Going Beyond Conventional

Mammographic Density to Discover

Novel Mammogram-Based Predictors of
Breast Cancer Risk John L Hopper
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 Journal of
Clinical Medicine

Commentary
Going Beyond Conventional Mammographic Density
to Discover Novel Mammogram-Based Predictors of
Breast Cancer Risk
John L Hopper *, Tuong L Nguyen, Daniel F Schmidt, Enes Makalic , Yun-Mi Song,
Joohon Sung, Gillian S Dite , James G Dowty and Shuai Li
Centre for Epidemiology & Biostatistics/Melbourne School of Population & Global Health, Faculty of Medicine,
Dentistry & Health Sciences, The University of Melbourne, Victoria 3010, Australia;
nguk@unimelb.edu.au (T.L.N.); daniel.schmidt@monash.edu (D.F.S.); emakalic@unimelb.edu.au (E.M.);
yunmisong@skku.edu (Y.-M.S.); jsung@snu.ac.kr (J.S.); g.dite@unimelb.edu.au (G.S.D.);
jdowty@unimelb.edu.au (J.G.D.); shuai.li@unimelb.edu.au (S.L.)
* Correspondence: j.hopper@unimelb.edu.au




Received: 31 January 2020; Accepted: 17 February 2020; Published: 26 February 2020


Abstract: This commentary is about predicting a woman’s breast cancer risk from her mammogram,
building on the work of Wolfe, Boyd and Yaffe on mammographic density. We summarise our efforts
at finding new mammogram-based risk predictors, and how they combine with the conventional
mammographic density, in predicting risk for interval cancers and screen-detected breast cancers
across different ages at diagnosis and for both Caucasian and Asian women. Using the OPERA
(odds ratio per adjusted standard deviation) concept, in which the risk gradient is measured on
an appropriate scale that takes into account other factors adjusted for by design or analysis, we
show that our new mammogram-based measures are the strongest of all currently known breast
cancer risk factors in terms of risk discrimination on a population-basis. We summarise our findings
graphically using a path diagram in which conventional mammographic density predicts interval
cancer due to its role in masking, while the new mammogram-based risk measures could have a
causal effect on both interval and screen-detected breast cancer. We discuss attempts by others to
pursue this line of investigation, the measurement challenge that allows different measures to be
compared in an open and transparent manner on the same datasets, as well as the biological and
public health consequences.

Keywords: breast cancer; cumulus; cirrocumulus; cirrus; mammogram-based risk; mammographic

density; OPERA

1. Introduction
This Special Issue calls for manuscripts that will help advance our understanding of mammographic
density to improve the detection and prevention of breast cancer. But the issue is much larger than
mammographic density, at least as it has been defined conventionally as the ‘white or bright areas
on a mammogram’. The last few decades had seen many efforts to measure this historical concept,
including automated measures now that digital mammography is widespread, building on the seminal
work of Boyd and Yaffe who created a semi-automated method [1]. But those efforts had missed a
fundamental point. The aim shouldn’t be to better measure mammographic density per se, but to better
predict risk.
This commentary is about finding ways to predict a woman’s breast cancer risk from her mammogram,
building on the work of Wolfe, Boyd and Yaffe on what has become known as mammographic density.
We summarise our efforts at finding new mammogram-based risk predictors, and how they combine

J. Clin. Med. 2020, 9, 627; doi:10.3390/jcm9030627 www.mdpi.com/journal/jcm

J. Clin. Med. 2020, 9, 627 2 of 13

with the conventional measure of mammographic density in predicting breast cancer risk for interval
cancers and screen-detected cancers, as well as across different ages at diagnosis and for both Caucasian
and Asian women. We also discuss more recent attempts by others to pursue this line of investigation,
the Measurement Challenge that allows different measures to be compared in an open and transparent
manner on the same datasets, as well as the biological and public health consequences.
Before we do so, we discuss the pivotal concept of OPERA (odds ratio per adjusted standard
deviation) which should not be relegated to an Appendix or Supplementary Material. It is fundamental
to understanding, and putting into proper perspective, the relative strengths of risk factors with one
another, and with other risk factors. It is particularly relevant to mammographic density due to its
negative confounding with the major breast cancer risk factor of age and, for some, another breast
cancer risk factor in body mass index. The reader might want to first skip this section and go straight
to the section on Wolfe and beyond, but it is essential to understanding the area.

2. OPERA: A Measure of the Ability of a Risk Factor to Identify Future Cases on a

Population Basis
Consider the issue of how to rank risk factors in terms of their ability to predict disease risk on a
population-basis; namely the extent to which they differentiate cases from controls. For simplicity, we
consider risk factors measured on a continuous scale, though this is not an essential assumption. The key
issue is to recognise that disease risk almost invariably depends on age, and often sex (dramatically so
for breast cancer), as well as many other factors. Some of these factors are measured and adjusted
for in analysis while others can be controlled for, at least to some extent, by design. Consequently,
studies of breast cancer and mammography measures typically consider women only, and use cases
and controls who are similar, or even matched for, age. Some family-based designs control for familial
factors—genetic and environmental. The result is an estimate of relative, not absolute, risk—the latter
can be determined by appropriate use of population age-specific incidences.
The point that is often not appreciated is that the resulting risk estimate applies to a change in the
risk factor of concern while holding all other factors constant. Therefore, the risk scale is not that of the
unadjusted risk factor. Expressing the risk gradient in terms of units of the unadjusted risk factor is
inappropriate, yet standard practice throughout epidemiology despite it giving misleading results.
For mammogram-based risk measures in particular, these considerations are particularly important.
Conventional mammographic density declines with age in the decades around the menopausal years.
Percent mammographic density has a mean of around 25–35% before age 40 years, then decreases over
the next two decades to plateau at about 15–25% in later life for Caucasian women [2]. A similar pattern
with age has been observed for Asian women [3]. After adjusting for age and body mass index, percent
density has a standard deviation of ~10% and is highly, though not completely, stable (correlation
between measures 10 years apart ~ 0.9) [2].
The commonly used concept of percent mammographic density (absolute density divided by
the total breast size) is also moderately, and negatively, correlated with body mass index. But
breast cancer risk increases substantially with age, and post-menopause, with body mass index.
Intriguingly, body mass index has a negative (i.e., protective) association with breast cancer risk prior to
menopause [4]. Consequently, almost all studies of mammogram-based risk measures use case-control
samples of similar ages, and adjust for age, body mass index, and often other measured risk factors
though the latter don’t explain much variance [5]. The fact that the adjustment has been made in the
analysis is typically denoted in the footnotes to Tables of results but rarely commented on in the text.
Major problems arise when the estimated risk gradient is presented in terms of the so-called
‘change in risk per standard deviation’ specifically as the increment in risk for a one-unit change of the
standard deviation of the unadjusted, raw measures [6]. These ‘per standard deviation’ risks are not
meaningful (they do not represent what has been estimated) and should not be used to compare risk
factors across measures.
J. Clin. Med. 2020, 9, 627 3 of 13

The considerations above when applied to mammogram-based risk factors were the driving force
in us developing the concept of OPERA (odds ratio per adjusted standard deviation) [7]. The word
“adjusted” refers to the use of the standard deviation of the risk factor (for the population about which
inference is being made) after it has been adjusted for other factors being taken into consideration by
the design and analysis.
OPERA is the correct way to interpret risk estimates in terms of risk discrimination on a population
basis because it recognises that a risk factor is in effect the risk measure adjusted for covariates, not
the raw measure itself. In addition, if it is assumed that the risk measure is approximately normally
distributed (typically researchers have transformed mammographic density measures to achieve
this) and that risk increases multiplicatively across the risk spectrum (which empirically appears
to be a reasonable assumption), then the log(OPERA) is approximately equal to the difference in
means (in terms of standard deviation for the population) between cases and controls, and so is
easily interpreted.
The measure log(OPERA), therefore, is an appropriate scale on which to discuss and compare
risk discrimination. There is a mathematical relationship between log(OPERA) and the area under the
receiver operating characteristic curve (AUC) which is approximately linear at least for AUC between
0.5 to 0.7 (OPERA between 1 and 2). Under these assumptions, the inter-quartile risk ratio (IQRR) is
approximately OPERA2.5 (see Supplementary Material [8]).
When OPERA is used, it emerges that conventional mammographic density is one of the stronger
risk factors for female breast cancer. The OPERA is typically around 1.4, substantially more than for
the classic epidemiological risk factors, as discussed previously [7]. The risk discrimination from
conventional mammographic density compares favourably with that achieved by the latest polygenic
risk scores for which the OPERA is about 1.6 [9]. Our new mammogram-based risk measures have
OPERAs of 1.6 or higher [8,10–14] and we will now discuss how this came about.

