chronic lymphocytic leukemia ( cll ) is the most commonly found leukemia in the adult population of the western world and of clinical interest because of its prevalence .
it is a neoplasm of monomorphic small round b lymphocytes which can be observed in peripheral blood , bone marrow , and/or lymph nodes .
it is likely that cll has a multifactorial mode of inheritance with both genetic and environmental components .
however , of all hematologic neoplasms , cll is reported to have the highest genetic predisposition and like many other hematologic malignancies , development of cll is found to be much higher in males than in females with a corresponding m / f sex ratio of at least 1.5 or higher .
although the clinical ( phenotypic ) m / f sex ratio for cll has been well documented , the genetic ( genotypic ) m / f sex ratios associated with abnormal fluorescence in situ hybridization ( fish ) probes have not . for these reasons
, cll represents an attractive cancer entity in which to evaluate what genetic impact , if any , gender may have on its development . genetic studies of sex ratio disparities in human neoplasms have been few , perhaps , because of the relative inaccessibility of investigational materials including appropriate databases which could be informative .
it follows that the genetic basis for this phenomenon remains largely unknown and our understanding of it very limited .
since our laboratory has collected clinical and laboratory data over many years on major categories of cancers including cll , review and analyses of these data presented an opportunity to examine certain aspects of this question . in order to better study the nature of the multifactorial components in cll , we determined the representation of genetic abnormalities detected by four defined fish probes , with respect to male and female cll patients . by this approach ,
our study addressed not only what the m / f ratio is in cll patients having the clinical phenotype , but also the m / f ratio in patients who have a genotype which included specific fish abnormalities .
these results provided a genetic basis for the notion that the fish abnormalities found underlie the phenotypic m / f sex ratio and also that they may be sex chromosomes ( x and/or y ) influenced .
cll fish results in the database are derived from testing done on cytological preparations made from peripheral blood or bone marrow specimens submitted for cll evaluation .
fish analyses were conducted using a fish panel of dna probes specific for the atm gene ( 11q22.3-q23.1 ) , chromosome 12 centromere ( d12z3 locus ) , the d13s319 locus ( 13q14.3 ) , and the tp53 gene ( 17p13.1 ) commercially available from vysis ( an abbott company ) , downers grove , il .
the prognostic value and characterization of these dna probes have been reported in the literature .
the technical protocols for fish testing ( hybridization of probes and detection of hybridization signals ) were those recommended by the manufacturer .
microscopic studies were conducted by experienced technologists , then reviewed and interpreted by board - certified cytogeneticists .
these fish test results were then archived in a computer - based database from which data were retrieved and statistically analyzed .
data of fish results from all integrated oncology laboratories , a business unit of esoterix genetic laboratories , llc , using the same dna fish probes to evaluate cll patients were collected , reviewed , and statistically evaluated .
the data contained entries of cll - fish panels performed during the period of 11/13/2005 through 10/20/2009 .
these panels were specifically used for cll studies , and results were used only when the entire cll panel was applied in order to clearly identify only those patients having or suspected of having cll .
also , cll panel probe results were those used only for the initial fish evaluation of newly diagnosed cll patient - and did not involve repeat studies on the same patient . this investigation was conducted in a fashion in which laboratory results had no patient - identity associations , in order to conform to privacy guidelines and patient confidentiality .
this data was then imported into sas9.2 and formatted by sas data step and analyzed by sas procedures for one side binomial test and chi - square test .
the one - side binomial tests were used to evaluate the sex ratio with respect to the abnormalities found for each of the probes used .
the first category represented all abnormal panels having only a single abnormality per panel , for each probe .
the second category included those having any abnormality with respect to a given probe whether it appeared as a single abnormality or in combination with other abnormalities .
the binomial test took into account the frequency of an abnormality or abnormalities ( according to the categories describe above ) in a pool of data ( total number of normal and abnormal panels ) for each of the fish probes considered , and compared the results obtained for the male group with results obtained for the female group .
essentially , the mean value for positive results ( all having the same abnormality + ) versus total results ( positive results plus normal having 0 abnormalities ) , was compared between males and females for each of the probe categories described above . the odds ratio ( or ) and 95% confidence interval ( ci ) values were derived from the one side binomial test while the p - values were determined by the chi - square tests .
cll fish results in the database are derived from testing done on cytological preparations made from peripheral blood or bone marrow specimens submitted for cll evaluation .
fish analyses were conducted using a fish panel of dna probes specific for the atm gene ( 11q22.3-q23.1 ) , chromosome 12 centromere ( d12z3 locus ) , the d13s319 locus ( 13q14.3 ) , and the tp53 gene ( 17p13.1 ) commercially available from vysis ( an abbott company ) , downers grove , il .
