Prognostic significance of the digit ratio after hormone therapy for prostate cancer: a prospective multicenter study

The digit ratio has been used as a retrospective noninvasive biomarker to investigate the putative effects of prenatal exposure to androgens. In recent years, many scholars have paid attention to the association between 2D:4D (the second and fourth digits) and prostatic cancer. This study explored the prognostic significance of digit ratio in prostate cancer patients. We reviewed the progressive status and survival of 382 prostate cancer patients who had received hormone therapy at our institutions. Survival of clinicopathological variables analyzed as categorical variables were determined by the log-rank test. According to Cox’s proportional hazards analysis, R2D:4D, L2D:4D, PSA at 6 month,bone metastasis were significant independent factors for prostate cancer. The risk of any progression of prostate cancer similarly depressed with increasing 2D:4D, for any progression (R2D:4D HR = 0.71, p = 0.003; L2D:4D HR = 0.67, p = 0.001), for cancer-specific death (R2D:4D HR = 0.67, p = 0.025; L2D:4D HR = 0.74, p = 0.036). Digit ratio may not only have predictive value in risk but also prognosis of prostatic cancer. This finding suggests that low 2D:4D can be used as prognostic factors to identify patients with a poor prognosis. These patients may benefit from more aggressive management.

However, no correlative literature has investigated the association between digit ratio and survival in prostate cancer patients, whether digit ratio can be used to predict the progressive status and survival has not been reported as far as we know.
Based on such considerations, this study was undertaken to explore the prognostic significance of digit ratio in prostate cancer patients who had received hormone therapy at our institutions.

Methods
Participants. This study was performed in three large academic medical centers in china. Patients with prostate cancer were recruited from the oncology and urology department of The Second Affiliated Hospital Of XinJiang Medical University, Shandong Provincial Hospital, China-Japan Union Hospital of Jilin University, and met the following inclusion criteria: having a diagnosis of prostate adenocarcinoma and undergoing wholecourse endocrine therapy at our institution.
The study population consisted initially of 926 patients who underwent endocrine therapy from march 2011 to march 2014 at our institutions. Patients were excluded for the following reasons: History of related radiation or surgery (n = 435); Histologic slides, Gleason score, PSA data or digit ratio values not available (n = 25); Not right-handed, skeletal dysplasias, or who had suffered from any kind of injuries to the fingers (n = 43); Lack of research authorization (n = 41). Nevertheless, sedatives or analgesic medication, limited operations to remove lower urinary tract obstructions (cystostomy or catheterization) were permitted.
The final study group consisted of 382 prostate cancer patients who were diagnosed pathologically at our institutions from march 2011 to march 2014. Among these participants, 337 patients were treated with endocrine therapy, using medical castration (using a luteinizing hormone-releasing hormone analogue) with antiandrogens, and the remaining 45 patients were treated with simple medical castration.
Our study was approved by The Ethics Committee of The XinJiang Medical University and all of the relevant hospitals. Informed consent was provided according to the Declaration of Helsinki and all of the patients' guardians informed written consent. We confirming that all experiments were performed in accordance with relevant guidelines and regulations that promote respect for all human subjects and protect their health and rights.
Follow-up information. The median time of follow-up and the follow-up range were 45.0 months and 1-72 months, respectively. All these patients were followed every month for the first 3 months and once every 3 months thereafter. Serum PSA levels, complete blood cell counts, blood chemistry studies, rectal examinations were recorded and analyzed. Serum levels of PSA were assayed by CanAg PSA ElA (CanAg Diagnostics AB). Meanwhile, chest x-rays and radionuclide bone scans (injection of 25 mCi Tc-99m methylene diphosphonate) were performed every 6 months. If the patient complained of subjective symptoms, computerized tomography of the abdomen and pelvis or abdominal ultrasonography was also performed to measure the disease's progression. The TNM classification system was used for staging and the Gleason system was used for grading.
Any progression of the prostate cancer was defined as: (1) Progression to hormone refractory prostate cancer, when PSA increased after hormone treatment twice in succession and continued to increase even after suspension of flutamide; (2) Local recurrence, cancer on biopsy of prostatic bed without evidence of systemic recurrence. (3) Systemic progression, involved demonstrable metastatic deposits on biopsies, abdominal CT scan or bone scan. Prostate cancer death was defined as death in any patient with metastasis that showed any progression following hormonal therapy.
Digit measurement. Figure 1 shows how to measure the digit length. The 2D and 4D were measured by unified specially-trained investigators with a standard vernier caliper (recorded to 0.01 mm) from the basal palmar crease of the finger to the tip. This measurement has been previously reported providing a high degree of repeatability 21,22 . When two creases were visible at the base of the digit, the crease proximal to the palm was chosen. The measurers had no information about the progression and therapy about the patients. All measurements were made twice by two investigators and the mean value was adopted. We got the 2D:4D values at the beginning of the treatment mostly. The intraclass correlation coefficient (ICC) of two repeated measures of digit ratio by a single investigator was 0.918. This direct measurement method is probably better, as indirect finger measurements (e.g. from photocopies or scans) may lead to distortions that result in consistently lower 2D:4D values 23, 24 . Statistical analysis. For the analysis of the results, the patients were divided into groups according to 2D:4D (<0.950 versus ≥0.950), pathological Gleason score (<6 or 7 versus 8-10), initial PSA (<20, 20-99.9, 100-1000, >1000), PSA at 3 month (<4 versus ≥4), PSA at 6 month (<4 versus ≥4), bone metastasis (yes versus no), lung metastasis (yes versus no), lymph node metastasis (yes versus no), therapy (monotherapy versus combined) and pathological tumor stage (TX, T0, T1 or T2 or T3 versus T4).
All statistical analyses were performed by using the SPSS software (SPSS Inc., Chicago, Illinois) with a significance level of 0.05.
Variables of the different groups were compared using Student's t test or Fisher's exact test. Survival curves were constructed by the Kaplan-Meier method, survival of clinicopathological variables analyzed as categorical variables were determined by the log-rank test. Multivariate analysis was then performed according to the Cox proportional hazards regression model to determine the significant factor in predicting disease outcome. To obtain a multivariate model with the maximum precision of the important variables, a stepwise selection procedure was used.