3. Wolfe and Beyond

In the 1970s, John Wolfe demonstrated that certain aspects of a mammogram were predicting, for
women deemed at a mammographic screen to not have a breast cancer, which of them were more likely
to be diagnosed with breast cancer at subsequent screens [15]. An advocate of early mammographic
screening, Wolfe developed a classification scheme that initially apparently failed to gain adoption by
radiologists due to lack of replication in measurement and inconsistent results [16], though the latter
might have been a consequence of the former. It was recognised, however, that the dense regions of
mammograms were implicated in a major weakness of mammographic screening-the failure to detect
cancers hidden, or ‘masked’, by the white or bright regions that are mammographically ‘dense’.
The seminal work of Boyd and Yaffe, who created a semi-automated way to measure
mammographic density called CUMULUS [17] and applied it to prospective studies of large screening
populations, consistently found evidence that percent mammographic density—adjusted by design
and/or analysis for age and body mass index—was predicting both interval and screen-detected cancers
in the long-term [18]. Use of the OPERA concept confirmed that this risk gradient was stronger for
interval cancer [19], consistent with a role of masking. For example, Krishnan and colleagues [20]
found that for percent density adjusted for age and body mass index, the OPERA was 2.05 (95%
confidence interval (CI) 1.65 to 2.56) for interval cancer compared with 1.37 (95% CI 1.21 to 1.56) for
screen-detected cancer (p for difference = 0.002). Moreover, percent mammographic density was the
best predictor of whether a woman was to develop interval versus screen-detected cancer, with an
OPERA of 1.51 (95% CI 1.23 to 1.86; p = 0.00005).
Therefore, percent mammographic density was clearly predicting something specific to interval
cancers, presumably masking, and was also associated with intrinsic breast cancer risk, but to a far
lesser extent. While its association with interval cancer was by its nature likely to be causal, in the sense
that changing percent density could lead to a reduction in risk, it was still unclear if the conventional
measure of density, or even density per se, is causal for breast cancer. It is possible that the conventional
J. Clin. Med. 2020, 9, 627 4 of 13

concept of density as a risk factors is correlated with other mammogram-or lifestyle-related causal risk
factors. To examine these hypotheses, we took two approaches to discovering new mammogram-based
measures of risk, and importantly, considered their risk predictions concurrently with those arising
from conventional mammographic density when fitted together.

4. Redefining Mammographic Density in Terms of Brightness: Cumulus, Altocumulus

and Cirrocumulus
Initially, mammographic density was defined as the light or bright area and calculated using
the CUMULUS software [17]. We call the resultant measure Cumulus in honour of the method and
its creators, and because it aligns with the meteorological use of this word to describe clouds often
described as puffy, cotton-like or fluffy and based on the Latin word “cumulo”, meaning heap or pile.
We challenged this convention by defining mammographic density at, in effect, two substantially
higher pixel-brightness thresholds as the brighter (Altocumulus) and brightest (Cirrocumulus) regions.
We used the CUMULUS software but chose the bright regions within the white areas to define
Altocumulus, and the brighter regions among the bright areas to define Cirrocumulus. These words
also align with their meteorological use by matching pixel intensity to height (the Latin word “alus”
means high). Altocumulus are middle altitude clouds typically higher than cumulus clouds and have
elements that are larger than those of cirrocumulus clouds which typically appear at an even higher
altitude—the Latin word “cirro” means curl and is used to describe a high cloud.
Operators of CUMULUS have traditionally been trained to be highly repeatable in their measures
of Cumulus, both with themselves and with other operators. We similarly trained operators to ensure
measures of Altocumulus and Cirrocumulus were highly repeatable.
We found from multiple studies of Australian and Korean women [10–14] that the new measures
of mammographic density, Altocumulus and Cirrocumulus, did better than Cumulus at predicting risk.
One notable exception is risk of interval breast cancer [14].
Tables 1 and 2 show that the risk gradients for Altocumulus and/or Cirrocumulus were comparable
to those for the genetic risk scores developed from genome-wide association studies (OPERA = 1.6; [9])
and often better than for Cumulus (OPERA = 1.4). The risk estimates for Altocumulus were often little
different to those for Cumulus, but the risk estimates for Cirrocumulus in particular were in general
stronger, especially for Korean women (who are diagnosed on average a decade or so earlier than
Australian women) [10] and for Australian women diagnosed at a younger age [11]. These findings
applied to film and well as digital mammograms, and for the latter, appeared to depend on the
manufacturer [12].

Table 1. Odds ratio per unadjusted standard deviation (OPERA1 ) for dense area mammographic
density measures in predicting younger age at diagnosis breast cancer for women with a family history
of breast cancer (Nguyen et al. (2017)) [11].

Univariable Multivariable
Cirrocumulus 1.73 (1.52–1.97) 1.41 (1.21–1.65) 1.44 (1.20–1.72)
Altocumulus 1.74 (1.53–1.99) 1.43 (1.22–1.68) 1.48 (1.21–1.81)
Cumulus 1.62 (1.42–1.83) – 0.95 (0.76–1.18)
1standard deviation is calculated for each measure adjusted for age and body mass index using the controls sample
and, for the multivariable analysis, in effect adjusted for the other measure.

Table 2. Odds ratio per unadjusted standard deviation (OPERA1 ) for dense area mammographic
density measures in predicting screen-detected breast cancer (Nguyen et al. (2018)) [13].

Univariable Multivariable
Cirrocumulus 1.32 (1.18–1.48) 1.19 (1.06–1.34) 1.19 (1.04–1.36)
Altocumulus 1.25 (1.12–1.40) – 0.99 (0.85–1.14)
Cumulus 1.25 (1.12–1.40) 0.98 (0.87–1.10) 0.99 (0.85–1.15)
1standard deviation is calculated for each measure adjusted for age and body mass index using the controls sample
and, for the multivariable analysis, in effect adjusted for the other measure.
J. Clin. Med. 2020, 9, 627 5 of 13

5. Revisiting Texture using Agnostic Machine Learning: Cirrus

Wolfe originally described ‘parenchymal patterns’ using verbal descriptions and subjective
measures of textural features [15]. We instead used machine learning to try to find agnostically if
there are textural patterns (defined mathematically) that predict breast cancer risk [8]. We studied film
mammograms from two samples of Australian women and from one sample of American-Hawaiian
women living in Hawaii. We measured 20 textural features and found that the major principal
component was based on 11 of these features. Risk measures created from these 11 features and learnt
on one sample did well in predicting breast cancer in another sample (OPERAs ~ 1.7), even when
learnt on the small sample of Japanese women and applied to Australian women.
We call the risk measure based on fitting these 11 features to the combined sample Cirrus. These
features were highly correlated with one another, and the best predicting individual feature was
‘homogeneity’, a measure of the degree of “scatteredness” of the texture within an image; images with
large areas of similar intensity pixels have a higher degree of homogeneity than those composed of a
large number of small dissimilar regions.

6. What Happens When the New Measures are Fitted Together with the Conventional Measure?
We now consider what happens when the new measures are fitted with and without taking into
account the conventional measure, and vice versa. This allows inference to be made about the extent
to which associations are due to confounding with one another from examining what happens to
the estimates.

6.1. Cirrocumulus and Altocumulus

Except for interval cancer, when the new measures Cirrocumulus and Altocumulus were fitted with
the conventional measure Cumulus, the Cumulus risk estimate attenuated towards the null and was
often no longer statistically significant. In contrast, the new measures generally remained significant.
This happened consistently, despite these measures all being correlated with one another (e.g., ~0.8 for
Cumulus and Altocumulus; ~0.6 for Cumulus and Cirrocumulus). These general findings have been
replicated [21].
Table 1 shows an example in which the different density measures (in absolute terms) were
considered together as predictors of, on average, younger age at diagnosis breast cancer for women
with a family history of breast cancer [11]. All three measures gave strong and somewhat similar risk
predictions, with OPERAs of about 1.6–1.7, when considered alone (univariable analyses). When fitted
together, the Altocumulus and Cirrocumulus measures both attenuated to be about 1.4 but remained
highly significant. (The average of these two measures had an OPERA of about 1.8). The Cumulus
measure, however, went to the null and was no longer significant. This happened for each combination
that included Cumulus. The dense area measures gave a better fit than the percent mammographic
density areas for which a similar pattern of results were observed (the univariable association for
Cumulus as a percentage had a smaller OPERA of 1.52 (95% CI 1.34–1.73)).
Table 2. shows another example, this time for screen-detected breast cancer [13]. All three
measures predicted risk on their own, with the risk gradient for Cirrocumulus being 24% greater on the
log(OPERA) scale. When Cirrocumulus was fitted, the Cumulus measure went to the null and was no
longer significant whether or not Altocumulus was also fitted.
Table 3 shows an example for interval breast cancer [13]. This time the percent mammographic
density measures gave better fits than did the dense area measures. All three measures predicted
risk on their own, with the risk gradient for Cumulus being the greatest. Contrary to the previous
examples, it was the Cumulus measure that remained significant in the multivariable analyses whereas
both Altocumulus and Cirrocumulus were no longer significant once Cumulus was also fitted.
J. Clin. Med. 2020, 9, 627 6 of 13

Table 3. Odds ratio per unadjusted standard deviation (OPERA1 ) for percent mammographic density
measures in predicting interval breast cancer (Nguyen et al. (2018)) [13].

