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Cell cycle gene alterations in 4864 tumors analyzed by next generation

sequencing: Implications for targeted therapeutics

Authors

Teresa Helsten1*, Shumei Kato2*, Maria Schwaederle1, Brett N. Tomson3, Timon P.H.

Buys3, Sheryl K. Elkin3, Jennifer L. Carter3, Razelle Kurzrock1

*These authors contributed equally

Facility

1Center for Personalized Cancer Therapy, UC San Diego Moores Cancer Center, La Jolla, CA

2Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX

3N-of-One®, Inc., Lexington, MA

Corresponding author’s contact information:

Shumei Kato, M.D. Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center 1400 Holcombe Blvd, Houston, TX 77030 Email: [email protected] Phone: 713-795-9246 Fax: 713-745-3885 Running head:

Cell cycle gene aberrations in cancer

Conflicts of Interest and financial disclosure: Dr. Kurzrock has research funding from Genentech, Pfizer, Merck Serono and Foundation Medicine, consultant fees from Sequenom, and ownership interest in RScueRx. Drs. Tomson, Buys, Elkin and Carter are employees of N-of-One®, Inc., which is a for-profit company.

Acknowledgments:

R Kurzrock is funded in part by the Joan and Irwin Jacobs philanthropic fund.

on March 6, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 11, 2016; DOI: 10.1158/1535-7163.MCT-16-0071

http://mct.aacrjournals.org/

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ABSTRACT

Alterations in the cyclin-dependent kinase (CDK)-retinoblastoma (Rb) machinery

disrupt cell cycle regulation and are being targeted in drug development. In order to

understand the cancer types impacted by this pathway, we analyzed frequency of

abnormalities in key cell cycle genes across 4864 tumors using next-generation

sequencing (182 or 236 genes) (Clinical Laboratory Improvement Amendments

[CLIA] laboratory). Aberrations in the cell cycle pathway were identified in 39% of

cancers, making this pathway one of the most commonly altered in cancer. The

frequency of aberrations was as follows: CDKN2A/B (20.1% of all patients), RB1

(7.6%), CCND1 (6.1%), CCNE1 (3.6%), CDK4 (3.2%), CCND3 (1.8%), CCND2

(1.7%), and CDK6 (1.7%). Rates and types of aberrant cell cycle pathway genes

differed between cancer types and within histologies. Analysis of co-existing and

mutually exclusive genetic aberrations showed that CCND1, CCND2 and CCND3

aberrations were all positively associated with CDK6 aberrations (odds ratio [OR] and

p-values, multivariate analysis: CCND1 and CDK6 [OR: 3.5, p-value: <0.0001];

CCND2 and CDK6 [OR: 4.3, p-value: 0.003]; CCND3 and CDK6 [OR: 3.6, p-value:

0.007]). In contrast, RB1 alterations were negatively associated with multiple gene

anomalies in the cell cycle pathway including: CCND1 (OR: 0.25, p-value: 0.003);

CKD4 (OR: 0.10, p-value: 0.001); and CDKN2A/B (OR: 0.21, p-value: <0.0001). In

conclusion, aberrations in the cell cycle pathway were very common in diverse

cancers (39% of 4864 neoplasms). The frequencies and types of alterations differed

between and within tumor types, and will be informative for drug development

strategies.




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INTRODUCTION

The cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma (Rb) pathway is

a key gatekeeper for the G1 phase of the cell cycle. Aberrations in the cell cycle

pathway have been implicated in human cancer that leads to tumor proliferation,

chromosomal instability, and attenuation of genomic integrity (1-3).

The Rb tumor suppressor protein is crucial in regulating the G1 phase. The G1

phase is also controlled by the cyclin D and CDK4/6 complex, leading to

phosphorylation of the Rb protein and subsequent E2F-mediated transcription of

target genes that are required for G1 cell-cycle progression (4-6). Recent literature

suggests that the cyclin D1 and CDK4/6 complex adds just one phosphate group to

the Rb (out of fourteen different phosphate binding sites) and subsequent phosphate

groups are added by the cyclin E and CDK2 complex, to further hyperphosphorylate

the Rb protein before entering into the next phase of cell cycle (7) (Figure 1). This

pathway can be altered through multiple mechanisms including loss of RB1, increased

signaling through CDK4/6 amplification, aberrations in CCND1/CCNE1, or

inactivation of pathway inhibitors including CDKN2A/B (5, 6).

Alterations in the cell cycle pathway have been described in multiple cancers

(8-12) and are associated with poorer clinical outcome in some tumor types (13-15)

including acute lymphoblastic leukemia (9), ovarian (10), colon cancer (12), and

medulloblastoma (11).

To overcome cyclin pathway abnormalities in cancers, there are several

inhibitors in clinical trials, with varying selectivity for specific members of the CDK

family (2). Among them, palbociclib (PD0332991), a potent and selective CDK4/6

inhibitor, demonstrated prolonged progression-free survival (PFS) in patients with

hormone receptor-positive, human epidermal growth factor receptor 2 (Her2) negative




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metastatic breast cancer, in combination with the hormone modulator letrozole, as

compared to letrozole alone (PALOMA-1 trial, median progression-free survival of

20.2 versus 10.2 months, respectively [p-value = 0.0004]) (16). These observations

led to accelerated Food and Drug Administration (FDA) approval in February 2015.

Additional CDK4/6 inhibitors are in clinical trials including LEE011(17), abemaciclib

(LY-2835219) (4) and the CDK1/2/5/9 inhibitor dinaciclib (MK-7965, formerly SCH-

727965) (18, 19).

With novel promising CDK inhibitors in clinical trials (4, 16-19), the

comprehensive analysis of cell cycle gene aberrations amongst diverse cancer types is

of interest. Next-generation sequencing (NGS) technology makes rapid and accurate

identification of these aberrations feasible. Herein, we report the molecular

characteristics of CCND1, CCND2, CCND3, CDK4/6, CCNE 1, CDKN2A/B and RB1

in 4864 patients with diverse cancers interrogated by NGS.




