November 2016, Vol. 5, No. 9

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A One Pathway “Two-Different Hit” Hypothesis: Symbiosis in Transformation

Steven Sorscher, MD; Boris Pasche, MD, PhD, FACP; Shakti Ramkissoon, MD, PhD


The Knudson Two-Hit Hypothesis is the accepted common mechanism for transformation in patients with most familial cancer syndromes. A germline deleterious mutation in a tumor suppressor gene (TSG) increases the risk of developing specific malignancies. The gatekeeper event is loss of heterozygosity (LOH) or loss of expression of normal protein from the second copy of that same gene and resulting transformation. Here, we describe a patient with a documented germline variant of a TSG, the partner and localizer of BRCA2 (PALB2). Inheritance of an altered PALB2 is associated with an increased risk of breast and pancreatic cancer, both of which this patient had. Next-generation sequencing (NGS) demonstrated the same PALB2 variant (c.721A>G) in both the patient’s pancreatic tumor and germline and a co-occurring BRCA2 alteration, each of which showed a mutation allelic frequency (MAF) of roughly 50% in the pancreatic adenocarcinoma.

We propose that these 2 alterations together allowed for the transformation and/or progression of her pancreatic cancer. NGS has shown great promise in identifying actionable molecular abnormalities that might serve as therapeutic targets. This case appears to underscore that the “target” might be the result of partial loss of function of 2 distinct products that normally act together to maintain a nonmalignant phenotype.

Case Report

LL is a 71-year-old woman who underwent right breast-conserving surgery followed by radiation therapy in 1990 (pathology report not available) followed by no adjuvant systemic therapy. In June 2014, she underwent a right mastectomy that demonstrated a 1-cm, grade 3, invasive ductal carcinoma without lymph node sampling. By immunohistochemistry, the tumor was estrogen and progesterone receptor and HER2 negative. Her sister had had breast cancer at age 62 years. She declined adjuvant therapy.

She underwent germline testing by Myriad myRisk assay sequencing and large gene rearrangement analysis for 26 genes (including BRCA1 and BRCA2). A PALB2 (c.721A>G) variant of uncertain clinical significance was identified.

On August 10, 2015, she underwent a Whipple surgical procedure for a 3.8 × 3.4-cm lymph node–positive (3 of 15 lymph nodes) pancreatic adenocarcinoma with negative surgical margins. She received 6 cycles of adjuvant gemc­i­tabine therapy (1000 mg/m2 every 3 of 4 weeks × 6).

A chest/abdomen/pelvis CT on June 6, 2016, showed enlarged portal lymphadenopathy, an enlarging soft tissue nodule encasing the superior mesenteric artery, and a subcutaneous enhancing soft tissue nodule in the abdominal wall. A percutaneous fine-needle aspiration was “highly suspicious for adenocarcinoma.”

FoundationOne NGS clinical testing was performed on her pancreatic adenocarcinoma that analyzed 315 genes as well as introns of 28 genes involved in rearrangements and revealed alterations within ERBB3 (p.Q281H), KRAS (p.G12V), and TP53 (p.C238F). These variants occurred with low MAF in the tumor of well less than 50%, inconsistent with LOH. In contrast, for an identified altered BRCA2 (p.R2842C) gene and the PALB2 variant (c.721A>G), MAF was roughly 50% for each.


Inherited mutations resulting in loss of function of PALB2 are a relatively common cause of hereditary breast cancer. Biallelic loss of function either through germline mutations in each copy (Fanconi’s anemia) or a germline mutation and subsequent loss of function (eg, through somatic cell inactivation or LOH) in a breast or pancreatic cell is the presumed mechanism of transformation to breast or pancreatic cancer. Inheritance of a germline deleterious mutation in BRCA2 predisposes individuals to a high lifetime risk of developing pancreatic or breast cancer through a similar mechanism. The normal PALB2 gene product is critical for BRCA2 function.1-4

From her personal and family history, the patient had a high pretest probability of harboring a germline mutation in either PALB2 or BRCA2. A germline PALB2 variant that until recently has been described as being of uncertain significance was documented.5 Both her tumor and normal tissue demonstrated MAF = 50% for the PALB2 variant. However, NGS showed a somatic alteration in BRCA2, MAF = roughly 50%, occurring in tumor cells as well.