Univariable Multivariable
Cirrocumulus 2.06 (1.67–2.54) – 1.10 (0.90–1.36)
Altocumulus 1.92 (1.55–2.36) 0.88 (0.72–1.07) -
Cumulus 2.33 (1.85–2.92) 1.67 (1.36–2.07) 1.48 (1.19–1.95)
1standard deviation is calculated for each measure adjusted for age and body mass index using the controls sample
and, for the multivariable analysis, in effect adjusted for the other measure.

6.2. Cirrus
For Cirrus, Table 4 shows what happened when Cirrus was fitted together with absolute and
percentage Cumulus measures [8]. The Cirrus risk estimate remained high and attenuated by only 16%
on the log(OPERA) scale, while the Cumulus risk estimate attenuated by more than 50%. Most of the
data used these analyses came from a prospective cohort study that involved about 70% screen-detected
and 30% interval cancers. Note that the approach used to create Cirrus only used relative brightness
between pixels, not the absolute brightness of pixels as does the CUMULUS-based measures above.

Table 4. Odds ratio per unadjusted standard deviation (OPERA1 ) from Schmidt et al. combined data
set combined data set [8].

Univariable Multivariable
Cirrus 1.90 (1.73–2.09) 1.76 (1.59–1.95) 1.74 (1.57–1.93)
Cumulus (absolute) 1.34 (1.25–1.43) 1.16 (1.08–1.24) –
Cumulus (percent) 1.38 (1.29–1.48) – 1.16 (1.07–1.25)
1standard deviation is calculated for each measure adjusted for age and body mass index using the controls sample
and, for the multivariable analysis, in effect adjusted for the other measure.

7. Some Other New Mammogram-Based Measures of Breast Cancer Risk

Other researchers have also been trying to find aspects within a mammogram, other than
mammographic density, that contain information about risk of breast cancer. But rarely have these
researchers considered their new mammogram-based measure together with the conventional measure
of mammographic density as we have done.
One recent example is by Dembrower and colleagues [22] who developed a new risk measure
using deep learning, which they call DL. Their DL measure was “moderately” correlated with
conventional mammographic density measures (Spearman correlations between 0.25 and 0.42). Using
a prospective cohort design, they combined interval and screen-detected cancers as outcome (278 cases
and 2005 controls not matched for age, average 3.6 years follow-up, mean age at baseline mammogram
55 years) and calculated the OR per unadjusted standard deviation; see Table 5. They used LIBRA
automated software [23] to calculate conventional dense area and percentage density and adjusted the
mammogram-based measures for age (but not for body mass index, which might explain the poor
predictive performance of their percent mammographic density measure, as is predicted by the earlier
discussion about the OPERA concept).

Table 5. Odds ratio per unadjusted standard deviation1 from Dembrower et al. [22].

Univariable Multivariable
DL 1.56 (1.48–1.64) 1.52 (1.42–1.59) 1.55 (1.47–1.64)
Dense area 1.31 (1.24–1.38) 1.15 (1.09–1.22) -
Percent density 1.18 (1.11–1.25) - 1.02 (0.95–1.08)
1standard deviation is calculated for each measure adjusted for age and body mass index using the controls sample
and, for the multivariable analysis, in effect adjusted for the other measure.
J. Clin. Med. 2020, 9, 627 7 of 13

Table 5 shows that the risk gradient for the DL measure was unchanged by adjusting for the
conventional mammographic density measures. The risk gradient for the absolute dense area measure
halved after adjusting for the DL measure. Wanders and colleagues also considered together a
conventional mammographic density measure and a texture method assessment method they had
developed [24], but they adjusted the texture measure for mammographic density (but not vice versa).
The risk gradient for the texture measure attenuated by about 15–35%, and remained highly significant.
Unfortunately, they did not report what happened to the mammographic density risk after adjustment
for texture.

8. Interpretation
Figure 1 shows an interpretation, in terms of a path diagram, of the general findings above.
Unmeasured causes are shown as circles and the arrows represent causes in a given direction.
The conventional and new measures are moderately correlated, so there must be causal factors specific
to both measures (or even a causal relationship between the conventional and new measures, though
we haven’t shown that on the figure).

Figure 1. Path diagram indicating causal pathways implicated in conventional and new mammogram-based
predictors of interval and screen-detected breast cancers.

Because there is no convincing evidence that conventional mammographic density predicts

screen-detected breast cancer once the other mammogram-based risk factors are taken into account,
it appears to be a predictor of interval cancers due only (or mostly) to its role in masking. The new
mammogram-based risk measures appear to be predicting interval cancer through their association
with conventional mammographic density, particularly the other mammographic density measures
defined by higher pixel brightness thresholds which attenuate once the conventional measure is fitted
(see Table 3). But the new measures could also be having a direct effect given that their risk association
is still evident even after adjustment for conventional mammographic density (see Tables 1 and 2 and
Tables 4 and 5). The data are consistent with the new mammogram-based risk measures (rather than
conventional mammographic density) having a direct causal effect on both interval and screen-detected
breast cancer, and this hypothesis now needs to be addressed.

9. Importance of Mammogram-Based Risk Factors as Breast Cancer Risk Predictors

Following the concept of OPERA, Figure 2 shows that the new mammogram-based measures are
the strongest of the currently known risk factors in terms of risk discrimination on a population-basis.
This Figure is an update of Table 1 in the original paper on OPERA [7]. The next best risk factor is
derived from multi-generational family history using the tools BOADICEA and BRCAPRO; the OPERA
in Figure 2 pertains to risk of breast cancer diagnosed before age 50 years for women under the age of
50 years [25]. Given familial risks are highly dependent on age at diagnosis, and age at risk, this is
J. Clin. Med. 2020, 9, 627 8 of 13

likely to be greater than it would be for breast cancer diagnosed at any age. The OPERA for polygenic
risk scores is based on the latest publication from the Breast Cancer Association Consortium [9].

Figure 2. Risk discrimination for breast cancer based on the odds ratio per adjusted standard deviation
(OPERA) (see [7]) and equivalent inter-quartile risk ratio (IQRR).

Conventional mammographic density, adjusted for age and body mass index, is at most weakly
associated with the other known lifestyle-based breast cancer risk factors [5], as mentioned in Section 1.
Familial and genetic factors are perhaps the strongest; about 10–15% of the increased risk of breast
cancer associated with having at least one affected first-degree relative family history is explained by
the fact that mammographic density adjusted for age and body mass index has a correlation of about
0.3 in first-degree relatives and 0.6 in monozygotic twin pairs [5,26,27] Indeed, a similar proportion of
the known genetic variants associated with breast cancer risk are also implicated in explaining the
variance of mammographic density for age and body mass index [28,29]. The relationships between the
new mammogram-based risk factors and known breast cancer risk factors have not yet been published.

10. Discussion
Wolfe was right, and wrong. There are aspects of a mammographic image that predict risk of
breast cancer—but it isn’t all about parenchymal patterns, breast density, or the percentage of the
breast covered by white areas. Wolfe’s initial strong predictions were, on face value correct, but they
were mostly due to the strong role of conventional mammographic density in predicting masking, not
so much as risk per se.
Risk of interval cancers is an important issue, but it is not all due to density and masking. We have
found that it can be greatly improved by treating density as a continuum (not as a binary construct
as in current clinical practice) and by including other risk factors such as family history, and a new
measure based on breast tissue ageing [14]. It will likely improve with the addition of polygenic risk
scores and the other mammogram-based risk factors related to risk per se.
Our work has successfully identified new insights into breast cancer risk, but we would not have
been able to do this without the enormous contributions of others. In particular, Professor Norman
Boyd kept alive the ideas of Wolfe by developing the technology and resources, and inspiring the
workforce, to pursue this line of investigation. The CUMULUS software has been an incisive tool for
understanding breast cancer risk. We trained observers, not necessarily radiologists or physicians,
to use CUMULUS in novel ways and achieved high repeatability and better prediction of breast cancer
than the latest polygenic risk scores found from a massive expenditure on genetics applied to very
large-scale epidemiological studies. These new mammogram-based risk factors are also much better
J. Clin. Med. 2020, 9, 627 9 of 13

