GDF15 linked to maternal risk of nausea and vomiting during pregnancy.

GDF15, a hormone acting on the brainstem, has been implicated in the nausea and vomiting of pregnancy, including its most severe form, hyperemesis gravidarum (HG), but a full mechanistic understanding is lacking1-4. Here we report that fetal production of GDF15 and maternal sensitivity to it both contribute substantially to the risk of HG. We confirmed that higher GDF15 levels in maternal blood are associated with vomiting in pregnancy and HG. Using mass spectrometry to detect a naturally labelled GDF15 variant, we demonstrate that the vast majority of GDF15 in the maternal plasma is derived from the feto-placental unit. By studying carriers of rare and common genetic variants, we found that low levels of GDF15 in the non-pregnant state increase the risk of developing HG. Conversely, women with ß-thalassaemia, a condition in which GDF15 levels are chronically high5, report very low levels of nausea and vomiting of pregnancy. In mice, the acute food intake response to a bolus of GDF15 is influenced bi-directionally by prior levels of circulating GDF15 in a manner suggesting that this system is susceptible to desensitization. Our findings support a putative causal role for fetally derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by prepregnancy exposure to the hormone, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

Fetally-encoded GDF15 and maternal GDF15 sensitivity are major determinants of nausea and vomiting in human pregnancy.

Human pregnancy is frequently accompanied by nausea and vomiting that may become severe and life-threatening, as in hyperemesis gravidarum (HG), the cause of which is unknown. Growth Differentiation Factor-15 (GDF15), a hormone known to act on the hindbrain to cause emesis, is highly expressed in the placenta and its levels in maternal blood rise rapidly in pregnancy. Variants in the maternal GDF15 gene are associated with HG. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We found that the great majority of GDF15 in maternal circulation is derived from the feto-placental unit and that higher GDF15 levels in maternal blood are associated with vomiting and are further elevated in patients with HG. Conversely, we found that lower levels of GDF15 in the non-pregnant state predispose women to HG. A rare C211G variant in GDF15 which strongly predisposes mothers to HG, particularly when the fetus is wild-type, was found to markedly impair cellular secretion of GDF15 and associate with low circulating levels of GDF15 in the non-pregnant state. Consistent with this, two common GDF15 haplotypes which predispose to HG were associated with lower circulating levels outside pregnancy. The administration of a long-acting form of GDF15 to wild-type mice markedly reduced subsequent responses to an acute dose, establishing that desensitisation is a feature of this system. GDF15 levels are known to be highly and chronically elevated in patients with beta thalassemia. In women with this disorder, reports of symptoms of nausea or vomiting in pregnancy were strikingly diminished. Our findings support a causal role for fetal derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to GDF15, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

Liquid chromatography/mass spectrometry based detection and semi-quantitative analysis of INSL5 in human and murine tissues.

RATIONALE: Insulin-like peptide 5 (INSL5) is a hormone produced by enteroendocrine L-cells in the colon that has recently been implicated in the control of metabolic homeostasis. However, research into its physiology has been hindered by the reported unreliability of commercially available immunoassays and additional detection assays would benefit this emerging field. METHODS: Peptides from purified murine L-cells and homogenates from both human and mouse colonic tissues were extracted by precipitating larger proteins with acetonitrile. Untargeted liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses, followed by database searching, were used to detect and identify various INSL5 gene derived peptides and characterise their precise sequence. A similar approach was developed to quantify INSL5 levels in primary intestinal culture supernatants after purification and concentration by solid-phase extraction. RESULTS: Mass spectral analysis of purified enteroendocrine cells and tissue homogenates identified the exact sequence of A and B chains of INSL5 endogenously expressed in L-cells. Differences in the endogenously processed peptide and the Swissprot database entry were observed for murine INSL5, whereas the human sequence matched previous predictions from heterologous expression experiments. INSL5 was detected in the supernatant of human and mouse primary colonic cultures and concentrations increased after treatment with a known L-cell stimulus. CONCLUSIONS: The first LC/MS/MS-based method capable of the detection and semi-quantitative analysis of endogenous INSL5 using MS-based techniques has been demonstrated. The methodology will enable the identification of stimulants for INSL5 secretion from murine and human primary colonic epithelial cultures.

High-throughput ultra-high-performance liquid chromatography/tandem mass spectrometry quantitation of insulin-like growth factor-I and leucine-rich alpha-2-glycoprotein in serum as biomarkers of recombinant human growth hormone administration.