the prognostic value and characterization of these dna probes have been reported in the literature .
the technical protocols for fish testing ( hybridization of probes and detection of hybridization signals ) were those recommended by the manufacturer .
microscopic studies were conducted by experienced technologists , then reviewed and interpreted by board - certified cytogeneticists .
these fish test results were then archived in a computer - based database from which data were retrieved and statistically analyzed .
data of fish results from all integrated oncology laboratories , a business unit of esoterix genetic laboratories , llc , using the same dna fish probes to evaluate cll patients were collected , reviewed , and statistically evaluated .
the data contained entries of cll - fish panels performed during the period of 11/13/2005 through 10/20/2009 .
these panels were specifically used for cll studies , and results were used only when the entire cll panel was applied in order to clearly identify only those patients having or suspected of having cll .
also , cll panel probe results were those used only for the initial fish evaluation of newly diagnosed cll patient - and did not involve repeat studies on the same patient . this investigation was conducted in a fashion in which laboratory results had no patient - identity associations , in order to conform to privacy guidelines and patient confidentiality .
this data was then imported into sas9.2 and formatted by sas data step and analyzed by sas procedures for one side binomial test and chi - square test .
the one - side binomial tests were used to evaluate the sex ratio with respect to the abnormalities found for each of the probes used .
the first category represented all abnormal panels having only a single abnormality per panel , for each probe .
the second category included those having any abnormality with respect to a given probe whether it appeared as a single abnormality or in combination with other abnormalities .
the binomial test took into account the frequency of an abnormality or abnormalities ( according to the categories describe above ) in a pool of data ( total number of normal and abnormal panels ) for each of the fish probes considered , and compared the results obtained for the male group with results obtained for the female group . essentially , the mean value for positive results ( all having the same abnormality + ) versus total results ( positive results plus normal having 0 abnormalities ) , was compared between males and females for each of the probe categories described above .
the odds ratio ( or ) and 95% confidence interval ( ci ) values were derived from the one side binomial test while the p - values were determined by the chi - square tests .
a characterization of the study population evaluated by the cll - fish panel is given in table 1 , and a summary of general observations are provided in table 2 .
for example , table 2 shows that there were a total of 2773 ( 59.0% ) individual abnormal fish panels in the 4698 total fish panels applied to the cll patient study group . of the abnormal panels ,
1711 were male and 1062 were female for an m / f sex ratio of 1.61 .
other comparisons of the genders concerning data collected by this study are also reported in table 2 .
table 3 shows the m / f ratios of fish results for the study population of cll patients in categories of abnormal results ( either as a single abnormality or as a single abnormality plus multiple abnormalities ) , normal results and total results ( normal and abnormal ) with respect to gender .
there were 2035 results that appeared as only single abnormalities , and 3606 results that appeared as single plus other abnormalities combined ( artificially inflated since abnormalities were used multiple times ) . the most frequently found single abnormality was the deletion of 13q14.3 ( 1327 abnormal results : 65.2% ) , followed by trisomy 12 ( 472 abnormal : 23.2% ) , then deletion of 11q22.3 ( 138 abnormal : 6.8% ) , and least frequent was deletion of 17p13.1 ( 98 abnormal : 4.8% ) .
the sex ratio values for the fish probes ranged from a low value of 1.39 to a high value of 2.54 . with the exception of the sex ratio found for the atp gene ( 2.54 ) , the rest of the sex ratios for fish abnormalities ranged from 1.39 to 1.54 , clustering around 1.5 . from table 3
, it should be noted that the ab / nor ratio for each of the probes was not significantly different from each other except for the atm probe .
this suggest that proportionality of abnormal results reflect the phenotypic representation of cll clinically in our study population except for abnormalities of the atm gene which is over - represented in male from that expected .
figure 1 presents the distribution of patients ages in the cll study population in which the age range for males and females is very similar . at early ( less than 30 years ) and
late age groups ( greater than 80 years ) the sex ratios between male and females do not appear to be significant .
results from the chi - square tests and odds ratio analyses are presented in table 4 .
of the four fish probes , only the deletion of the 11q22.3 ( atm gene ) demonstrated statistical significance in the chi - square test ( p - value of 0.0049 ) .
the or value of 1.7045 is also significantly different from the or values found for the rest of the fish probes , indicating the male has 70.45% or 64.35% higher risk to have abnormal fish test results for the atm - fish probe .
this was true for deletions of the atm gene when found as a sole abnormality ( or=1.7045 ) or in combination with other abnormalities which included the deletion of the atm gene ( or=1.6435 ) .