than the other known epidemiological risk factors (see Figure 2) and complement pedigree-based risk
models like BOADICEA and BRCAPRO [25].
What differentiates our work over the last few years has been its focus on the aspects of a
mammographic image that predict risk, rather than on mammographic density alone. We have not
been alone in this pursuit, at least not recently, and now that digital mammography is widespread,
the prospect of finding even stronger risk predictors using agnostic computer-learning approaches is
a reality.
For example, Perutz and colleagues created OpenBreast [30], by applying logistic regression
analyses to multiple features as we did in creating Cirrus, this time using digital mammograms but
with a much smaller dataset. They recently reported that OpenBreast had an OPERA of 2.5, equivalent
to an extraordinarily high IQRR of almost 10 but with a wide 95% CI (given the small dataset) for
which the lower bound was 4 [30]. From the Supplementary Material [30], the most prominent
feature contributing to Open Breast was ‘maximum gray-level value’, which could be picking up
the aspects captured by Cirrocumulus (the amount of brightest areas). The next most prominent
feature was ‘high gray-level run’ followed by ‘run-percentage’ and then both ‘short-run emphasis’
and ‘long-run emphasis’, separated by ‘gray-level range’. Therefore, OpenBreast could be utilising
aspects of ‘homogeneity’ and the major principal component on which Cirrus is based, combined with
measures of mean and range of brightness implicated in Cirrocumulus.
It might be that, by applying machine learning to digital mammograms, essentially the same
risk-associated aspects of a mammogram are being discovered, but with greater power and more
precision. A similar approach to identify texture-based risk predictors agnostically also performed well
with an IQQR of about 6, with a lower bound of the 95% CI < 4 (again reflecting the small data set) [31].
Nevertheless, these two recent studies, combined with our discoveries using mostly film
mammograms, are very encouraging for finding very strong, and easily automated, breast cancer risk
prediction from applying machine leaning to digital mammograms. Our studies were detecting signals
despite considerable noise from digitising film mammograms across different machines. Despite this,
we appear to have been detecting true signals. Discovery of more and better signals might be much
easier using digital mammography.
Another attempt to achieve this aim using ‘deep learning’ applied to a large data set [32] failed to
use cases and controls of the same age, so they in effect learnt how to measure age from a mammogram,
derived inflated estimates of risk discrimination based on naïve use of AUC statistics, and might not
have discovered anything new. We know age predicts breast cancer risk, and is easily measured, so we
want to know what it is about a mammogram that predicts risk for women of the same age. Reuse of
that large data set to apply learning and replication using case-control sets matched for age could be
worthwhile, especially if it was made openly available.
To help achieve the aim of determining the optimal measures of the different aspect of breast
cancer risk from different mammographic images, we and others have created the Measurement
Challenge [33]. This is an open international resource which offers sets of anonymised full-field
digital mammogram images for analysis so that unbiased and transparent comparisons can be made
between measures.
These new discoveries have important implications for understanding the causes of breast
cancer. We have applied a new method for making inference about causation by examining familial
confounding, called ICE FALCON [34], to data on twin pairs. We found no evidence that conventional
DNA methylation in blood is having a causal effect on conventional mammographic density (Cumulus),
or vice versa [35]. We will apply this approach to study our new mammogram-based risk factors.
That conventional mammographic density might not necessarily be having a direct causal effect
on breast cancer risk has serious implications for biological research. At this stage it is very difficult to
identify which components of breast tissue are implicated in mammogram-based risk, but this will be
an area of likely productive aetiological research once the new generation of risk factors emerging
from agnostic approaches to digital mammograms are established and automated.
J. Clin. Med. 2020, 9, 627 10 of 13

There are also major public health implications, especially given the magnitude of breast cancer
over-diagnosis associated with mammographic screening [36]. It is interesting to reflect on Wolfe being
an advocate of early mammographic screening. These new findings play to that scenario by making
it possible to clarify with considerable precision, and at a young adult age, a woman’s risk of breast
cancer [37].
If screening was to be tailored to a woman’s risk using new mammogram-based measures, family
history and polygenic risk scores, large proportions of the population would be classified as well
below ‘average’ risk while small proportions (but substantial numbers given the number of women
screened) of women with risks as high as BRCA1 and BRCA2 mutation could be readily identified [37].
New automated risk predictors based on mammograms, improved genetic risk prediction and proper
collection and utilisation of family history data could greatly change the landscape of breast cancer risk
prediction without needing to collect data from women about lifestyle and other factors with small
impact on risk discrimination, making implementation far more practical and realistic.
These advances in breast cancer risk prediction based on aspects of mammograms could also be
used to help alleviate racial and ethnic disparities. For example, in the U.S., black women are more
likely to be diagnosed with more aggressive interval breast cancer. Asian women also have on average
higher percent mammographic density and are at increased risk of this type of breast cancer. Better
risk prediction for interval breast cancer using multiple risk factors as in [14] is, therefore, a priority.
To be effective, however, it will need to be automated, include new measures such as Cirrus, and be
validated for use in racially and ethnically diverse populations.
In summary, our research has directly challenged the historical paradigm of mammographic
density research that there is one concept of mammographic density, defined as the light of bright
regions on a mammogram, and that this measure is predictive of both masking of existing tumours
and risk. We have drastically changed the conventional view by finding evidence that the conventional
density is implicated in breast cancer risk—specifically interval cancers—by masking, not directly
with risk per se. Other aspects of a mammogram, based on brightness and texture, appear to be more
directly implicated in risk, and this applies to Caucasian as well as Asian women. The prospect of
automated mammogram-based risk measures, combined with other major risk factors, could change
population screening to be based more on relative risk than just age, make screening more effective,
reduce its negative aspects, and hopefully save more lives.

Author Contributions: Conceptualization: J.L.H., T.L.N., D.F.S., E.M., Y.-M.S., J.S., J.G.D., S.L.; methodology:
J.L.H., T.L.N., D.F.S., E.M., Y.-M.S., J.S., G.S.D., J.G.D., S.L.; software: D.F.S., E.M., J.S.; formal analysis: J.L.H.,
T.L.N., D.F.S., E.M., G.S.D., J.S.; resources: J.L.H., T.L.N., Y.-M.S., J.S.; writing—original draft preparation: J.L.H.,
T.L.N.; writing—review and editing: J.L.H., T.L.N., D.F.S., E.M., Y.-M.S., J.S., G.S.D., J.G.D., S.L.; visualisation:
J.L.H., T.L.N.; supervision: J.L.H., T.L.N.; project administration: J.L.H., T.L.N., Y.-M.S., J.S. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was supported by the National Health and Medical Research Council (251533, 209057,
and 504711), the Victorian Health Promotion Foundation, Cancer Council Victoria, Cancer Council NSW, Cancer
Australia, and the National Breast Cancer Foundation. It has also been supported by the Breast Cancer Network
Australia, the National Breast Cancer Foundation, Victoria Breast Cancer Research Consortium and was further
supported by infrastructure provided by the Cancer Council Victoria and the University of Melbourne. We thank
the Victorian Cancer Registry, BreastScreen Victoria, and the Australian Mammographic Density Research
Facility. TLN has been supported by Cure Cancer Australia Foundation through Cancer Australia Priority-Driven
Collaborative Cancer Research Scheme (1159399). TLN and SL has been supported by a Victorian Cancer Council
Post-Doctoral Fellowship and a grant from the Picchi Foundation—Victorian Comprehensive Cancer Centre. JLH
is a NHMRC Senior Principal Research Fellow.
Acknowledgments: We thank the participants in our studies and the Victorian Cancer Registry, BreastScreen
Victoria, the Australian Mammographic Density Research Facility.
Conflicts of Interest: The authors have no conflicts of interest.
J. Clin. Med. 2020, 9, 627 11 of 13