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MATERIALS AND METHODS

Patients

We investigated the CCND1/2/3, CDK4/6, CCNE 1, CDKN2A/B and RB1 aberration

status of patients with diverse malignancies that were referred for NGS from

December 2011 to November 2013 (N=4864) (Table 1 and Supplemental Table 1

and 2).

Tissue samples and mutational analysis

We collected sequencing data from 4864 cancers whose formalin-fixed, paraffin-

embedded (FFPE) tumor samples were submitted to a clinical laboratory

improvement amendments (CLIA)-certified lab for genomic profiling (Foundation

Medicine, Cambridge, MA). Samples were required to have a surface area ≥ 25 mm2,

volume ≥ 1 mm3, nucleated cellularity ≥ 80%, and tumor content ≥ 20% (20). The

methods used in this assay have been validated and previously reported (20-22).

Briefly, 50-200ng of genomic DNA was extracted and purified from the submitted

formalin-fixed paraffin-embedded (FFPE) tumor samples. This whole-genome DNA

was subjected to shotgun library construction and hybridization-based capture before

paired-end sequencing on the Illumina HiSeq2000 platform. Hybridization selection

is performed using individually synthesized baits targeting the exons of 182 or 236

cancer-related genes and the introns of 14 or 19 genes frequently re-arranged in

cancer. Sequence data were processed using a customized analysis pipeline (20).

Sequencing was performed with an average sequencing depth of coverage greater

than 250x, with > 100x at > 99% of exons. This method of sequencing allows for

detection of copy number alterations, gene rearrangements, and somatic mutations

with 99% specificity and > 99% sensitivity for base substitutions at ≥ 5 mutant allele




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frequency and > 95% sensitivity for copy number alterations. A threshold of ≥ 8

copies for gene amplification with >6 copies considered equivocal (except for ERRB2,

which is considered equivocally amplified with ≥ 5 copies) was used. Synonymous

mutations were not included in the analysis. Also of note, promoter/enhancer regions

for the CDKN2A/B and RB1 loci were not interrogated and only coding regions were

evaluated in the current report. The submitting physicians provided specification of

tumor types. The database was de-identified with only diagnosis available. NGS data

were collected and interpreted by N-of-One®, Inc. (Lexington, MA). For this study,

the dataset of 4864 sequenced tumors was queried for alterations in CCND1, CCND2,

CCND3, CDK4/6, CCNE 1, CDKN2A/B and RB1 genes. This study and data analysis

herein was performed in accordance with UCSD IRB guidelines.

cBio Cancer Genomics Portal data

Publically availfoable data sets containing genomic information from a diverse array

of cancer types were investigated for cell cycle pathway genes (CCND1, CCND2,

CCND3, CDK4/6, CCNE 1, CDKN2A/B and RB1) using cBio Cancer Genomics

Portal data (cBioPortal) (http://cbioportal.org, March 2016) to compare with current

study (23, 24) (see Supplemental Methods for additional information).

Endpoints and statistical methods

Descriptive statistics were used to summarize the baseline patient characteristics. The

Fisher’s exact test was used to assess the association between categorical variables.

All tests were 2-sided. Statistical analyses were carried out using SPSS version 22.0

(Chicago, IL, USA).




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RESULTS

Overview of frequency of alterations in the key cell cycle pathway genes (CCND1,

CCND2, CCND3, CDK4/6, CDKN2A/B, CCNE1 and RB1) (Table 1, Figure 2)

Amongst the 4864 patients, the most common cancers were breast (10.4%)

and lung adenocarcinoma (8.4%) (Table 1). Thirty-nine percent of patients had at

least one cell cycle pathway anomaly. Amongst 1918 patients with aberrant cell cycle

pathway genes, CDKN2A/B alterations were the most common (51.0% [978/1918]),

followed by aberrations in RB1 (19.2% [369/1918]), CCND1 (15.4% [295/1918]),

CCNE1 (9.2% [176/1918]) and CDK4 (8.1% [156/1918]). CCND3 (4.5% [86/1918]),

CCND2 (4.4% [84/1918]) and CDK6 (4.4% [84/1918]) were less common. Fourteen

percent (274/1918) of patients had more than one abnormality in these cell cycle

pathway genes (Table 1 and Figure 2).

Overview of cancer diagnosis and abnormalities in cell cycle pathway genes

Aberrations in the cell cycle pathway were most frequently detected among

glioblastoma (73.8% [62/84]), followed by those with squamous cell carcinoma

(SCC) of the esophagus (71.4% [15/21]) and transitional cell carcinoma of the bladder

(67.8% [61/90]). Papillary thyroid carcinoma was less frequently associated with cell

cycle aberrations (9.5% [2/21]) (Figure 3 and Table 1). Overall, the frequencies of

cell cycle pathway aberrations among cancer diagnoses from our current report were

similar to those found in the cBioPortal data (Figure 4).

Distribution of specific gene alterations

CCND1: CCND1 amplification was seen in 6.1% of patients (295/4864). CCND1

amplification was most commonly identified in patients with SCC of esophagus




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(42.9% [9/21]) (Supplemental Figure 1.A and Supplemental Table 1 and 2).

Overall, the results from the cBioPortal data were similar, with 7.8% (466/6009) of

cancer cases having CCND1 aberrations (Supplemental Figure 2.A).

CCND2: CCND2 aberrations were documented in only 1.7% (84/4864) of all cancer

types. The most common alteration was amplification (96.4% [81/84]); mutations

were infrequent (3.6% [3/84]). CCND2 aberrations were most commonly seen in

patients with adrenal carcinoma (11.5% [3/26]) (Supplemental Figure 1.B and

Supplemental Table 1 and 2). This data is consistent with the data derived from the

cBioPortal, where CCND2 alterations across all cancers were also infrequent, being

aberrant in 2.5% (152/6009) of cases, including a few cases of deletion (0.3%

[20/6009]) (Supplemental Figure 2.B).

