cells to recover from sublethal damage following irradiation in vivo and in vitro has been measured using a single tumour system. The methods of assay were tumour growth delay, local tumour control and tumour cell survival in vitro following treatment in vivo or in vitro. Tumours in vivo or cells in vitro rendered acutely hypoxic during irradiation were irradiated with either single doses or two doses 24 hours apart. Cells left in situ had a greater capacity for recovery than those treated either in vivo or in vitro and then assayed in vitro. It is suggested that tumours may not show a systematically reduced capacity for recovery relative to normal tissues, unless chronically hypoxic tumour cells have a reduced capacity for recovery and determine the response. However, the results imply that deductions as to the ability of tumour cells to recover from sublethal damage (whether chronically hypoxic or not), which rely on in vitro assays, may underestimate the extent of recovery.

Interaction between electron-affinic sensitizers.

Measurements of effect of pairs of the sensitizers PNAP, misonidazole and oxygen have shown that if one sensitizer is giving an enhancement ratio (ER) of about 1.6 or less and the other is giving a larger ER, the combination behaves as if only the latter is present. There is only an additive effect of the two if they both give ERs less than 1.6 or greater than 1.6. These results show that at low concentrations sensitizers only act on damage associated with the "fast component" of the oxygen effect. This could result from interaction with two distinct species, or there could be only one critical species formed initially but more than one step by which it could be converted to a lethal form.

The effect of repeated small doses of radiation on recovery from sub-lethal damage by Chinese hamster cells irradiated in the plateau phase of growth.

Unfed plateau-phase cells have been irradiated with either single doses or up to ten fractions of X-rays 6 hours apart. The single-dose survival curve had an extrapolation number of 11-4, and the oxygen-enhancement ratio (o.e.r.) was 3-1. Cells were exposed to multiple fractions of 200 rad or 150 rad in air and 600 rad or 450 rad in hypoxia. The resulting survival curves did not fit a multi-target, single-hit model of cell survival, being much steeper than that would predict. The curves were exponential up to five fractions of X-rays, but tended to bend downwards with increasing number of fractions. Cells that had survived five fractions of 200 rad (or 600 rad in hypoxia) 6 hours apart, were less able to absorb damage as sub-lethal than those which had not previously been exposed to radiation. The ratio of the initial slopes of the fractionated survival curves for irradiation in air and hypoxia was 2-1, implying that the o.e.r. "on the shoulder" may be less than that in the exponential region of survival.