References
1. Byng, J.W.; Yaffe, M.J.; Jong, R.A.; Shumak, R.S.; Lockwood, G.A.; Tritchler, D.L.; Boyd, N.F. Analysis
of mammographic density and breast cancer risk from digitized mammograms. Radiographics 1998, 18,
1587–1598. [CrossRef] [PubMed]
2. Krishnan, K.; Baglietto, L.; Stone, J.; Simpson, J.A.; Severi, G.; Evans, C.F.; MacInnis, R.J.; Giles, G.G.;
Apicella, C.; Hopper, J.L. Longitudinal Study of Mammographic Density Measures That Predict Breast
Cancer Risk. Cancer Epidemiol. Biomarkers Prev. 2017, 26, 651–660. [CrossRef] [PubMed]
3. Shia, W.C.; Wu, H.K.; Huang, Y.L.; Lin, L.S.; Chen, D.R. Mammographic Density Distribution of Healthy
Taiwanese Women and its Naturally Decreasing Trend with Age. Sci. Rep. 2018, 8, 14937. [CrossRef]
[PubMed]
4. Hopper, J.L.; Dite, G.S.; MacInnis, R.J.; Liao, Y.; Zeinomar, N.; Knight, J.A.; Southey, M.C.; Milne, R.L.;
Chung, W.K.; Giles, G.G.; et al. Age-specific breast cancer risk by body mass index and familial risk:
Prospective family study cohort (ProF-SC). Breast Cancer Res. 2018, 20, 132. [CrossRef] [PubMed]
5. Nguyen, T.L.; Schmidt, D.F.; Makalic, E.; Dite, G.S.; Stone, J.; Apicella, C.; Bui, M.; Macinnis, R.J.; Odefrey, F.;
Cawson, J.N.; et al. Explaining variance in the cumulus mammographic measures that predict breast cancer
risk: A twins and sisters study. Cancer Epidemiol. Biomarkers Prev. 2013, 22, 2395–2403. [CrossRef] [PubMed]
6. Eng, A.; Gallant, Z.; Shepherd, J.; McCormack, V.; Li, J.; Dowsett, M.; Vinnicombe, S.; Allen, S.;
dos-Santos-Silva, I. Digital mammographic density and breast cancer risk: A case-control study of six
alternative density assessment methods. Breast Cancer Res. 2014, 16, 439. [CrossRef]
7. Hopper, J.L. Odds per adjusted standard deviation: Comparing strengths of associations for risk factors
measured on different scales and across diseases and populations. Am. J. Epidemiol. 2015, 182, 863–867.
[CrossRef]
8. Schmidt, D.F.; Makalic, E.; Goudey, B.; Dite, G.S.; Stone, J.; Nguyen, T.L.; Dowty, J.G.; Baglietto, L.;
Southey, M.C.; Maskarinec, G.; et al. Cirrus: An automated mammography-based measure of breast cancer
risk based on textural features. JNCI Cancer Spectrum 2018, 2. [CrossRef]
9. Mavaddat, N.; Michailidou, K.; Dennis, J.; Lush, M.; Fachal, L.; Lee, A.; Tyrer, J.P.; Chen, T.H.; Wang, Q.;
Bolla, M.K.; et al. Polygenic Risk Scores for Prediction of Breast Cancer and Breast Cancer Subtypes. Am. J.
Hum. Genet 2019, 104, 21–34. [CrossRef]
10. Nguyen, T.L.; Aung, Y.K.; Evans, C.F.; Yoon-Ho, C.; Jenkins, M.A.; Sung, J.; Hopper, J.L.; Song, Y.M.
Mammographic density defined by higher than conventional brightness threshold better predicts breast
cancer risk for full-field digital mammograms. Breast Cancer Res. 2015, 17, 142. [CrossRef]
11. Nguyen, T.L.; Aung, Y.K.; Evans, C.F.; Dite, G.S.; Stone, J.; MacInnis, R.J.; Dowty, J.G.; Bickerstaffe, A.;
Aujard, K.; Rommens, J.M.; et al. Mammographic density defined by higher than conventional brightness
thresholds better predicts breast cancer risk. Int. J. Epidemiol. 2017, 46, 652–661. [CrossRef] [PubMed]
12. Nguyen, T.L.; Choi, Y.H.; Aung, Y.K.; Evans, C.F.; Trinh, N.H.; Li, S.; Dite, G.S.; Kim, M.S.; Brennan, P.C.;
Jenkins, M.A.; et al. Breast cancer risk associations with digital mammographic density by pixel brightness
threshold and mammographic system. Radiology 2018, 286, 433–442. [CrossRef] [PubMed]
13. Nguyen, T.L.; Aung, Y.K.; Li, S.; Trinh, N.H.; Evans, C.F.; Baglietto, L.; Krishnan, K.; Dite, G.S.; Stone, J.;
English, D.R.; et al. Predicting interval and screen-detected breast cancers from mammographic density
defined by different brightness thresholds. Breast Cancer Res. 2018, 20, 152. [CrossRef] [PubMed]
14. Nguyen, T.L.; Li, S.; Dite, G.S.; Aung, Y.K.; Evans, C.F.; Trinh, H.N.; Baglietto, L.; Stone, J.; Song, Y.M.;
Sung, J.; et al. Interval breast cancer risk associations with breast density, family history and breast tissue
aging. Int. J. Cancer 2019. [CrossRef]
15. Wolfe, J.N. Risk for breast cancer development determined by mammographic parenchymal pattern. Cancer
1976, 37, 2486–2492. [CrossRef]
16. Rafferty, E.A. Mammographic breast density: From Wolfe and beyond. Menopause 2014, 21, 920–921.
[CrossRef] [PubMed]
17. Byng, J.W.; Boyd, N.F.; Fishell, E.; Jong, R.A.; Yaffe, M.J. The quantitative analysis of mammographic densities.
Phys. Med. Biol. 1994, 39, 1629–1638. [CrossRef]
18. Boyd, N.F.; Guo, H.; Martin, L.J.; Sun, L.; Stone, J.; Fishell, E.; Jong, R.A.; Hislop, G.; Chiarelli, A.;
Minkin, S.; et al. Mammographic density and the risk and detection of breast cancer. N. Engl. J. Med. 2007,
356, 227–236. [CrossRef]
J. Clin. Med. 2020, 9, 627 12 of 13

19. Baglietto, L.; Krishnan, K.; Stone, J.; Apicella, C.; Southey, M.C.; English, D.R.; Hopper, J.L.; Giles, G.G.
Associations of mammographic dense and nondense areas and body mass index with risk of breast cancer.
Am. J. Epidemiol. 2014, 179, 475–483. [CrossRef]
20. Krishnan, K.; Baglietto, L.; Apicella, C.; Stone, J.; Southey, M.C.; English, D.R.; Giles, G.G.; Hopper, J.L.
Mammographic density and risk of breast cancer by mode of detection and tumor size: A case-control study.
Breast Cancer Res. 2016, 18, 63. [CrossRef]
21. Wang, C.; Brentnall, A.R.; Cuzick, J.; Harkness, E.F.; Evans, D.G.; Astley, S. Exploring the prediction
performance for breast cancer risk based on volumetric mammographic density at different thresholds.
Breast Cancer Res. 2018, 20, 49. [CrossRef] [PubMed]
22. Dembrower, K.; Liu, Y.; Azizpour, H.; Eklund, M.; Smith, K.; Lindholm, P.; Strand, F. Comparison of a Deep
Learning Risk Score and Standard Mammographic Density Score for Breast Cancer Risk Prediction. Radiology
2020, 294, 265–272. [CrossRef] [PubMed]
23. Keller, B.M.; Chen, J.; Daye, D.; Conant, E.F.; Kontos, D. Preliminary evaluation of the publicly available
Laboratory for Breast Radiodensity Assessment (LIBRA) software tool: Comparison of fully automated area
and volumetric density measures in a case-control study with digital mammography. Breast Cancer Res. 2015,
17, 117. [CrossRef] [PubMed]
24. Wanders, J.O.P.; van Gils, C.H.; Karssemeijer, N.; Holland, K.; Kallenberg, M.; Peeters, P.H.M.; Nielsen, M.;
Lillholm, M. The combined effect of mammographic texture and density on breast cancer risk: A cohort
study. Breast Cancer Res. 2018, 20, 36. [CrossRef] [PubMed]
25. Dite, G.S.; MacInnis, R.J.; Bickerstaffe, A.; Dowty, J.G.; Allman, R.; Apicella, C.; Milne, R.L.; Tsimiklis, H.;
Phillips, K.A.; Giles, G.G.; et al. Breast cancer risk prediction using clinical models and 77 independent
risk-associated SNPs for women aged under 50 years: Australian breast cancer family registry.
Cancer Epidemiol. Biomarkers Prev. 2016, 25, 359–365. [CrossRef]
26. Boyd, N.F.; Dite, G.S.; Stone, J.; Gunasekara, A.; English, D.R.; McCredie, M.R.; Giles, G.G.; Trichler, D.;
Chiarelli, A.; Yaffe, M.J.; et al. Heritability of mammographic density, a risk factor for breast cancer. N. Engl.
J. Med. 2002, 347, 886–894. [CrossRef]
27. Martin, L.J.; Melnichouk, O.; Guo, H.; Chiarelli, A.M.; Hislop, T.G.; Yaffe, M.J.; Minkin, S.; Hopper, J.L.;
Boyd, N.F. Family history, mammographic density, and risk of breast cancer. Cancer Epidemuiol. Biomakers
Prev. 2010, 19, 456–463. [CrossRef]
28. Stone, J.; Thompson, D.J.; Dos Santo Silva, I.; Scott, C.; Tamimi, R.M.; Lindstrom, S.; Kraft, P.; Hazra, A.;
Li, J.; Eriksson, L.; et al. Novel assocaitions between common breast cancer susceptibility variants and
risk-predicting mammographic density measures. Cancer Res. 2015, 75, 2457–2467. [CrossRef]
29. Lindstrom, S.; Thompson, D.J.; Paterson, A.D.; Li, J.; Gierach, G.L.; Scott, C.; Stone, J.; Douglas, J.A.;
dos Santos-Silva, I.; Fernando-Navarro, O.; et al. Genome-wide association study identifies multiple loci
associated with both mammographic density and breast cancer risk. Nat. Commun. 2014, 5, 5303. [CrossRef]
30. Pertuz, S.; Sassi, A.; Holli-Helenius, K.; Kamarainen, J.; Rinta-Kiikka, I.; Laaperi, A.L.; Arponen, O. Clinical
evaluation of a fully-automated parenchymal analysis software for breast cancer risk assessment: A pilot
study in a Finnish sample. Eur. J. Radiol. 2019, 121, 108710. [CrossRef]
31. Tan, M.; Mariapun, S.; Yip, C.H.; Ng, K.H.; Teo, S.H. A novel method of determining breast cancer risk using
parenchymal textural analysis of mammography images on an Asian cohort. Phys. Med. Biol. 2019, 64,
035016. [CrossRef] [PubMed]
32. Yala, A.; Lehman, C.; Schuster, T.; Portnoi, T.; Barzilay, R. A Deep Learning Mammography-based Model for
Improved Breast Cancer Risk Prediction. Radiology 2019, 292, 60–66. [CrossRef]
33. Dench, E.; Bond-Smith, D.; Darcey, E.; Lee, G.; Aung, Y.K.; Chan, A.; Cuzick, J.; Ding, Z.Y.; Evans, C.F.;
Harvey, J.; et al. Measurement challenge: Protocol for international case-control comparison of
mammographic measures that predict breast cancer risk. BMJ Open 2019, 9, e031041. [CrossRef] [PubMed]
34. Li, S.; Wong, E.M.; Bui, M.; Nguyen, T.L.; Joo, J.E.; Stone, J.; Dite, G.S.; Dugue, P.A.; Milne, R.L.; Giles, G.G.; et al.
Inference about causation between body mass index and DNA methylation in blood from a twin family
study. Int. J. Obes. 2019, 43, 243–252. [CrossRef] [PubMed]
35. Li, S.; Dugue, P.A.; Baglietto, L.; Severi, G.; Wong, E.M.; Nguyen, T.L.; Stone, J.; English, D.R.; Southey, M.C.;
Giles, G.G.; et al. Genome-wide association study of peripheral blood DNA methylation and conventional
mammographic density measures. Int. J. Cancer 2019, 145, 1768–1773. [CrossRef]
J. Clin. Med. 2020, 9, 627 13 of 13