CCND3: CCND3 aberrations were seen in 1.8% (86/4864) of all cancer types;

amplification (94.2% [81/86]), mutations (3.5% [3/86]) and fusions (2.3% [2/86])

were observed. CCND3 anomalies were most commonly found in esophageal

adenocarcinoma (11.6% [8/69]). (Supplemental Figure 1.C and Supplemental

Table 1 and 2). According to cBioPortal, CCND3 was aberrant in 2.5% (149/6009)

of cancer cases, with 7.5% (14/186) of esophageal adenocarcinoma harboring CCND3

aberration (Supplemental Figure 2.C).

CDK4: CDK4 amplification was seen in 3.2% (156/4864) of all cancer types. CDK4

amplification was most commonly found in neuroblastoma (17.9% [5/28]) followed

by glioblastoma (11.9% [10/84]) (Supplemental Figure 1.D and Supplemental

Table 1 and 2). According to the cBioPortal, CDK4 aberrations were also seen in




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3.2% (191/6009) of cancer cases (Supplemental Figure 2.D). Since neuroblastoma

data in cBioPortal only reflects mutations and not amplification, we are unable to

directly compare cBioPortal alterations to our incidence of CDK4 amplification in

neuroblastoma. However, CDK4 amplification was seen in 16.7% (47/281) of

glioblastoma cases, which is similar to the incidence in our cases (Supplemental

Figure 2.D).

CDK6: CDK6 amplification was seen in 1.7% (84/4864) of all malignancies, with the

most common tumor type affected being gastroesophageal junction carcinoma (20.7%

of patients [6/29]) followed by esophageal adenocarcinoma (11.6% [8/69])

(Supplemental Figure 1.E and Supplemental Table 1 and 2). This was consistent

with cBioPortal data, where CDK6 is aberrant in 2.7% (165/6009) of cancer cases,

including CDK6 amplification in 14.5% (27/186) of esophageal adenocarcinoma

(Supplemental Figure 2.E).

CDKN2A/B: CDKN2A/B aberrations were seen in 20.1% (978/4864) of patients.

These aberrations were most commonly CDKN2A/B loss (63.4% [620/978]) followed

by CDKN2A alterations (36.6% [358/978]). Amongst the 358 samples analyzed for

CDKN2A alteration, 49.4% (177/358) harbored at least one frameshift or nonsense

alteration, 30.2% (108/258) harbored at least one structural alteration (including loss:

20.1% [72/358], splice site alterations: 5.9% [21/358], partial loss: 2.2% [8/358],

indel: 1.1% [4/358] and rearrangement: 0.8% [3/358]) and 25.1% (90/358) harbored

at least one missense alteration (including inactivating alterations: 15.9% [57/358],

variant of unknown significance: 8.7% [31/258] and no effect: 0.6% [2/358])

(Supplemental Figure 1.F and Supplemental Table 1 and 2). As opposed to




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CDKN2A/B aberrations, CDKN2C alterations were rare, seen in only 0.25% (12/4864)

of cases (Supplemental Table 2). According to the cBioPortal, CDKN2A/B was

aberrant in 18.0% (1084/6009) of cases, with 44.8 % (13/29) of cutaneous SCC

harboring CDKN2A/B aberrations; these data are similar to the observations in the

current report (Supplemental Figure 2.F).

CCNE1: CCNE1 was amplified in 3.6% (176/4864) of patients with all cancer types.

CCNE1 amplifications were most frequent in those with ovarian epithelial carcinoma

(15.1% [8/53]) and adenocarcinoma of esophagus (14.5% [10/69]) (Supplemental

Figure 1.G and Supplemental Table 1 and 2). According to cBioPortal, CCNE1

aberrations were also seen in 5.0% (303/6009) of cancer cases (Supplemental Figure

2.G).

RB1: Aberrations in the RB1 tumor suppressor gene were documented in 7.6%

(369/4864) of all cancer types. Amongst 369 cases with RB1 alteration, 53.9%

(199/369) harbored at least one nonsense or frameshift alteration, 45.3% (167/369)

harbored at least one structural alteration (including loss: 29.0% [107/369], partial

loss: 1.9% [7/369], rearrangement: 0.5% [2/369], partial duplication: 0.5% [2/369],

amplification: 0.3% [1/369], and splice site alterations: 13.0% [48/369]), and 2.7%

(10/369) harbored at least one missense alteration (including inactivating alterations:

1.6% [6/369] and variants of unknown significance: 1.1% [4/369]). RB1 aberrations

were commonly identified in patients with small cell lung cancer (60.5% [26/43])

(Supplemental Figure 1.H and Supplemental Table 1 and 2). The data in our

current report is similar to the incidence reported in cBioPortal, where 7.6%

(456/6009) of cancer cases have RB1 aberrations. Consistent with our findings, small




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cell lung cancer is the diagnosis most commonly associated with RB1 aberration in

the cBioPortal (74.5% [82/110]) (Supplemental Figure 2.H).

Distribution of specific gene alterations by cancer type

Breast cancers: Forty percent of patients (203/507) with breast cancer had alterations

in cell cycle pathway genes; these alterations were strikingly common in the small

subset of patients with metaplastic breast carcinoma (70.6% [12/17]) (Table 1 and

Supplemental Table 1). Metaplastic breast cancer was more commonly associated

with alterations in RB1 (23.5% [4/17]) as opposed to 7.5% [38/507] in other breast

cancers. CDKN2A/B aberrations were also more commonly seen in patients with

metaplastic breast cancer (23.5% [4/17]) when compared to those with other breast

cancers (7.9% [40/507]) (Table 1 and Supplemental Table 1).

Lung cancers: Aberrations in cell cycle elements were relatively common in patients

with non-small cell lung cancer (NSCLC) (adenocarcinoma, 41.8% [171/409]; SCC,

58.7% [54/92]; large cell carcinoma, 60.0% [18/30]) and small cell lung cancer

(SCLC) (62.8% [27/43]). Alterations in RB1 were more commonly associated with

SCLC (60.5% [26/43]) as opposed to NSCLC (5.4-16.7%) (Table 1). On the other

hand, CDKN2A/B aberrations were more commonly observed in NSCLC (24.0-

43.5%) when compared to SCLC (4.7% [2/43]) (Table 1).