both these two categories of single and multiple probe abnormalities strongly indicated that the deletion of the atm gene was disproportionately over - represented in male cll patients than in female cll patients , and that this difference could not be explained by chance alone .
these data lead to the conclusion that this mutation was not only found in a higher percentage in males compared to females , but that it was distinct from the m / f sex ratio of ~1.5 found for the other fish probes [ trisomy 12 , del(13q14.3 ) , del(17p13.1 ) ]
. the or values for the m / f sex ratios of trisomy 12 , deletion 13q , and deletion 17p ( tp53 gene ) fish probe abnormalities in either single or multiple combinations were not significantly different from 1.0 and could be explained by chance alone .
therefore , the m / f sex ratio for each of the cll - fish probe abnormalities appeared to be about the same and not significantly different from an m / f sex ratio of 1.5 .
although sex ratios in lymphoid neoplasms and other tumors show male predominance , there remains an incomplete understanding of these observations . our study makes this m / f ratio determination based on a study population of patients clinically diagnosed with , or suspected of having cll for whom fish studies were conducted .
based on this clinical assignment , a ratio of 1.51 was found , which is congruent with previous studies , indicating similar values or higher .
these observations suggest that cll may have a sex chromosomeinfluenced component determining its transmission .  
we also examined the question of what the m / f ratio is in those cll patients who had fish abnormalities detected by specific genetic probes and found that 3 of the 4 probes had values close to 1.5 and that 1 probe ( atm gene ) had a significantly higher m / f ratio of 2.54 .
the coincidence that , like the clinical m / f ratio , the genetic m / f ratio for 3 probes may simply suggest that in the cll study population which already has a disproportionately high number of male patients , the likelihood of detecting a genetic abnormality is equally high ( in males ) and that it may be sex chromosome influenced .
however , for the atm - fish probe , the m / f ratio was significantly higher ( ~2.5 ) than the ~1.5 ratio found for the other probes and suggests that for this mutation , a special set of conditions necessary for the development of cll are more effectively enhanced in male than in female patients .
m / f values vary depending on the type of cancer and the age of the patient
. there are three possible categories of cancers ( hematologic and non - hematologic ) with respect to altered m / f ratios having values ranging from less than 0.50 to over 20.0 .
generally , these categories include cancers in which : i ) males have a higher risk , ii ) females are more susceptible ,   and iii ) males and females are equally represented .
however , these reported m / f values are phenotypic m / f values which do not take genetic aspects into account .
our survey confirms that in our cll study population , as indicated in earlier reports the phenotypic m / f sex value is very close to 1.5 .
m / f sex ratio corresponds to cancers in the first category in which common mechanisms of cancer development may exist .
the purpose for using the fish data from the cll study population was to initiate a characterization of what the genotypic m / f sex ratio is for each of the fish probes used .
surprisingly , very much like the phenotypic m / f ratio of ~1.5 found , our study demonstrated a genotypic m / f sex ratio higher than 1.0 ( 1.39 - 2.5 ) with respect to all cll fish probes .
it is important to note that the genotypic m / f sex ratio of ~2.5 found for the deletion of the atm gene , is markedly skewed in males , and suggests a special mechanism which may involve atm gene functions which could include dna fidelity , homologous recombinational repair and chromosomal stability .
losses and mutations of the atm gene have been demonstrated in a number of neoplasms including t - lymphocytic leukemia ( t - pll ) , b - cell chronic lymphocytic leukaemia ( b - cll ) and in mantle cell lymphoma ( mcl ) .
these reports suggest that loss of a tumor suppressor gene [ or loss of heterozygosity ( loh ) ] may be one of many steps leading to a cancerous state in complex diseases with multifactorial contributions .
gender dependent susceptibility to complex diseases could include polygenic mechanisms , epigenetic modulations , and sex - chromosome linked genes .
two possible explanations can account for the skewed m / f ratios found in our study ( including the atm fish probe showing especially high level of deletions in male cll patients ) .
explanation # 1 involves the non pseudoautosomal regions ( non - par ) of the x and y chromosome , and explanation # 2 which concerns the pseudoautosomal region ( par ) of the sex chromosomes .
explanation 1 : the genotypic m / f sex ratio of ~1.5 was found for trisomy 12 , deletion of d13s319 at chromosome 13q14.3 , and deletion of the tp53 gene .
these hypothetical x - chromosome linked genes appear as a single dose in male cells ( hemizygous ) and as two ( non - par , and not x - chromosome inactivated ) doses in female cells . if there is a predisposing mutation for any of the three fish autosomal markers in either males or females , the cll - x gene product will continue normal function with no cll development .