36. Glasziou, P.P.; Jones, M.A.; Pathirana, T.; Barratt, A.L.; Bell, K.J. Estimating the magnitude of cancer
overdiagnosis in Australia. Med. J. Aust. 2019. [CrossRef]
37. Hopper, J.L. Genetics for population and public health. Int. J. Epidemiol. 2017, 46, 8–11. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
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Another random document with
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 Fig. 41.—Spawn of a
species of Natica
(from a specimen in
the British Museum) ×
½.

Fig. 42.—Ianthina fragilis Lam. FL, float; O, ova; Pr,

proboscis; Br, branchiae; F, foot. (Quoy and Gaimard.)

Chiton marginatus, when kept in captivity, has been noticed[243] to

elevate the posterior part of the girdle, and to pour out a continuous
stream of flaky white matter like a fleecy cloud, which proved to be of
a glutinous nature. It then discharged ova, at the rate of one or two
every second, for at least fifteen minutes, making a total of 1300 to
1500, each being about 1/100 inch diameter. The ova were shot into
the glutinous cloud, which seemed to serve as a sort of nidus to
entangle the ova and prevent them being carried away. The
subsequent development was rapid, and in seven days the young
Chiton was hatched, being then about 1/20 inch long. Lovén has
described the same species as laying its eggs, loosely united in
clusters of seven to sixteen, upon small stones. There is probably
some mistake about the identification, but the observation illustrates
the varying methods of oviposition among allied forms.

Fig. 43.—Egg-capsules of A, Sepia elegans

Orb., and B, Octopus vulgaris Lam.
Not very much is known with regard to the ovipositing of the
Cephalopoda, especially those which inhabit deep water. Masses of
ova arranged in very various forms have occasionally been met with
floating in the ocean, but it is next to impossible to determine to what
species, or even genus, they belong.[244]
In Loligo punctata the ova are contained in small cylindrical cases
measuring 3 to 4 in. by ½ in., to the number of about 250 ova in each
case. Hundreds of these cases are attached together like a bundle of
sausages or young carrots, and the movements of the embryos
within can be distinctly noted. Sepia officinalis lays large black pear-
shaped capsules, each of which is tied to some place of attachment
by a kind of ribbon at the upper end of the capsule, the whole
forming a large group like a bunch of grapes. Octopus vulgaris
deposits thousands of small berry-shaped ova, attached to a string
which runs along the centre of the mass (Fig. 43).
The so-called shell of the female Argonauta is nothing more than
a form of protection for the ova, and is in no sense homologous to
the ordinary molluscan shell. The ova consist of a large granulated
mass, attached to a many branched stem; they are contained in the
spire of the shell, in contact with the posterior part of the body of the
mother, but sometimes project externally beyond the coil of the spire.
Certain species possess the curious property of laying their eggs
on the outside of their own shells. Buccinopsis Dalei is not
unfrequently found decorated with its own egg-capsules. Possibly
this species, which lives on oozy ground, finds this the only secure
place of attachment for its progeny. Neritina fluviatilis has a similar
habit, and so have many other species of Neritina and Navicella. It is
not quite clear, in the latter cases, whether the eggs are laid by the
specimens on whose shell they are found, or whether they are
deposited by others. In either case, perhaps the shell is the safest
place for them in the rapid streams which both genera frequent.
Specimens of Hydrobia ulvae taken on the wet sands at the mouth of
the Dee, are found to have several little rounded excrescences
scattered over the surface of the shell. These, on examination, are
found to be little masses of small sand-grains, in the centre of which
is a clear jelly containing segmenting ova or young embryos. Here
again, in all probability, the shell is the only comparatively stable
object, in the expanse of shifting sands, on which the eggs can be
laid.[245]
The pulmonate genus Libera, which occurs on a few of the island
groups in the Central Pacific, is remarkable for the habit of laying its
eggs within its own cavernous umbilicus, which is narrowed at the
lower part. The eggs number from four to six, or the same number of
very young shells may be seen closely packed in the cavity, each
being in shape exactly like a young Planorbis. This constriction of the
umbilicus does not occur till the formation of the last two whorls, i.e.
till the animal is sexually mature. Some species, but not all, provide
for the safety of their eggs more completely by forming a very thin
shelly plate, which nearly closes the umbilical region, and breaks
away or is absorbed to facilitate the escape of the young shells.[246]
Union of Limax.—With regard to the act of union itself, the
method in certain species of Limax deserves special notice. L.
maximus has been observed at midnight to ascend a wall or some
perpendicular surface. A pair then crawl round and round one
another emitting a quantity of mucus which at length forms a patch, 2
to 2½ inches in diameter. When this acquires consistency the pair
begin to twist round each other in corkscrew form, and detach
themselves from the wall, hanging by a cord of the thickened mucus,
about 8–15 inches long, and still twisting round each other. The
external generative organs are then protruded and copulation takes
place, after which the bodies untwist, separate, and crawl up the
cord again to the wall.[247]
Periodicity in Breeding.—In the marine Mollusca, the winter
months appear to be the usual time for the deposition of eggs.
Careful observations have been made on the Mollusca occurring at
Naples,[248] and the general result seems to be that for all Orders
alike the six winter months from November to April, roughly
speaking, are the breeding time. Scarcely any forms appear to breed
habitually in August, September, or October. On our own coasts,
Nudibranchiata come in shore to deposit their ova from January to
April. Purpura lapillus may be observed depositing ova all the year
round, but is most active from January to April. Buccinum undatum
breeds from October to May; Littorina all the year round.
The land Mollusca exhibit rather more periodicity than the marine.
In temperate climates they breed exclusively in the summer months.
In the tropics their periods are determined by the dry and rainy
seasons, where such occur, otherwise they cohabit all the year
round. According to Karl Semper, the snails of the warm
Mediterranean region arrive at sexual maturity when they are six
months old, i.e. before they are fully grown. After a rest of about
three months during the heat of summer, a second period of
ovipositing occurs.[249] Helix hortensis and H. nemoralis ascend
trees, sometimes to a height of forty feet, when pairing.[250]
Hybridism as the result of union between different species of
Mollusca is exceedingly rare. Lecoq once noticed[251] on a wall at
Anduze (Gard) as many as twenty specimens of Pupa cinerea united
with Clausilia papillaris. No offspring seem to have resulted from
what the professor calls ‘this innocent error,’ for the wall was
carefully scrutinised for a long time, and no hybrid forms were ever
detected.
The same observer noticed, in the Luxembourg garden at Paris,
and M. Gassies has noticed[252] at various occasions, union
between Helix aspersa and nemoralis, H. aspersa and vermiculata,
between Stenogyra decollata and a Helix (sp. not mentioned), H.
variabilis and pisana, H. nemoralis and hortensis. In the two latter
cases a hybrid progeny was the result. It has been noticed that these
unions generally took place when the air was in a very electric
condition, and rain had fallen, or was about to fall, abundantly.
Of marine species Littorina rudis has been noticed[253] in union
both with L. obtusata and with L. littorea, but no definite facts are
known as to the result of such unions.
Self-impregnation (see p. 44).
Development of the Fertilised Ovum.—The first stages in the
development of the Mollusca are identical with those which occur in
other classes of animals. The fertilised ovum consists of a vitellus or
yolk, which is surrounded with albumen, and is either contained in a
separate capsule, or else several, sometimes many, ova are found in
the same capsule, only a small proportion of which ultimately
develop. The germinal vesicle, which is situated at one side of the
vitellus, undergoes unequal segmentation, the result of which is
usually the formation of a layer of small ectoderm cells overlying a
few much larger cells which contain nearly the whole of the yolk. The
large cells are then invaginated, or are simply covered by the growth
of the ectoderm cells. The result in either case is the formation of an
area, the blastopore, where the inner cells are not covered by the
ectoderm. The blastopore gradually narrows to a circular opening,
which, in the great majority of cases, eventually becomes the mouth.
The usual differentiation of germinal layers takes place, the epiblast
eventually giving rise to the epidermis, nervous system, and special
sense organs, the hypoblast to the liver and to the middle region of
the alimentary tract, the mesoblast to the muscles, the body cavity,
the vascular, the excretory and reproductive systems. The next, or
trochosphere (trochophora) stage, involves the formation of a circlet
of praeoral cilia, dividing the still nearly spherical embryo into two
unequal portions, the smaller of which consists simply of the
prostomium, or part in front of the mouth, the larger bearing the
mouth and anus.
So far the series of changes undergone by the embryo are not
peculiar to the Mollusca; we now come to those which are definitely
characteristic of that group. The stage next succeeding the
development of the trochosphere is the definitive formation of the
velum, a process especially characteristic of the Gasteropoda and
Pelecypoda, but apparently not occurring in the great majority of land
Pulmonata.