Central nervous system (CNS) tumors: Amongst CNS tumors, glioblastoma was

most frequently associated with aberrations in cell cycle pathway genes (73.8% of

patients [62/84]), followed by anaplastic astrocytoma (50% [11/22]), glioma (38.5%

[5/13]), oligodendroglioma (35.3% [6/17]), astrocytoma (26.1% [6/23]) and




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meningioma (22.2% [4/18]). Among patients with CNS tumors, CCND1 aberrations

were only discerned in meningioma (5.6% [1/18]). Alterations in RB1 were more

commonly detected in glioblastoma (11.9% [10/84]) and oligodendroglioma (11.8%

[2/17]), and were less regularly observed in astrocytoma (4.3% [1/23]); they were not

detected in individuals with anaplastic astrocytoma, glioma and meningioma (Table 1

and Supplemental Table 1).

Squamous cell carcinoma (SCC): Amongst SCC histology in various primary cancer

types, esophageal SCC was most commonly associated with aberrations in cell cycle

pathway genes (71.4% [15/21]). Cervical SCC was the least frequently associated

with molecular anomalies in cell cycle pathway genes (22.7% [5/22]) (Table 1 and

Supplemental Table 1).

Adenocarcinoma: Amongst adenocarcinomas, esophageal adenocarcinoma was most

commonly associated with alterations in the cell cycle pathway (56.5% [39/69]) while

such alterations were least common in colorectal cancer (10.1% [30/298]) (Table 1

and Supplemental Table 1).

Co-existing genomic aberrations amongst cell cycle gene aberrations

We investigated if certain cell cycle gene aberrations were correlated with

one another using the Fisher’s exact test and multivariate logistic regression analysis

(Figure 5 and Supplemental Table 3). CCND1, CCND2, and CCND3 aberrations

were all positively associated with CDK6 aberrations (odds ratio [OR] and p-values

after multivariate analysis: CCND1 and CDK6 [OR: 3.5, p-value: <0.0001]; CCND2

and CDK6 [OR: 4.3, p-value: 0.003]; CCND3 and CDK6 [OR: 3.6, p-value: 0.007])




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(Figure 5 and Supplemental Table 3.E.). On the other hand, RB1 mutations/loss

were negatively associated with multiple aberrations in the cell cycle pathway

including CCND1 (OR: 0.25, p-value: 0.003), CKD4 (OR: 0.10, p-value: 0.001) and

CDKN2A/B (OR: 0.21, p-value: <0.0001) (Figure 5 and Supplemental Table 3.H.).




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DISCUSSION

This study represents a comprehensive overview of aberrations in relevant cell

cycle pathway genes in a large number of cancer tissues interrogated by NGS

(N=4864). Overall, 39.4% of cancers (1918/4864) had alterations in the key cell cycle

pathway genes, making the CDK/CCND/RB1 axis one of the most frequently

disturbed in cancer (Figure 2). Our findings were also comparable to the data from

cBioPortal (23, 24) where 38.7 % (2326/6009) of cases harbored aberrations in these

cell cycle pathway genes (Figure 4 and Supplemental Figures 2).

The frequency of cell cycle pathway aberrations varies by disease, with

glioblastoma having the highest rate of abnormalities (73.8%), while colorectal

adenocarcinoma has a much lower rate (10.1%), consistent with previous reports on

smaller numbers of patients (Figure 3) (4, 8, 13).

Certain cancer diagnoses were significantly associated with different cell cycle

pathway aberrations. For example, esophageal SCC was highly associated with

CCND1 aberrations (OR: 14.5, p-value: <0.0001); gastroesophageal junction

carcinoma, with CDK6 aberration (OR: 19.7, p-value: <0.0001); and small cell lung

cancer, with RB1 aberration (OR: 21, p value <0.0001) (Supplemental Table 3).

The selective CDK4/6 inhibitor palbociclib has been FDA-approved in

hormone receptor-positive, Her2-negative metastatic breast cancer. To date, a

correlation between cell cycle gene aberrations and response of breast cancer to

palbociclib has not been shown; however the question remains of interest. Up-

regulation of CDK4/6 may occur via CDK4 or CDK6 amplification, inactivation of

CDKN2A/B, or CCND1 amplification, which subsequently augments phosphorylation

and inactivation of RB1 (4, 5). Meanwhile, resistance to CDK4/6 inhibitors may be




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mediated by RB1 or CCNE1 aberrations, which subsequently leads to activation of

E2F (25, 26).

In our dataset, amongst cancers originating from breast, aberrations in cell

cycle pathway were more commonly seen in patients with metaplastic breast cancer

when compared to breast cancer patients in general (70.6% [12/17] versus 40.0%

[203/507]) (Table 1 and Supplemental Table 1). Metaplastic breast cancer is a rare

subtype that is typically negative for either estrogen/progesterone receptors or Her2

(27) and thus is usually treated as triple-negative breast cancer. However, women

with metaplastic breast cancer experience detrimental clinical outcomes when

compared to patients with triple-negative breast cancer, (28) and thus there is no

standard therapy for metaplastic breast cancer. However, recent data suggest

susceptibility to a combination of liposomal doxorubicin, bevacizumab and

temsirolimus, perhaps because these tumors also commonly harbor PI3K/AKT/mTOR

pathway aberrations (27, 29, 30). Our results suggest that, for patients with

metaplastic breast cancer, an aberrant cell cycle pathway may be a potential

therapeutic target to be considered, with the caveat being that about one-third of the

anomalies were in the CCNE1 and RB1 gene, which would confer resistance to

CDK4/6 inhibitors.