however , if the cll - x gene is subsequently mutated in males ( hemizygous ) , gene function of the cll - gene markers would also be impaired resulting in cll . however , in females ( heterozygous ) the second non - mutated cll - x gene salvages the gene dosage requirement with no development of cll .
mutations of both cll - x genes in females would be necessary for subsequent cll development .
this explanation is consistent with the observation that cll typically has an adult age onset , that relatively fewer females succumb to cll given the salvaging effect of the double doses of the xlinked cll genes they possess , and offers a plausible biological / genetic basis for the ~1.5 sex ratio found for these three important cll genetic markers . since atm loss is considerably higher in males than females , with a genotypic sex ratio of ~2.5 , additional factors are implicated .
other highly skewed phenotypic m / f sex ratios reported in hematological , nonhematological cancers , and in certain solid tumors , can be as high as 28.7 such as for kaposi sarcoma .
for these entities it may be that mutations are not necessarily disabled by 100% loss of function . in males after the first atm mutation , a partially functioning cll - x gene might contribute to the loss of the 2 atm gene .
however , in females the two partially functioning cll - x genes may be result in sufficient dna repair to prevent atm loss at a higher level than that found in males .
this secondary salvage pathway would provide for a lower rate of atm gene loss and may represent a relative protective mechanism favoring females .
there may also be other models that could explain gender ratio distortions involving multiple dna - repair related gene families .  
our hypothesis of x - linked dna repair - related genes is supported by recent mapping of dna repair genes ( which include the ape1 , cetn2 , trex2 , ube2a , and rpa4 genes ) on the x chromosome .
explanation 2 : the par is homologous in the human x and y chromosomes where they pair during meiosis , and where there are 2 doses for every gene found in this region .
compelling evidence shows that the par plays a role in the m / f ratio found in mantle cell lymphoma ( mcl ) which may also apply to cll .
the study demonstrated male predominance in mantle cell lymphoma ( mcl ) with loss of the y chromosome and homozygous deletions within the par .
in all except 3 of the 21 mcl cases studied , the loss of the y chromosome was demonstrated .
furthermore , 2 of the 3 cases ( which showed no y chromosome loss ) had biallelic losses of par1 in xp / yp , and of 16 cases in which there was y chromosome loss , an additional case showed biallelic loss of par1 .
the genotypic 1.5 m / f ratio for the 3 probes used in our study could also be ex plained by mutations / losses of the par region of the x and y chromosomes .
a similar linkage to the par has been proposed for hodgkin s lymphoma in which the risk for brothers of affected males or sisters of affected females was higher than the risk for siblings of the opposite gender . also supporting
this explanation is that chemokines and chemokine receptors may play a role in b - cell malignancies ,   and that related cytokine - receptor genes map to the par of sex chromosomes .
increasingly , it appears that gender plays a major role in defining not only the identity and nature of some neoplasms but also the mechanisms involved in their origin and progression .
 distorted sex ratios occur in hematologic disorders . 
 for example , chronic lymphocytic leukemia ( cll ) displays disproportionate sex ratios with a large male excess . 
 however , the underlying genetics for these disparities are poorly understood , and gender differences for specific cytogenetic abnormalities have not been carefully investigated . 
 we sought to provide an initial characterization of gender representation in genetic abnormalities in cll by using fluorescence in situ hybridization ( fish ) . 
 we confirm the well known skewed male - tofemale ( m / f sex ratio ) of ~1.5 in our cll study population , but also determine the genotypic m / f sex ratio values corresponding to specific fish dna probes . genetic changes in cll detectable by four fish probes were statistically compared with respect to gender . 
 initial fish evaluations of 4698 cll patients were retrospectively examined and new findings of the genotypic m / f sex ratios for these probes are reported . 
 this study represents the largest cll survey conducted in the united states using fish probes . 
 the cll database demonstrated that fish abnormalities ( trisomy 12 , 13q14.3 deletion and 17p13.1 deletion ) probes had skewed m / f ratios of ~1.5 . 
 also , by statistical analysis it was shown that atm gene loss ( 11q22.3q23.1 deletion ) solely or with other abnormalities was considerably higher in males with an m / f ratio of 2.5 and significantly different from m / f ratios of 1.0 or 1.5 . 
 we hypothesize that interactions involving these autosomal abnormalities ( trisomy 12 , and deletions of 11q22.3 , 13q14.3 , and 17p13.1 ) , and the sex chromosomes may provide the genetic basis for the altered phenotypic m / f ratio in cll . 