Fig. 44.—Veligers of Dentalium entalis L.: A, longitudinal section of a larva 14

hours old, × 285; B, larva of 37 hours, × 165; C, longitudinal section of
larva of 34 hours, × 165; m, mouth; v, v, velum. (After Kowalewsky.)
The circlet of cilia becomes pushed more and more towards the
anterior portion of the embryo, the cilia themselves become longer,
while the portion of the body from which they spring becomes
elevated into a ridge or ring, which, as a rule, develops on each side
a more or less pronounced lobe. The name velum is applied to this
entire process of ciliated ring and lobes, and to the area which they
enclose.
Fig.45.—Veliger of Patella vulgata
L., 130 hours old: f,
rudimentary foot; op,
operculum; sh, shell; v, v,
velum. (After Patten, highly
magnified.)
 Fig. 46.—Developed larva of Cyclas
cornea L.: br, rudimentary
branchiae; by, byssus; f, foot;
m.e, mantle edge; sh, shell.
(After Ziegler, highly magnified.)

Fig. 47.—A, Advanced veliger of Dreissensia: f, foot; m,

mouth; sh, shell; v, v, velum. (After Korschelt and
Heider, much enlarged.) B, Veliger of a Pteropod
(Tiedemannia): op, operculum; sh, shell; v, velum.
(After Krohn, much enlarged.)
In this so-called veliger stage, the velum serves, in the first place,
to cause rotation of the larva within the egg-capsules, and, after
hatching, as an organ of locomotion. As a rule, the velum disappears
entirely in the adult mollusc after the free-swimming stage is over,
but in the common Limnaea stagnalis it persists, losing its cilia, as
the very prominent circum-oral lobes. Simultaneously with the
development of the velum, and in some cases earlier, appear the
rudiments of the shell-gland and of the foot, the latter being situated
on the ventral side, between the mouth and anus, the former on the
dorsal side, behind the velum, and above the surface of the eventual
visceral sac. Thus the prime characteristics of the veliger stage,
subsequent to the appearance of the velum itself, are the
development of the visceral sac and shell-gland on the upper, and of
the foot on the under side. According to Lankester the primitive shell-
gland does not, as a rule, directly give rise to the shell of the adult
mollusc, but becomes filled up by a horny substance, and eventually
disappears; the permanent shell then forms over the surface of the
visceral hump from the original centre of the shell-gland. It is only in
Chiton, and possibly in Limax, that the primitive shell-sac is retained
and developed into the final shell-forming area, which is much wider,
and extends to the edges of the mantle. Within the velar area first
appear the rudiments of the tentacles and eyes; when these become
developed the velum atrophies and disappears.
Several of these veligers when captured in the open sea have
been mistaken for perfect forms, and have been described as such.
Thus the larva of Dolium has been described as Macgillivrayia, that
of a Purpura as Chelotropis and Sinusigera, that of Aporrhais pes
pelecani as Chiropteron, that of Marsenia conspicua as Brownia,
Echinospira, and Calcarella.
Cephalopoda.—The embryonic development of the Cephalopoda
is entirely distinct from that of all other Mollusca. The segmentation
of the vitellus is partial, and the embryo is furnished with a vitelline
sac, which is very large in the majority of cases (Fig. 48). There is no
free-swimming stage, but the embryo emerges from the egg fully
developed.
Differences of Sex.—In the Mollusca there are two main types of
sexual difference: (i) sexes separate (dioecious type), (ii) sexes
united in the same individual (hermaphrodite type).
 Fig. 48.—Two stages in the development of
Loligo vulgaris Lam.: a1, a1, first, and a2,
a2, second pairs of arms; br, branchiae,
seen through m, mantle; e, e, eyes; fi,
fins; fu, funnel; v.s, vitelline sac. (After
Kowalewsky.)
In some cases—e.g. certain Pelecypoda—what is practically a
third type occurs. The animal is hermaphrodite, but the male and
female elements are not developed simultaneously, i.e. the same
individual is at one time female, at another male.
1. The sexes are separate in
All Cephalopoda.
Gasteropoda Amphineura (except Neomeniidae).
Gasteropoda Prosobranchiata (except Valvata and some species of
Marsenia).
Scaphopoda.
Many Pelecypoda.

2. The sexes are united in

Gasteropoda Opisthobranchiata.
Gasteropoda Pulmonata.
Certain Pelecypoda.[254]
 In the dioecious Mollusca, sexual union is the rule, but is by no
means universal. In some instances,—e.g. Vermetus, Magilus,
Patella, Haliotis, Crepidula, Chiton, the Scaphopoda—the form and
habits of the animal do not admit of it; in others (many Trochus) a
male copulative organ is wanting. When this is the case, the male
scatters the spermatozoa freely; the majority must perish, but some
will be carried by currents in the direction of the female.
When the sexes are separate, the female is frequently larger than
the male. This is markedly the case in Littorina, Buccinum, and all
the Cephalopoda; in Argonauta the difference is extreme, the male
not being more than ¼ the size of the female.
Those hermaphrodite Mollusca which are capable of sexual union
(Gasteropoda, Pulmonata, and Opisthobranchiata) are conveniently
divided into two sections, according as (1) there are separate orifices
for the male and female organs, or (2) one orifice serves for both. To
the former section (Digonopora[2]) belong the Limnaeidae,
Vaginulidae, and Onchidiidae, and many Opisthobranchiata,
including all the Pteropoda; to the latter (Monogonopora[255]) nearly
all the Nudibranchiate Opisthobranchiata, and all the rest of the
Pulmonata. In the latter case during union, mutual impregnation
takes place, and each of the two individuals concerned has been
observed (compare p. 42) to deposit eggs. In the former, however,
no such reciprocal act can take place, but the same individual can
play the part of male to one and female to another, and we
sometimes find a string of Limnaea thus united, each being at once
male and female to its two adjacent neighbours.
The Reproductive System.—Broadly speaking, the complicated
arrangements which are found in Mollusca resolve themselves into
modifications of three important factors:—
(a) The gonads or germ-glands, in which are developed the ova
and the spermatozoa. These glands are generally known as the
ovary in the female, the sperm-gland or testis in the male.
(b) The channels which provide for the passage of the seminal
products; namely, the oviduct in the female, the vas deferens or
sperm-duct in the male.
(c) The external generative organs.
Fig. 49.—Generative and other organs
of Littorina obtusata L., female.
A, anus.
Br, branchia.
Buc, buccal mass.
H, heart.
Hep, hepatic duct.
I, continuation of oesophagus.
Ki, kidney.
Li, liver.
M, muscle of attachment.
O´, female orifice.
Od, oviduct.
Oes, oesophagus.
Ov, ovary.
Ra, radula.
St, stomach.
U, uterus.

(After Souleyet.)
 Fig. 50.—Generative and other organs
of Littorina obtusata L., male.
A, anus.
Br, branchia.
H, heart.
I, intestine.
Li, liver.
M, muscle of attachment.
Pe, penis.
Te, testis.
VD, vas deferens.