Amongst patients with lung cancer, frequencies of aberrations in cell cycle

pathway genes were similar between NSCLC (adenocarcinoma, 41.8% [171/409];

squamous cell carcinoma, 58.7% [54/92]; large cell carcinoma, 60.0% [18/30]) and

SCLC (62.8% [27/43]) (Table 1). However, RB1 aberrations were a hallmark of

SCLC (60.5% [26/43]) as opposed to NSCLC (5.4-16.7%). In contrast, CDKN2A/B

aberrations were more common amongst NSCLC (24.0-43.5%) when compared to




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SCLC (4.7%) (Table 1), consistent with previous reports (31-33). It is interesting to

note that both SCC of lung and SCLC are associated with tobacco exposure, yet the

aberration pattern in cell cycle genes are different.

We have also evaluated if the same histology amongst different cancer types

share similar patterns of aberrations (Table 1 and Supplemental Table 1). When

focusing on cancer with SCC or adenocarcinoma histologies in various cancer types,

aberrations in cell cycle elements occurred frequently, but rates still differed (Table

1). These results suggest that histologic classification may be further sub-stratified (34). Of interest in this regard, head and neck and other tumors infected with human

papilloma virus (HPV) do not generally exhibit cell cycle pathway abnormalities,

while HPV-negative tumors do harbor these alterations, which are often associated

with TP53 mutations (35).

Understanding the mutual exclusivity among some genomic aberrations has

been therapeutically important. For example, ALK rearrangements, EGFR or KRAS

mutations are mutually exclusive in patients with NSCLC (36). We have reported

here a comprehensive analysis of co-existing genetic aberrations and mutually

exclusiveness amongst cell cycle gene aberrations. We observed that RB1 alterations

were significantly less frequently associated with CCND1 (OR: 0.25, p-value: 0.003),

CDK4 (OR: 0.10, p-value: 0.001) and CDKN2A/B (OR: 0.21, p-value: <0.0001)

aberrations (Figure 5 and Supplemental Table 3.H). Aberrations in CDKN2A/B

were also less associated with CDK4 aberrations (OR: 0.20, p-value: <0.0001)

(Figure 5 and Supplemental Table 3.F). These data are consistent with previous

smaller studies reporting that CDKN2A, CDK4 and RB1 were mutually exclusive in

glioblastomas, and CDKN2A, CDK6 and RB1 were mutually exclusive in lung

adenocarcinomas (37). In contrast, we have observed that CDK4/6 aberrations were




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positively associated with cyclin D abnormalities (Figure 5 and Supplemental

Tables 3.A-E). The latter observations support the notion that CDK4/6 and cyclin D

cooperate closely for cell cycle progression. However, RB1 or CDKN2A/B

aberrations alone may be sufficient to perturb cell cycle progression.

There are several limitations to these data. First, the dataset was not clinically

annotated; hence, correlation with phenotypic characteristics and outcome was not

feasible. Second, methylation status (which could change gene expression) and

correlation between gene amplification and protein expression were not evaluated in

this study. Since we have not evaluated normal tissues, the impact of germline

mutations is not addressed. Similarly, as multiple samples were not analyzed from

individual cases, we were unable to evaluate intratumoral heterogeneity. Third, the

number of cases with each malignancy relied on the number of specimens submitted

by physicians for molecular profiling; this introduces the possibility of sample size

bias. In this regard, it is important to note that the data set only included small

numbers of hematologic malignancies (Supplemental Table 1). This is important

because cell cycle genes may be affected in them as well. For instance, CCND1 (also

known as BCL1) is rearranged in about 60 percent of mantle cell lymphomas (38),

and translocations are also seen in about 15-20% of multiple myeloma specimens (39)

Interestingly, a preliminary study showed activity for the CDK4/6 inhibitor in mantle

cell lymphoma (40). Further study of this family of genes is needed in hematologic

malignancies. Fourth, other genes including CDKN2D, RBL1, RBL2, E2F, CCNE2,

CCNA1, CCNA2 and CDK2 that are known to influence cell cycle pathway were not

evaluated in the current report, and further investigation is required as to their

impact. A final limitation of the data was that diagnosis was determined based on the




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attending physician designation. Yet despite these limitations, this study provides a

very large and comprehensive analysis of cell cycle gene alterations in a wide-range

of human cancers.

In conclusion, we have interrogated 4864 patients with cancer, and

demonstrated that aberrations in cell cycle pathway genes are extremely common,

being discerned in 39% of tissues across cancer types (Figures 2 and 3). However,

the frequencies and the types of aberrant cell cycle pathway differ between and within

cancer types. Of interest in this regard, we and others have previously shown that

patients with metastatic cancer mostly have distinct molecular portfolios, highlighting

the heterogeneity of cancer, and that matching patients with genomically targeted

therapy can have salutary effects (41-47). Aberrations in the cell cycle pathway may

also have prognostic value. For instance, it has been reported that cell cycle pathway

gene alterations are independently associated with poor clinical outcomes including

overall survival (13-15). Further study will be required to elucidate the impact of each

of the diverse cell cycle pathway genes and their correlation to response with the

various inhibitors being deployed in the clinic, including but not limited to palbociclib

(PD0332991) (16), LEE011(17), abemaciclib (LY-2835219) (4) and the CDK1/2/5/9

inhibitor, dinaciclib (MK-7965, formerly SCH-727965) (18, 19). Based on the

frequent finding of aberrations in the cell cycle pathway across diverse malignancies,

prosecution of this pathway in multiple tumor types merits further exploration.