Insulin-like growth factor-I (IGF-I) is a known biomarker of recombinant human growth hormone (rhGH) abuse, and is also used clinically to confirm acromegaly. The protein leucine-rich alpha-2-glycoprotein (LRG) was recently identified as a putative biomarker of rhGH administration. The combination of an ACN depletion method and a 5-min ultra-high-performance liquid chromatography/tandem mass spectrometry (uHPLC/MS/MS)-based selected reaction monitoring (SRM) assay detected both IGF-I and LRG at endogenous concentrations. Four eight-point standard addition curves of IGF-I (16-2000 ng/mL) demonstrated good linearity (r(2) = 0.9991 and coefficients of variance (CVs) <13%). Serum samples from two rhGH administrations were extracted and their uHPLC/MS/MS-derived IGF-I concentrations correlated well against immunochemistry-derived values. Combining IGF-I and LRG data improved the separation of treated and placebo states compared with IGF-I alone, further strengthening the hypothesis that LRG is a biomarker of rhGH administration. Artificial neural networks (ANNs) analysis of the LRG and IGF-I data demonstrated an improved model over that developed using IGF-I alone, with a predictive accuracy of 97%, specificity of 96% and sensitivity of 100%. Receiver operator characteristic (ROC) analysis gave an AUC value of 0.98. This study demonstrates the first large scale and high throughput uHPLC/MS/MS-based quantitation of a medium abundance protein (IGF-I) in human serum. Furthermore, the data we have presented for the quantitative analysis of IGF-I suggest that, in this case, monitoring a single SRM transition to a trypsin peptide surrogate is a valid approach to protein quantitation by LC/MS/MS.

Effects of vertical-banded gastroplasty on bone and mineral metabolism in obese patients.

The surgical treatment of obesity can have adverse effects on bone, but there are few published data on the effects of vertical-banded gastroplasty. Serial measurements of bone mineral density at the lumbar spine and three upper femoral sites, using dual-energy X-ray absorptiometry, and also of biochemical indices of bone and mineral metabolism at intervals up to 2 years after operation were performed in 18 patients with morbid obesity who had vertical-banded gastroplasty. Bone mineral density measurements were also made in age- and sex-matched non-obese controls. Bone density before operation was significantly greater in the obese than in the controls (P < 0.02 at all sites). The obese patients lost weight rapidly after vertical-banded gastroplasty (mean weight loss 29 kg at 1 year, P < 0.001). This was accompanied by a measurable loss of bone density from the trochanter and Ward's triangle sites in the upper femur (P < 0.05), but not from the lumbar spine. Bone density values remained stable over 14 months in the controls. Hydroxyproline excretion increased significantly (P < 0.005), indicating an increase in bone resorption. Alkaline phosphatase levels decreased significantly (P < 0.001), but this probably represents the reversal of hepatic steatosis. There was no evidence of hyperparathyroidism or vitamin D deficiency. In conclusion, vertical-banded gastroplasty causes modest bone density loss from femoral sites, but not the lumbar spine. The difficulties of assessing bone density changes in the obese are discussed.

Treatment of breast cancer in the Auckland region 1976-85.

AIM: A descriptive study of the treatment of breast cancer in Auckland between the years 1976 to 1985. METHODS: A database was constructed utilising information from all new breast cancer cases recorded in the Auckland region from September 1976 to September 1985. Details of treatment were obtained at the time of diagnosis and the database was updated every 9 months. Patient survival was measured and changes in the pattern of treatment were assessed. RESULTS: After a median follow up of 9 years 41% of patients were alive without evidence of breast cancer, 9% were alive with recurrence and 50% had died, 38% having died of breast cancer. Survival of node positive patients at 5 years of follow up who received adjuvant tamoxifen or adjuvant chemotherapy was 57 (SE 4)% and 63 (4)% respectively. The proportion of less than mastectomy surgical procedures increased over the study period, and local recurrence in these patients was reduced by postoperative radiotherapy. CONCLUSIONS: Between 1976 and 1985 there was an increasing rate of conservative surgery for breast cancer in Auckland. Overall survival of patients was comparable to that reported in international studies, with increasing use of adjuvant endocrine therapy but a decline in adjuvant chemotherapy over the duration of the study.

The relationship of urinary and plasma androgens to steroid receptors and menopausal status in breast cancer patients and their influence on survival.