The MAF estimates the percentage of mutated copies of a particular gene in the tumor. Mutated copies would be expected to express nonfunctioning protein. LOH in a tumor is evidence of all or nearly all copies of a particular gene being altered, and the MAF would be expected to be 100% for that gene when LOH is detected. For TSGs, the resulting complete or near complete loss of expression of normal functioning TSG protein due to LOH is a typical driver event of the malignant phenotype. For example, if nearly all gene copies of PALB2 were altered in a tumor, insufficient PALB2 normal functioning protein would be expressed, and a malignant phenotype would be expected to result. Similarly, if nearly all copies of BRCA2 were altered in a tumor, there would be insufficient BRCA2 normal functioning protein expressed and the malignant phenotype would result. However, PALB2 and BRCA2 act in a common signaling pathway, and their protein products are known to be symbiotic and necessary in maintaining a benign phenotype. For the patient presented, although there was LOH for neither PALB2 nor BRCA2 (ie, MAF was roughly only 50% for each), it seems reasonable to conclude that the estimated 50% normal functioning protein expressed from both PALB2 and BRCA2 in combination was insufficient expression of normal functioning PALB2/BRCA2 proteins to prevent the resulting malignant phenotype from occurring. In this case, the partial loss of expression of normal protein from each gene was tantamount to complete loss of expression of normal functioning protein from either gene alone.

The PALB2 variant has not been proved to be deleterious. However, given the development of the pancreatic cancer with this molecular profile, it would appear to have been “deleterious” in that the BRCA2 somatic alteration with MAF = 50% in tumor cells would seem unlikely, without the loss of normal PALB2 gene product (50% variant PALB2), to be the transforming event. It remains very possible that the gatekeeper mutational event was the expression of mutated KRAS, as more than 90% of pancreatic cancers show this oncogenic abnormality, and it is generally thought to be an early event in transformation.6 Still, because of the personal and family history, it seems reasonable to conclude that PALB2/BRCA2 alterations might be fundamentally involved, either by increasing the risk of the somatic RAS mutation occurring or by contributing to the cancer phenotype as a later driver event. The identified ERBB3 and TP53 alterations may also play a role in the transformation and/or maintenance of the cancer phenotype in this tumor.

Mutualism is the form of symbiosis that refers to different organisms interacting to the benefit of both. Essentially all cancers are characterized by a constellation of many molecular abnormalities that together contribute to the cancer phenotype. The protein products (or loss of normal products) interact symbiotically, resulting in oncogenesis or progression of the cancer. Recently, NGS has shown great promise in identifying actionable molecular abnormalities that might serve as therapeutic targets. This case appears to underscore that the “target” might be the result of partial loss of function of 2 distinct products that normally act together to maintain a nonmalignant phenotype.

Olaparib is a PARP inhibitor, approved by the FDA for patients with ovarian tumors that are derived from the LOH of BRCA1/2.7 In a sense, olaparib presumably “replaces” lost TSG functions by blocking PARP activity that the cancer cell relies on in the setting of lack of BRCA1/2 wild-type gene product function. The probable partial loss of the PALB2 (which is critical for BRCA2 function) and BRCA2 wild-type gene products could similarly suggest activity for the PARP inhibitor in this case.

Regarding TSGs, molecular alterations that result in complete loss of function are generally held as the “driver” or even “gatekeeper” event. However, expression of an abnormal protein product from a single mutated copy can mitigate the tumor suppressor effect of the expressed wild-type gene product, so that LOH is not necessary for a TSG to be an important contributor to the cancer phenotype. For example, mutant p53 protein can act as an oncogene.8 The case presented appears to represent a variation on that theme. Rather than an abnormal protein expressed from the mutated TSG mitigating TSG function, an expressed abnormal protein for a gene product from the same pathway (BRCA2) “complements” the lack of normal TSG product resulting from the germ­line copy of a different gene (PALB2). The presented case reminds us that the molecular abnormalities (and their consequent expressed proteins) need to be taken together and in the context of the host genome.


  1. Antoniou AC, Casadei S, Heikkinen D, et al. Breast-cancer risk in families with mutations in PALB2. N Engl J Med. 2014;371:497-506.
  2. Sy SM, Huen MS, Zhu Y, et al. PALB2 regulates recombinational repair through chromatin association and oligomerization. J Biol Chem. 2009;284:18302-18310.
  3. Zhang F, Ma J, Wu J, et al. PALB2 links BRCA1 and BRCA2 in the DNA-damage response. Curr Biol. 2009;19:524-529.
  4. Guidugli L, Pankratz VS, Singh N, et al. A classification model for BRCA2 DNA binding domain missense variants based on homology-directed repair activity. Cancer Res. 2013;73:265-275.
  5. National Center for Biotechnology Information. ClinVar.
  6. Cerny WL, Mangold KA, Scarpelli DG. K-ras mutation is an early event in pancreatic duct carcinogenesis in the Syrian golden hamster. Cancer Res. 1992;52:4507-4513.
  7. Kaufman B, Shapira-Frommer R, Schmutzler RK, et al. Olaparib monotherapy in patients with advanced cancer and a germline BRCA1/2 mutation. J Clin Oncol. 2015;33:244-250.
  8. Strano S, Dell’Orso S, Di Agostino S, et al. Mutant p53: an oncogeneic transcription factor. Oncogene. 2007;26:2212-2219.
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