19

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22

Table 1: Summary of Cell Cycle Gene Aberrations By Tumor Type: Primary cancer diagnosis with N ≥ 20*

Tumor Type Any Alteration

CCND1 CCND2 CCND3 CDK4 CDK6 CDKN2A/B CCNE1 RB1

Breast carcinoma (N=507)

203 (40.0%) 87 (17.2%) 13 (2.6%) 15 (3.0%) 12 (2.4%) 10 (2.0%) 40 (7.9%) 28 (5.5%) 38 (7.5%)

Lung adenocarcinoma (N=409)

171 (41.8%) 14 (3.4%) 5 (1.2%) 7 (1.7%) 19 (4.6%) 4 (1.0%) 98 (24.0%) 20 (4.9%) 27 (6.6%)

Sarcoma (N=348) 186 (53.4%) 9 (2.6%) 6 (1.7%) 13 (3.7%) 40 (11.5%) 0 (0%) 70 (20.1%) 14 (4.0%) 61 (17.5%)

Colorectal adenocarcinoma (N=298)

30 (10.1%) 2 (0.7%) 8 (2.7%) 4 (1.3%) 1 (0.3%) 2 (0.7%) 6 (2.0%) 3 (1.0%) 8 (2.7%)

Carcinoma of unknown primary (N=269)

112 (41.6%) 10 (3.7%) 3 (1.1%) 3 (1.1%) 6 (2.2%) 5 (1.9%) 70 (26.0%) 7 (2.6%) 23 (8.6%)

Ovarian serous carcinoma (N=169)

45 (26.6%) 2 (1.2%) 5 (3.0%) 6 (3.6%) 2 (1.2%) 1 (0.6%) 7 (4.1%) 20 (11.8%) 6 (3.6%)

Pancreatic ductal adenocarcinoma (N=159)

65 (40.9%) 2 (1.3%) 4 (2.5%) 2 (1.3%) 2 (1.3%) 3 (1.9%) 49 (30.8%) 5 (3.1%) 4 (2.5%)

Melanoma (N=135) 62 (45.9%) 5 (3.7%) 1 (0.7%) 2 (1.5%) 4 (3.0%) 1 (0.7%) 49 (36.3%) 0 (0%) 6 (4.4%)

Gastric adenocarcinoma (N=134)

47 (35.1%) 10 (7.5%) 1 (0.7%) 4 (3.0%) 3 (2.2%) 8 (6.0%) 22 (16.4%) 6 (4.5%) 2 (1.5%)

Non-small cell lung carcinoma (NSCLC) (N=124)

47 (37.9%) 7 (5.6%) 0 (0%) 0 (0%) 2 (1.6%) 3 (2.4%) 27 (21.8%) 2 (1.6%) 13 (10.5%)

Cholangiocarcinoma (N=114)

36 (31.6%) 3 (2.6%) 1 (0.9%) 2 (1.8%) 2 (1.8%) 4 (3.5%) 27 (23.7%) 2 (1.8%) 2 (1.8%)

Head and neck squamous cell carcinoma (HNSCC) (N=107)

59 (55.1%) 26 (24.3%) 1 (0.9%) 0 (0%) 1 (0.9%) 5 (4.7%) 47 (43.9%) 0 (0%) 5 (4.7%)

Lung squamous cell carcinoma (N=92)

54 (58.7%) 9 (9.8%) 4 (4.3%) 1 (1.1%) 1 (1.1%) 4 (4.3%) 40 (43.5%) 2 (2.2%) 5 (5.4%)

Bladder urothelial (transitional cell) carcinoma (N=90)

61 (67.8%) 18 (20.0%) 0 (0%) 3 (3.3%) 5 (5.6%) 2 (2.2%) 32 (35.6%) 1 (1.1%) 14 (15.6%)

Glioblastoma (N=84) 62 (73.8%) 0 (0%) 1 (1.2%) 0 (0%) 10 (11.9%) 4 (4.8%) 45 (53.6%) 0 (0%) 10 (11.9%)

Endometrial adenocarcinoma (N=79)

18 (22.8%) 2 (2.5%) 0 (0%) 1 (1.3%) 1 (1.3%) 1 (1.3%) 3 (3.8%) 7 (8.9%) 7 (8.9%)

Esophageal adenocarcinoma (N=69)

39 (56.5%) 9 (13.0%) 1 (1.4%) 8 (11.6%) 2 (2.9%) 8 (11.6%) 17 (24.6%) 10 (14.5%) 1 (1.4%)

Prostate adenocarcinoma (N=63)

14 (22.2%) 6 (9.5%) 0 (0%) 1 (1.6%) 2 (3.2%) 0 (0%) 4 (6.3%) 1 (1.6%) 2 (3.2%)

Renal cell carcinoma (N=58)

12 (20.7%) 2 (3.4%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 10 (17.2%) 0 (0%) 2 (3.4%)

Ovarian epithelial carcinoma (N=53)

20 (37.7%) 3 (5.7%) 3 (5.7%) 1 (1.9%) 1 (1.9%) 1 (1.9%) 8 (15.1%) 8 (15.1%) 1 (1.9%)

Head and neck adenoid cystic carcinoma (N=48)

6 (12.5%) 0 (0%) 0 (0%) 0 (0%) 1 (2.1%) 0 (0%) 4 (8.3%) 0 (0%) 1 (2.1%)

Gallbladder adenocarcinoma (N=45)

16 (35.6%) 1 (2.2%) 1 (2.2%) 1 (2.2%) 0 (0%) 0 (0%) 10 (22.2%) 5 (11.1%) 2 (4.4%)

Small cell lung carcinoma (N=43)

27 (62.8%) 1 (2.3%) 0 (0%) 1 (2.3%) 1 (2.3%) 0 (0%) 2 (4.7%) 1 (2.3%) 26 (60.5%)

Hepatocellular carcinoma (N=43)

13 (30.2%) 2 (4.7%) 0 (0%) 0 (0%) 1 (2.3%) 1 (2.3%) 6 (14.0%) 0 (0%) 5 (11.6%)




23

*Pathologic diagnosis per submitting physician

Tumor Type Any Alteration

CCND1 CCND2 CCND3 CDK4 CDK6 CDKN2A/B CCNE1 RB1

Neuroendocrine carcinoma (N=40)

18 (45.0%) 2 (5.0%) 3 (7.5%) 0 (0%) 1 (2.5%) 0 (0%) 3 (7.5%) 3 (7.5%) 7 (17.5%)

Mesothelioma (N=36) 16 (44.4%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 16 (44.4%) 1 (2.8%) 0 (0%)

Cutaneous squamous cell carcinoma (N=35)

23 (65.7%) 1 (2.9%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 19 (54.3%) 0 (0%) 3 (8.6%)