Results
Follow up results. Patient characteristics of the study are summarized in Table 1, all of the participants were Chinese Han patients.
This study did not find a relationship between Gleason score and 2D:4D, but encountered significant negative correlation between 2D:4D and PSA at 3 months, 6 months after treatment (Table 2).
Univariate and multivariate analysis. Individual parameters were evaluated using univariate analysis for possible correlation with any progression of prostate cancer, R2D:4D, L2D:4D, initial PSA, PSA at 6 month after treatment, bone metastasis, lymph node metastasis influenced the progression (Table 3). However, by multivariate analysis, stepwise inclusion of variables in the Cox's proportional hazards model showed that the significant prognostic factors were R2D:4D, L2D:4D, initial PSA, PSA at 6 month, bone metastasis were significant independent factors (Table 4).

Discussion
Several converging lines of evidence suggest that prenatal hormone exposure is associated with digit ratio, and much of relevant evidence has been previously reviewed 25 . A direct evidence, Lutchmaya et al. reported that L2D:4D is negatively correlated with the ratio of testosterone to oestradiol measured by amniocentesis in the second trimester 3 . HOX genes regulate the embryogenesis, morphogenesis and cell differentiation of human, including the genitals and digits, and they have been suggested to be deregulated in malignant tumors 26,27 . In primary prostate cancer cells, HOX8 or MSX2 gene expressions are greatly reduced, followed by a gradual increase in more aggressive prostate cancer cell lines 28 . Despite the controversy and criticism 29 , it has been postulated that digit ratio was associated with the CAG repeats number in the AR (androgen receptors) genes 30 . It is generally known that testosterone and AR play crucial roles in prostate growth and the progress of prostate cancer. The short CAG repeats of AR have been reported to be associated with the pathogenesis of prostate cancer 31,32 . Furthermore, the polymorphisms of AR gene may occur in advanced prostate cancer, which became resistant to hormonal treatment 33,34 .
Recently, many experts and scholars had a strong interest in the relation between 2D:4D and prostate. In a case-control study, compared with low 2D:4D men, abnormal development (persistence of mid-line cyst formation) of the prostate was more common in men with high 2D:4D. This suggests that 2D:4D may relate to the normal development of prostate 35 . In another convincing study, the researchers found that the digit ratio is a predictor of the response to dutasteride (one inhibitor of the action of 5α-reductase) treatment 36 . This suggests that the 2D:4D may be linked to the function of the prostate. In tumor research studies, prostate cancer is the cancer most connected to 2D:4D, which is considered to develop as a result of hormonal carcinogenesis. Whereby hormones promote cell division and cell proliferation, and the accumulation of genetic mutations that arise during the process of cell division eventually result in a malignant phenotype 37 .
In 2010, a significant study to evaluate the correlations between 2D:4D and prostate cancer found that individuals with a higher risk of prostate biopsy possessing high levels of PSA presented lower 2D:4D values 20 . Then, in a major cohort study in the United Kingdom, researchers have proposed that 2D:4D may be a simple marker of prostate cancer risk, with length of 2D greater than 4D suggestive of lower risk 9 . Another investigation in 2012, the Korean researchers found an association between 2D:4D, prostate cancer volume and Gleason score after investigating 770 men with lower urinary tract symptoms 38 . However, in 2013, one US study found lower right-hand digit ratios for African-Americans with prostate cancer in comparison to Caucasians, but no significant differences for Gleason scores 14 . Similarly, in our study, we found no correlation between 2D:4D and Gleason scores either. Such differences may reside in the different evaluation of digit ratios, technique use, data processing mode and the different backgrounds of the samples (patients condition and prognosis, ethnic). In short, prostate   Table 4. Cox proportional hazards model for predicting any cancer progression (Hazards ratio estimates and 95% CI). All estimates adjusted. *Variables were used as categorical, as in Table 2.  Table 5. Cox proportional hazards model for prostate carcinoma death (Hazards ratio estimates and 95% CI). All estimates adjusted. *Variables were used as categorical, as in Table 2.  cancer is a kind of multifactorial disease and influenced by both ethnic, environmental and genetic factors, larger multi-center prospective study may be necessary for further research.