The relationships between urinary 11-desoxy-17-oxo steroids (11-DOS), the ratio of 11-DOS to urinary 17-hydroxycorticosteroids (urinary discriminant ratio), plasma levels of the adrenal androgens dehydroepiandrosterone (DHA), DHA sulphate (DHAS), and 7 alpha-hydroxy DHA (7 alpha DHA), and tumour oestrogen receptor (ER) and progesterone receptor (PR) status were examined in pre, peri-, and postmenopausal women with breast cancer. Androgenic steroids and their metabolites decreased with age in women with breast cancer. In perimenopausal women there was a significant association of PR positive tumours and high androgen levels, whereas in postmenopausal women high androgen levels were associated with ER negative tumours. Survival was significantly related to plasma DHA level and tumour steroid receptor status. Thus, adrenal androgen levels below the group mean were associated with significantly decreased survival in women with postmenopausal receptor-positive tumours, and the association was particularly apparent in those who were axillary node negative. Since the number of patients studied was small these results should be regarded as provisional in nature. Nonetheless, the identification of this subgroup of node negative breast cancer women with reduced survival may be important when considering node negative patients for adjuvant therapy.

Detection of contralateral breast cancer by mammography in women with previous breast cancer and the impact of endocrine therapy.

AIMS: To determine the efficacy and extent of screening mammography for detection of contralateral breast cancer in a cohort of women with previous unilateral mammary carcinoma, and to assess the effect of endocrine therapy on the risk of developing cancer in the contralateral breast. METHODS: Women with previous breast cancer eligible for mammography were identified from the Auckland breast cancer data file and the extent and outcome of mammographic screening determined by questionnaire and survey of mammography reports. The extent of adjuvant hormonal therapy and development of contralateral breast cancer was ascertained from the ABCDF records. RESULTS: Of 703 eligible subjects, 59% had undergone screening mammography with a cancer detection rate of 17 per 1000 mammograms and a benign to malignant ratio was 1.7 to 1. Contralateral breast cancer developed in 2.9% of 1980 women with previous unilateral mammary tumours who did not receive endocrine therapy with 1.1% of 374 women who were given adjuvant hormonal treatment (p = 0.04). CONCLUSIONS: The efficacy of mammography in those screened was comparable to major overseas screening programs, but the proportion of women undergoing mammography in this high risk group was relatively low suggesting a need for greater promotion of mammography in Auckland. The use of adjuvant endocrine therapy significantly reduced the rate of development of contralateral breast tumours supporting the current development of formal trials of chemoprevention of breast cancer in women at high risk groups of the disorder.

Age and ethnicity as prognostic factors influencing overall survival in breast cancer patients in the Auckland region. Auckland Breast Cancer Study Group.

AIMS: to assess the effect of age at diagnosis and ethnicity on overall survival from breast cancer. METHODS: information was collected from 2706 breast cancer patients in the Auckland region, diagnosed between 1976 and 1985. Age at diagnosis was categorised into four groups: < 35 years, 35-49 years, 50-74 years and 75 years or older. Ethnicity was self reported in three categories: European, Maori and Pacific Island Polynesian. Independent effects were assessed by controlling for extent of disease, specifically metastases at presentation, nodal status and size of tumour. RESULTS: age and ethnic group were both significantly related to overall survival in univariate analyses. In multivariate analysis, age had an independent effect on survival mainly due to a significant survival difference between women aged less than 35 years and those aged 35 to 49 years (p < 0.0001; RR = 2.02). Survival was not significantly different between other age groups when adjustments were made for extent of disease. There was no significant effect of ethnicity on survival in the reduced dataset used for the multivariate analysis. Separate analyses suggested that ethnic differences in the extent of disease at diagnosis may be the cause of the apparent initial effect of ethnicity on survival. CONCLUSIONS: women < 35 years at diagnosis have a significantly poorer prognosis than women aged 35-49 years. Other differences between age groups were not significant when stage of disease was taken into account. Ethnicity was not an independent factor influencing survival after controlling for extent of disease but numbers in the Maori and Pacific Island groups were too small to conclusively evaluate any effect of ethnic group on prognosis.

Incidence and clinical features of breast cancer in the Auckland region.

Data on all new breast cancer cases in the Auckland area during the nine years September 1976 to September 1985 were used to obtain epidemiological information on breast cancer in the Auckland region. Breast tumours were found in 2706 women (300 per year), yielding a lifetime risk of breast cancer of one in 15. No significant difference in breast cancer incidence was detected between European, Maori and Pacific Island Polynesian women. Confidence limits for incidence were wide in the later groups. Fifty-one percent of women presented with intermediate sized (2-5 cm) tumours, and most (66%) were node negative. Eleven percent had evidence of metastatic disease at presentation. When the relationships between race, tumour size, nodal status and metastases were examined, Pacific Island women more frequently presented with large tumours and metastases, whereas Maori women were more frequently node positive. Eighty-five percent of tumours were invasive ductal carcinomas, 55% grade II, 35% grade III, and 10% grade I. Sixty-seven percent of tumours were oestrogen receptor positive (ER+ve) and ER status was significantly related to age; the proportion of ER+ve tumours was greater in older women. Fifty-seven percent of tumours were progesterone receptor positive (PR+ve), and PR distribution was bimodal with age. These data from the Auckland region are similar to breast cancer figures from other western countries, with some ethnic differences in tumour size and frequency of metastatic disease at presentation.