Squamous cell carcinoma (N=35)

19 (54.3%) 5 (14.3%) 2 (5.7%) 0 (0%) 0 (0%) 0 (0%) 15 (42.9%) 1 (2.9%) 1 (2.9%)

Salivary gland adenocarcinoma (N=31)

11 (35.5%) 2 (6.5%) 0 (0%) 1 (3.2%) 2 (6.5%) 0 (0%) 6 (19.4%) 0 (0%) 0 (0%)

Large cell lung carcinoma (N=30)

18 (60.0%) 2 (6.7%) 1 (3.3%) 0 (0%) 2 (6.7%) 0 (0%) 9 (30.0%) 1 (3.3%) 5 (16.7%)

Gastroesophageal junction carcinoma (N=29)

18 (62.1%) 4 (13.8%) 0 (0%) 0 (0%) 1 (3.4%) 6 (20.7%) 7 (24.1%) 4 (13.8%) 1 (3.4%)

GIST (Gastrointestinal stromal tumor) (N=29)

14 (48.3%) 0 (0%) 0 (0%) 0 (0%) 1 (3.4%) 0 (0%) 11 (37.9%) 0 (0%) 2 (6.9%)

Neuroblastoma (N=28)

10 (35.7%) 1 (3.6%) 0 (0%) 0 (0%) 5 (17.9%) 1 (3.6%) 2 (7.1%) 0 (0%) 1 (3.6%)

Adrenal carcinoma (N=26)

9 (34.6%) 0 (0%) 3 (11.5%) 0 (0%) 4 (15.4%) 0 (0%) 4 (15.4%) 0 (0%) 1 (3.8%)

Cervical adenocarcinoma (N=25)

5 (20.0%) 0 (0%) 0 (0%) 1 (4.0%) 0 (0%) 0 (0%) 2 (8.0%) 2 (8.0%) 0 (0%)

Head and neck carcinoma (N=24)

14 (58.3%) 5 (20.8%) 0 (0%) 1 (4.2%) 2 (8.3%) 0 (0%) 8 (33.3%) 1 (4.2%) 2 (8.3%)

Appendix adenocarcinoma (N=23)

8 (34.8%) 3 (13.0%) 1 (4.3%) 1 (4.3%) 0 (0%) 1 (4.3%) 1 (4.3%) 1 (4.3%) 1 (4.3%)

Anal squamous cell carcinoma (N=23)

7 (30.4%) 4 (17.4%) 0 (0%) 1 (4.3%) 0 (0%) 0 (0%) 2 (8.7%) 0 (0%) 1 (4.3%)

Astrocytoma (N=23) 6 (26.1%) 0 (0%) 0 (0%) 0 (0%) 1 (4.3%) 0 (0%) 4 (17.4%) 0 (0%) 1 (4.3%)

Clear cell renal cell carcinoma (N=23)

5 (21.7%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 5 (21.7%) 0 (0%) 0 (0%)

Anaplastic astrocytoma (N=22)

11 (50.0%) 0 (0%) 1 (4.5%) 0 (0%) 1 (4.5%) 1 (4.5%) 8 (36.4%) 0 (0%) 0 (0%)

Pancreatic neuroendocrine tumor (N=22)

9 (40.9%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 6 (27.3%) 1 (4.5%) 3 (13.6%)

Salivary gland carcinoma (N=22)

9 (40.9%) 0 (0%) 0 (0%) 0 (0%) 2 (9.1%) 1 (4.5%) 5 (22.7%) 0 (0%) 2 (9.1%)

Cervical squamous cell carcinoma (N=22)

5 (22.7%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 2 (9.1%) 0 (0%) 3 (13.6%)

Esophageal squamous cell carcinoma (N=21)

15 (71.4%) 9 (42.9%) 1 (4.8%) 0 (0%) 0 (0%) 1 (4.8%) 8 (38.1%) 0 (0%) 2 (9.5%)

Renal pelvis urothelial carcinoma (N=21)

12 (57.1%) 3 (14.3%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 8 (38.1%) 0 (0%) 2 (9.5%)

Papillary thyroid carcinoma (N=21)

2 (9.5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 2 (9.5%) 0 (0%) 0 (0%)




24

FIGURE LEGEND Figure 1. Function of cell cycle proteins and inhibitors. The Rb tumor suppressor protein plays a pivotal role in the negative control of the cell cycle. It is responsible for a major G1 checkpoint, blocking S-phase entry and cell growth. Phosphorylation leads to functional inactivation of Rb. Loss of Rb cell cycle suppressive functions can be mediated through multiple mechanisms: loss of RB1, increased signaling through CDK4 and CDK6 amplification, overexpression or aberration of cyclin D/E, and loss of the inhibitory function of gene products such as CDKN2A/B, the latter leading to CDK4/6 activity. Cyclin E1 and CDK2 complex further hyperphosphorylate Rb and facilitate G1-S progression. Cyclin dependent kinase inhibitors targeting CDK4/6 such as palbociclib, LY-2835219 and LEE011 as well as CDK1/2/5/9 inhibitors such as MK-7965 are under clinical investigation. Figure 2. Frequencies of alterations in cell cycle genes. Amongst 4864 patients with diverse malignancies, 1918 (39%) were found to have alterations in cell cycle pathway (CCND1/2/3, CDK4/6, CDKN2A/B, CCNE1 and Rb1). The most common aberration was in CDKN2A/B (978/1918, 51%) followed by RB1 (369/1918, 19%), CCND1 (295/1918, 15%), CCNE1 (176/1918, 9%) and CDK4 (156/1918, 8%). CDK6 (84/1918, 4%), CCND2 (84/1918, 4%) and CCND3 (86/1918, 4%) alterations were relatively uncommon. Fourteen percent (274/1918) of patients had more than one aberration in the cell cycle pathway. Figure 3. Frequencies of cumulative cell cycle gene aberrations (CCND1/2/3, CDK4/6, CDKN2A/B, CCNE1 and RB1) by cancer type. Figure included primary cancer diagnosis with N ≥ 20. Cell cycle gene aberrations were most commonly seen in patients with glioblastoma (73.8%) and least common in patients with papillary thyroid carcinoma (9.5%). Figure 4: Cumulative cell cycle gene aberrations (CCND1/2/3, CDK4/6, CDKN2A/B, CCNE1 and RB1) between current report and cBioPortal. In the current report, 39.4% (1918/4864) of patient samples were found to have aberrations in cell cycle pathway genes, while it was found in 38.7% (2326/6009) from cBioPortal (http://cbioportal.org). Figure 5. Co-altered cell cycle genes. Amongst multiple cell cycle-associated gene aberrations, positive and negative relationships for co-alterations were seen. For example, there was a positive correlation between CDK6 and CCND3 aberrations, with odds ratio of 4.5. In contrast, there was a negative correlation (odds ratio of 0.15) between RB1 and CDK4 anomalies. Solid black boxes indicate the association was statistically significant after multivariate analysis (Supplemental Tables 3).