Variables
In 2011, when we started this research, we measured the digit ratio and collected the relevant data of prostate cancer patients to examine whether 2D:4D is associated with prostate cancer risk. At that time, more and more attentions were paid to this subject in academia, and many major studies carried out. Then, we noted that all of these experiments restricted to investigations on the relationship between 2D:4D and risk (prevalence or severity) of prostate cancer, whether associations exist between 2D:4D and the disease prognosis was never mentioned. So, based on previous researches, we turned our attention to the prognostic significance of digit ratio in prostate cancer patients. During an average follow-up of 45.0 months, we found that digit ratio may not only have predictive value in risk but also prognosis of prostatic cancer finally.
Due to limited practical condition and research competence, our study has several potential limitations. Prostate cancer is indolent tumor that has long survival, even with metastases 39 . In our study, with relatively short-term following up, the number of outcome events was limited. Therefore, our results should be considered preliminary. So, longer-term studies are desperately needed to catch more significant differences in the progression.
Actually, the use of 2D:4D for outcome prediction may be not as straightforward as the result suggest. The main reason is that ethnic groups differ in mean 2D:4D. In a meta-analysis on Chinese 2D:4D, of the results of 28 studies (19,093 participants), the left 2D:4D was 0.951 in Chinese men, Q (26) = 658.61, P < 0.001, and the right 2D:4D was 0.948, Q (28) = 753.56, P < 0.001 40 . After analyzing the data of relevant research, it was found that the 2D:4D in Chinese individuals (Han ethnicity) was found to be higher than in Black populations but lower than in Caucasians and Hispanics [41][42][43] . These ethnic differences in 2D:4D further indicate that genetic factors likely affect the 2D:4D. Furthermore, the diagnosis, therapy and prognosis of prostatic cancer show significant differences in different countries and nationality 44,45 .For instance, the screening of prostate cancer in China is not as widespread as in Western countries, and the age-adjusted incidence and mortality rates of prostate cancer in Asia are much lower than Europe and the United States. In conclusion, due to all of the participants are Chinese Han in our study, the conclusion may be specific to the particular ethnic and regional composition of our study sample.
Since Huggins and Hodges found the beneficial effect of androgen ablation on metastatic prostate cancer in 1941 46 . androgen deprivation therapy, specifically medical castration, has been the first line treatment for advanced prostate cancer. And, there have been many experiments to study diverse prognostic factors for prostate cancer [47][48][49] . In our study, the results indicate that digit ratio may be associated with the prognosis of patients with prostate cancer, and 2D:4D can be used as prognostic factors to identify patients with a poor prognosis, who may benefit from more aggressive management. At present, detailed interpretation of this result is difficult due to the fact that we can only provide an indication that prenatal androgen exposure might be associated with the prognosis of prostatic cancer, the biochemical pathway or molecular mechanism cannot be explained systematically.
Here we offer two possible explanations: 1) differences in fetal exposure to androgens or the sensitivity of AR may be related to the appearance and proliferation of the receptor-negative tumour cells, and 2) by other possible mechanism of gene regulation, prenatal androgen exposure may be associated with the proliferation, invasion, migration, apoptosis and multi drug resistance of tumor cells.
In conclusion, despite this current limitation, 2D:4D remains a simple, easy to measure marker of prenatal androgen exposure which may not only have predictive value in risk but also prognosis of prostatic cancer.