Seasonal change in the concentration of progesterone receptor in breast cancer.

There are conflicting reports of seasonal changes in steroid hormone receptor levels in breast cancer tissue. Estrogen receptor and progesterone (PR) receptor levels from 1132 tumors were thus grouped according to month of initial tumor detection or month of tissue sampling/surgery. There was a significant circannual variation in the mean monthly PR receptor concentration in patients grouped according to month of tissue sampling/surgery with peak PR levels in April (late summer-early autumn) and nadir values in August and September (late winter-early spring). There was no significant cyclic variation in estrogen receptor values. A significant annual variation in tumor PR concentration was also seen when receptor levels from individual tumors were grouped according to month of initial tumor detection, with peak PR levels found in January and February. The time interval between tumor detection and biopsy/surgery was 3.3 +/- 5.3 months (mean +/- SD) which was close to the interval between the peak PR concentration expressed by month of tumor detection compared with month of tissue sampling for receptor assay. There was also a significant seasonal variation in the month of initial tumor detection, with peak detection occurring in December (summer). The close synchrony between month of maximum tumor detection and month of peak PR concentration suggests that seasonal changes in detection of breast cancer may in part relate to seasonal changes in hormone responsiveness within tumor tissue.

Does delay in diagnosis of breast cancer affect survival?

1675 breast cancer patients in the Auckland regional area have been divided into two major groups according to delay in diagnosis greater or less than six weeks. Overall there is no difference in survival although the variables tumour size, skin attachment, and nipple retraction are more common in the group with longer delay, and grade III tumours in those with short delay. Three important prognostic variables (the presence of tumour steroid receptors, positive axillary nodes, and distant metastases at diagnosis) are equally distributed and have a similar effect on survival within the two delay groups. However, in a subgroup of women with negative axillary nodes, short delay is associated with poorer survival, independent of tumour size. More tumours with grade III histology and a negative progesterone receptor status are found in this subgroup. Thus, short delay may constitute a new prognostic variable of some importance when in association with negative axillary nodes.

Season of initial discovery of tumour as an independent variable predicting survival in breast cancer.

The month of initial detection of tumour was recorded in 2,245 patients with breast cancer and correlated with survival over a follow-up period of 1.5-10 years. Women who initially detected their breast cancer in spring/summer had a significantly longer survival than those detecting their tumour at other times of the year. Overall, this relationship was independent of nodal status, tumour size and hormone receptor status. However, when patients were divided into groups the survival advantage was significantly associated with receptor status and age. Women aged greater than or equal to 50 years with ER-positive and PR-positive tumours who discovered their initial tumour in spring/summer had significantly better survival than those detecting their tumours at other times of the year. Survival was also longer in women aged less than 50 years with receptor-negative tumours who initially found their tumours in spring/summer compared with the rest of the year. This study suggests that the season of first detection of a breast cancer relates significantly to the later behaviour of the tumour, and may reflect seasonal changes in hormone dependent growth.

Season of tumour detection influences factors predicting survival of patients with breast cancer.

The rate of initial detection of breast tumours varies during the year in a seasonal fashion, more tumours being discovered in late spring/early summer than at other times of the year. This phenomenon is particularly pronounced in young women (less than 50 years) with progesterone receptor positive tumours. The present study investigates whether season of tumour detection influences the predictive capacity of several recognised prognostic and risk factors in patients with breast cancer. Axillary nodal status, tumour progesterone receptor status, and season of tumour detection significantly influenced survival in both older (greater than 50 yrs) and younger (less than 50 yrs) patients. Parity, lactational history, body mass index, tumour oestrogen receptor status, and patient age also influenced survival, but these effects were significant only in age groups less than 50 or greater than 50 yrs. Season of detection of tumour did not effect the prognostic significance of axillary nodal status. However, the effect of oestrogen receptor status on survival was more significant in patients who detected their tumours in the spring/summer compared with winter (odds ratio 0.52 and 0.73 respectively). Negative progesterone receptor status was associated with significant poorer survival only in patients with tumours found in the winter. There was a significant survival disadvantage for nulliparous compared with parous women with breast cancer who were greater than or equal to 50 years at diagnosis, and for women who had never lactated compared with those who had lactated, but this disadvantage was restricted to those who found their tumours in the summer. An increased body mass index (greater than or equal to 28) was associated with decreased survival, but this was significant only for those detecting tumours in winter. The increased incidence of detection of breast cancer in spring/summer may reflect cyclic influences on tumour growth. Such influences may be hormonal in nature and may underlie the effect of season of tumour detection on the prognostic influence of lactation, parity, body mass index, and oestrogen and progesterone receptor status in patients with breast cancer.