(After Souleyet.)
Dioecious Mollusca.—The common Littorina obtusata will serve
as a typical instance of a dioecious prosobranchiate, exhibiting the
simplest form of organs. In the female the ovary, a lobe-shaped
body, is embedded in the liver. An oviduct with many convolutions
conveys the ova into the uterus, an oblong chamber which consists
simply of a dilatation of the oviduct. The ova descend into the uterus,
which is sometimes furnished with a seminal pouch. In this seminal
pouch, or above it, in the oviduct, the ova come into contact with the
spermatozoa. The lower part of the uterus secretes a gelatinous
medium (or capsule, as the case may be) in which the fertilised ova
become enclosed previous to exclusion. In position the oviduct abuts
on the kidney, while the uterus is in close proximity to the rectum,
and the female external orifice is found close to the anus, within the
branchial cavity.
The male organs of Littorina are more simple. The testis is
lodged, like the ovary, in the liver; the vas deferens is, like the
oviduct, convoluted, and eventually traverses the right side of the
neck, emerging near the right tentacle, and terminating in the penis
or external copulative organ (Fig. 50).
This system prevails, with but slight modifications in detail,
throughout the prosobranchiate Gasteropoda. The most important
modification is the passage of the seminal products in certain cases
(many of the Diotocardia) through the right kidney, with which the
oviduct and vas deferens always stand in close relation. The same
arrangement occurs in the Scaphoda and some Pelecypoda.
The penis varies greatly in form and size. In the Strombidae (see
Fig. 99) and Buccinidae (Fig. 62) it is very large and prominent; in
Littorina it is somewhat spinulose at one side; in Paludina a portion
of it is lodged in the right tentacle, which becomes atrophied and
much more obtuse than the tentacle on the left side.
Spermatozoa.—The shape of the spermatozoa and of the ova in
Mollusca is of the usual type. In Paludina Ampullaria, and certain
species of Murex two types of spermatozoa occur, one hair-like, the
other worm-like, three times as long as the former, and not tapering
at one end. The former type alone take part in fertilisation, and
penetrate the ovum. It has been suggested that these worm-like
spermatozoa are a kind of incipient ova, and indicate a possible
stage in commencing hermaphroditism. And, since the nearest allies
of the Prosobranchiata (in which these types occur) are
hermaphrodite (i.e. the Opisthobranchiata and Pulmonata), it is not
unreasonable to suppose that the Prosobranchiata should show
some tendency towards hermaphroditism in their genital glands.[256]
Cephalopoda.—The special characteristic of the reproductive
organs in female Cephalopoda is the development of various glands,
some of considerable size, in connexion with the ovary and oviduct.
Sepia, Loligo, and Sepiola are furnished with two large nidamental
glands, which open into the mantle cavity independently of the
oviduct. Their purpose is to produce a viscid mucus, which envelops
the ova at the moment of their emission and eventually hardens into
the egg-capsules. A pair of accessory nidamental glands occur in
Sepia, as well as a pair of smaller glands situated on the oviduct
itself.
In many of the male Cephalopoda the vas deferens is long and
dilated at its outer end into a glandular reservoir, within which are
formed the spermatophores, or narrow cylindrical packets which
contain a very large number of spermatozoa. When charged, the
spermatophores pass into what is known as Needham’s sac, where
they remain until required for use. These spermatophores are a very
characteristic part of the reproductive arrangements in the
Cephalopoda. The male of Sepia has been noticed to deposit them,
during union, upon the buccal membrane of the female. During the
emission of the ova by the female, the spermatophores, apparently
through the agency of a kind of spring contained at one end, burst,
and scatter the spermatozoa over the ova.
The Hectocotylus Arm.—Perhaps the most remarkable feature
in the sexual relations of all the Mollusca is the so-called
hectocotylus of the Cephalopoda. In the great majority of the male
Cephalopoda, one of the ‘arms,’ which is modified for the purpose in
various ways and to a greater or less extent, becomes charged with
spermatophores, and sometimes, during union, becomes detached
and remains within the mantle of the female, preserving for some
considerable time its power of movement.
The hectocotylus is confined to the dibranchiate Cephalopoda,
and its typical form, i.e. when part of the arm becomes disengaged
and left with the female, occurs only in three genera of the
Octopodidae, viz.[Argonauta, Ocythoe (Philonexis), and
Tremoctopus. In all of these, the male is many sizes smaller than the
female. In Argonauta the third arm on the left side becomes
hectocotylised. At first it is entirely enveloped in a kind of cyst, in
such a way that only a small portion of the tip projects; subsequently
the cyst parts asunder, and allows the arm to become expanded to
its full length, which considerably exceeds that of the other arms. At
a certain point the acetabula or suckers terminate, and the
remainder of the arm consists of a very long, tapering, sometimes
thread-like filament, which is pointed at the extreme tip. It is not yet
known how the spermatophores find their way into the hectocotylus,
or how the hectocotylus impregnates the ova of the female. The arm
thus affected is not always the same. In Tremoctopus it is the third of
the right side, in the Decapoda the modification usually affects the
fourth of the left.
 Fig. 51.—Male of Ocythoe
tuberculata Raf. (= Philonexis
catenulatus Fér.),
Mediterranean, showing three
stages, A, B, and C, in the
development of the hectocotylus
arm: h.cy, hectocotylus still in
the cyst; c´y´, spoon-shaped cyst
at the end of the arm when
freed; th, thread-like organ freed
by the rupture of c´y´. Natural
size. From specimens in the
British Museum.
This singular property of the male Cephalopoda has only recently
been satisfactorily explained. It is true that Aristotle, more than
twenty-two centuries ago, distinctly stated that certain of the arms
were modified for sexual purposes. Speaking of what he calls the
polypus (which appears to represent the Octopus vulgaris of the
Mediterranean), he says: ‘It differs from the female in having what
the fishermen call the white sexual organ on its arm;’ again, ‘Some
say that the male has something of a sexual nature (αὶδοιῶδές τι) on
one of its arms, that on which the largest suckers occur; that this is a
kind of muscular appendage attached to the middle of the arm, and
that it is entirely introduced within the funnel of the female’.
Unfortunately the word translated by introduced is corrupt, and can
only be restored conjecturally. He again remarks, ‘The last of the
arms, which tapers to a fine point and is the only whitish arm, it uses
in sexual union.’[257]
The typical hectocotylus seems to have entirely escaped notice
until early in the present century, when both Delle Chiaje and Cuvier
described it, as detected within the female, as a parasite, the latter
under the name of Hectocotylus octopodis. Kölliker, in 1845–49,
regarded the Hectocotylus of Tremoctopus as the entire male
animal, and went so far as to discern in it an intestine, heart, and
reproductive system. It was not until 1851 that the investigations of
Vérany and Filippi confirmed a suggestion of Dujardin,[258] while H.
Müller, in 1853, completed the discovery by describing the entire
male of Argonauta.
In all genera of dibranchiate Cephalopoda except Argonauta,
Ocythoe, and Tremoctopus, one of the arms is sexually modified in
various ways, but never becomes so much prolonged, and is never
detached and left with the female. In Loligo Forbesii Stp. the fourth
arm on the left has 23 pairs of regularly developed acetabula, which
then lessen in size and disappear, being replaced by long
pedunculated papillae, of which there are about 40 pairs. In Loligo
vulgaris Lam. and L. Pleii Orb. 18 or 19 pairs of acetabula are
regularly formed, and then occur 40 pairs of papillae, as in Forbesii.
In other species of Loligo (gahi Orb., brevis Bl., brasiliensis Orb.)
only the outer row of suckers becomes modified into papillae after
about the 20th to the 22nd pair. In Sepioteuthis sepioides the
modification is the same as in the Loligo last mentioned, but the
corresponding arm on the right side is so covered with acetabula
towards its extreme end, that it is thought that it in some way co-
operates with the hectocotylised left arm.
In Octopus, the third arm on the right side is subject to
modification. This arm is always shorter than the corresponding arm
on the other side, and carries fewer suckers, but is furnished at the
extreme tip with a peculiar kind of plate, which connects with the
membrane at the base of the arm by a channel of skin, which
probably conveys the spermatophores up to the tip.
In Octopus vulgaris, the species referred to by Aristotle, the
hectocotylised arm is short, thin in its outer half and pointed at the
extremity, while the fold of skin is very white, and gives the arm an
appearance of being divided by a cleft at the side. At the same time,
an unusual development of one or two suckers on the arm is not
uncommon.[259]

Fig. 52.—Octopus lentus Baird, N. Atlantic,

showing the peculiar formation of the
hectocotylus arm, h.a. (After Verrill, × ½.)
It is believed that in the Tetrabranchiate Cephalopoda (Nautilus) a
union of the four inner ventral arms may correspond functionally to
the hectocotylising of the arm in the Dibranchiates.
Hermaphrodite Mollusca.—(a) Monogonopora.—The
reproductive system in the hermaphrodite Mollusca is far more
complicated than in the dioecious, from the union of the male and
female organs in the same individual. As a type of the