Figure 1. Cell cycle proteins and inhibitors




Figure 2: Frequencies of alterations in cell cycle genes.




Figure 3: Frequencies of cumulative cell cycle gene (CCND1/2/3, CDK4/6, CDKN2A/B, CCNE1 and RB1) aberrations by cancer

type.

9.5% 10.1% 12.5%

20.0% 20.7% 21.7% 22.2% 22.7% 22.8%

26.1% 26.6%

30.2% 30.4% 31.6%

34.6% 34.8% 35.1% 35.5% 35.6% 35.7%

37.7% 37.9%

40.0% 40.9% 40.9% 40.9% 41.6% 41.8%

44.4% 45.0% 45.9%

48.3% 50.0%

53.4% 54.3%

56.2% 56.5% 57.1% 58.3% 58.7% 60.0%

62.1% 62.8%

65.7% 67.8%

71.4% 73.8%

39.4%

0% 10% 20% 30% 40% 50% 60% 70% 80%

Papillary thyroid carcinomaColorectal adenocarcinoma

Head and neck adenoid cystic carcinomaCervical adenocarcinoma

Renal cell carcinomaClear cell renal cell carcinoma

Prostate adenocarcinomaCervical squamous cell carcinoma

Endometrial adenocarcinomaAstrocytoma

Ovarian serous carcinomaHepatocellular carcinoma

Anal squamous cell carcinomaCholangiocarcinoma

Adrenal carcinomaAppendix adenocarcinoma

Gastric adenocarcinomaSalivary gland adenocarcinoma

Gallbladder adenocarcinomaNeuroblastoma

Ovarian epithelial carcinomaNon-small cell lung carcinoma (NSCLC)

Breast carcinomaPancreatic ductal adenocarcinomaPancreatic neuroendocrine tumor

Salivary gland carcinomaCarcinoma unknown primary

Lung adenocarcinomaMesothelioma

Neuroendocrine carcinomaMelanoma

GIST (Gastrointestinal stromal tumor)Anaplastic astrocytoma

SarcomaSquamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC)Esophageal adenocarcinoma

Renal pelvis urothelial carcinomaHead and neck carcinoma

Lung squamous cell carcinomaLarge cell lung carcinoma

Gastroesophageal junction carcinomaSmall cell lung carcinoma

Cutaneous squamous cell carcinomaBladder urothelial (transitional cell) carcinoma

Esophageal squamous cell carcinomaGlioblastoma

All

Included primary cancer diagnosis with N ≥ 20. See Supplemental Table 1 for complete list of cancer diagnosis with N < 20. Frequencies of aberration were calculated as a percentage of the number of tumors of that type. on March 6, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from



Figure 4: Cell cycle gene (CCND1/2/3, CDK4/6, CDKN2A/B, CCNE1 and RB1) aberrations – comparison with cBioPortal

(http://cbioportal.org).

0% 10% 20% 30% 40% 50% 60% 70% 80% 90%

Breast Invasive Carcinoma (TCGA, Cell 2015)

Breast carcinoma

Pancreatic Adenocarcinoma (TCGA, Provisional)

Pancreatic ductal adenocarcinoma

Pancreatic Neuroendocrine Tumors (Johns Hopkins University,…

Pancreatic neuroendocrine tumor

Lung Adenocarcinoma (TCGA, Nature 2014)

Lung adenocarcinoma

Mesothelioma (TCGA, Provisional)

Mesothelioma

Skin Cutaneous Melanoma (TCGA, Provisional)

Melanoma

Sarcoma (TCGA, Provisional)

Sarcoma

Head and Neck Squamous Cell Carcinoma (TCGA, Nature 2015)

Head and neck squamous cell carcinoma (HNSCC)

Esophageal Carcinoma (TCGA, Provisional)

Esophageal adenocarcinoma

Lung Squamous Cell Carcinoma (TCGA, Nature 2012)

Lung squamous cell carcinoma

Small Cell Lung Cancer (U Cologne, Nature 2015)

Small cell lung carcinoma

Cutaneous squamous cell carcinoma (DFCI, Clin Cancer Res 2015)

Cutaneous squamous cell carcinoma

Bladder Urothelial Carcinoma (TCGA, Nature 2014)

Bladder urothelial (transitional cell) carcinoma

Glioblastoma (TCGA, Cell 2013)

Glioblastoma

All (cBioPortal)

All (current report)

*

*

* cBioPortal data available for mutation only. Included cancer diagnoses with cell cycle gene aberrations in ≥ 40% of patients from current report and when cBioPortal data were available for corresponding diagnosis.



http://cbioportal.org


Figure 5. Co-existing genetic aberrations among cell cycle gene aberrations.

Odds ratios are from univariate analysis (Fisher’s exact test).

Solid black boxes indicate the association was statistically significant after multivariate analysis.




Published OnlineFirst May 11, 2016.Mol Cancer Ther Teresa Helsten, Shumei Kato, Maria Schwaederle, et al. generation sequencing: Implications for targeted therapeuticsCell cycle gene alterations in 4864 tumors analyzed by next

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