Association between season of first detection of breast cancer and disease progression.

The season of recurrence of tumour was investigated by follow-up of 1324 patients with breast cancer and compared with the season of initial tumour detection. Unlike primary tumours, where an increased incidence of detection has previously been observed in late spring and early summer, there was no significant seasonal variation in the time of recurrence. However, women with oestrogen receptor positive or progesterone receptor negative primary tumours recurred significantly more frequently in the same season that their primary cancer was initially detected. Overall there was an increased frequency of recurrence one year from diagnosis. Women less than age 50 who initially found their tumour in winter or autumn had a significantly shorter disease-free interval before recurrence than those first detecting their tumour in summer or spring. This relationship was independent of nodal status and tumour size. Tumours initially detected in winter or autumn thus appeared to follow a more aggressive growth profile. This study indicates that the season of first detection of a breast cancer relates significantly to aspects of the future biologic behaviour of the tumour.

Influence of height, weight, and obesity on breast cancer incidence and recurrence in Auckland, New Zealand.

The relationship between obesity and breast cancer has been investigated in 1281 Auckland breast cancer patients. Using a definition of obesity as a Body Mass Index (BMI) of greater than or equal to 28 kg/m2, 179 (14%) breast cancer patients were classified as obese. The heights, weights, and BMI of 822 breast cancer patients aged 35-64 compared to 518 randomly selected Auckland women of similar age showed no significant difference. Within the breast cancer patients, there was no variation in nodal status or estrogen and progesterone receptor status between obese and non-obese women. However, tumours greater than 5 cm occurred significantly more often in obese patients. Time to recurrence was reduced in obese women with tumours less than or equal to 5 cm, no tumour in the axillary nodes, positive estrogen or progesterone receptor, and without metastases at the time of presentation of the disease. Although obesity has not been shown to influence breast cancer incidence, an effect on tumour recurrence is seen in patients with less advanced disease. This is similar to other reports which suggest that obesity is a weak but positive risk factor for recurrence.

Seasonal variation in breast cancer detection: correlation with tumour progesterone receptor status.

A significant circannual variation of the month in which patients detect the first sign or symptom of tumour has been defined in 1413 patients with breast cancer. The months of highest detection were in the late spring-early summer, and lowest detection was in late autumn-early winter. Analysis of subgroups indicates that this cyclic trend was most significant in younger women with small or moderate-sized tumours containing steroid hormone receptors, particularly progesterone receptors. It seems likely that this variation is related to the effect of cyclic hormonal changes on tumour growth, possibly mediated through the pineal.

Estrogen receptor status, adrenal androgens and 7 alpha-hydroxydehydroepiandrosterone in breast cancer patients.

Plasma concentrations of dehydroepiandrosterone (DHA), DHA-sulphate (DHAS) and 7 alpha hydroxy-DHA (7 alpha OHDHA) were measured and compared with tumor estrogen receptor (ER) status in 33 postmenopausal patients with breast cancer. Although the plasma concentrations of DHA, DHAS and 7 alpha OHDHA were not different between the ER-positive (ER+) and ER-negative (ER-) patient groups, the ratios of 7 alpha OHDHA/DHAS and of DHA/DHAS were significantly higher (P less than 0.001 and P less than 0.001 respectively) in the ER- group. Nine women (normal or with benign breast disease) of similar age and menopausal status had values for plasma 7 alpha OHDHA/DHAS and DHA/DHAS between those of ER+ and ER- patient groups. The measurement of these steroid ratios in the plasma of breast cancer patients thus provides an indirect estimate of ER status. Since DHA and 7 alpha OHDHA are major metabolites of precursor DHAS in mammary tumor tissues, changes in their relative quantities in plasma may reflect the influence of receptor-mediated events on mammary steroid metabolism. Alternatively, the relative increase in tumor metabolism of androgens inferred from high 7 alpha OHDHA/DHAS and DHA/DHAS ratios in the ER- group may disrupt the hormonal microenvironment of the estrogen receptor. These events may, in turn, predispose toward ER status and a poor response to endocrine therapy.